CN101132694A

CN101132694A - Medical device with coating for capturing genetically-altered cells and methods of using same

Info

Publication number: CN101132694A
Application number: CNA200580012581XA
Authority: CN
Inventors: M·J·B·库特凯; R·J·小科顿; S·M·罗兰
Original assignee: Orbus Medical Technologies Inc
Current assignee: Orbus Medical Technologies Inc
Priority date: 2004-04-30
Filing date: 2005-04-29
Publication date: 2008-02-27
Anticipated expiration: 2025-04-29
Also published as: CN102363051A; IL176853A0; CN102363051B; CN101132694B

Abstract

Therapeutic and drug delivery systems are provided in the form of medical devices with coatings for capturing and immobilizing target cells such as circulating progenitor or genetically-altered mammalian cells in vivo. The genetically-altered cells are transfected with genetic material for expressing a marker gene and at least one therapeutic gene in a constitutively or controlled manner. The marker gene is a cell membrane antigen not found in circulating cells in the blood stream and therapeutic gene encodes a peptide for the treatment of disease, such as, vascular disease and cancer. The coating on the medical device may be a biocompatible matrix comprising at least one type of ligand, such as antibodies, antibody fragments, other peptides and small molecules, which recognize and bind the target cells. The therapeutic and/or drug delivery systems may be provided with a signal source such as activator molecules for stimulating the modified cells to express and secrete the desired marker and therapeutic gene products.

Description

Medical treatment device and using method thereof with coating of cell that can be capturing genetically-altered

The application requires to be filed in the U.S. Provisional Patent Application No.60/566 on April 30th, 2004, and 829 and be filed in the U.S. Provisional Patent Application No.10/835 on April 30th, 2004,767 priority.

Technical field

The present invention relates to be used to implant patient vessel or hollow organ's medical treatment device, for example be used for the treatment of the support through applying, stent graft of various diseases, synthetic blood vessel graft, cardiac valves, conduit and blood vessel are repaired screen (vascular prosthetic filter).Particularly, the present invention relates to surface that blood contacts on have coating medical treatment device, described coating can be used for the surface of cell capture in described device through engineered.Captured cell can form individual layer and can be used for many therapeutical uses on the surface of described device, for example in drug delivery system and/or the treatment vascular disease.For example, the cell that is incorporated into the medical treatment device of implantation can be the natural endothelial progenitor cells of self-loopa blood and/or at external genetically modified cell, described cell has the molecule or the material of part or whole body therapeutic effect in patient's expression in vivo or secretion.

Background of invention

Atherosclerotic in the disease and cancer are two kinds of main causes that cause dead and disability in the world.Atherosclerotic produces relevant with the lip-deep fatty spew of lumen of artery.These fatty spews cause the lumen of artery narrow in cross-section.Finally cause blood flow to reduce, cause being subjected to the tissue generation ischemia injury of this artery blood supply at the diseased region far-end.

Coronary artery is to heart blood supply.In the U.S., coronary atherosclerosis or coronary heart disease (CAD) are modal life-threatening severe chronic diseases, suffer from this sick number and surpass 1,100 ten thousand people.Society that coronary atherosclerosis causes and economic loss are considerably beyond most of other diseases.The narrow influence to cardiac muscle of coronary arterial lumen at first causes angina pectoris, is miocardial infarction then, causes death at last, has 300,000 people above just dead before arriving at hospital among these patients.(" HarrisonShi internal medicine principle", Harrison ' sPrinciples of Internal Medicine, the 14th edition, 1998).

For coronary atherosclerosis, can adopt through percutaneous transluminal coronary internal shaping art (PTCA) and treat.The annual above PTCA of 400,000 examples that implements of the U.S. performs the operation.In PTCA, balloon catheter is inserted in the peripheral arterial, be advanced to the coronary artery that is blocked along arterial system.Air bag is expanded, this section arterial is strutted, flatten the fatty patch that blocks this place, thereby increase the blood flow in this impaired artery cross section of flowing through.Yet this treatment can not make impaired coronary artery keep open configuration enduringly usually.Nearly 50% the needs of patients of accepting the PTCA treatment was accepted repetitive therapy to correct ISR coronarius in 6 months.The narrow ISR that medically is called that this PTCA of acceptance artery takes place once again.Acute ISR relates to the resilience and the shrinkage of blood vessel.After blood vessel resilience and the shrinkage, propagation takes place to tackle by the caused arterial injury of PTCA in the middle level smooth muscle cell.The propagation part of smooth muscle cell is mediated by the various inflammatory factor that damage location discharges, and this class inflammatory factor comprises: thromboxane A2, platelet derived growth factor (PDGF) and fibroblast growth factor (FGF).Adopted multiple different technologies to overcome the ISR problem, these technology comprise: the patient is treated or keep artery to open with rack mechanical ground with various medicaments.(" HarrisonShi internal medicine principle ", Harrison ' s Principles of Internal Medicine, the 14th edition, 1998).

Proved that support is to overcome effective method in the various methods of treatments of ISR.Support is to be placed in the metal supporting frames that produces the normal blood vessels inner chamber in the diseased arteries section.Can prevent the resilience of this artery and the blood vessel graft that takes place then in the impaired artery section with propping up to be placed on.Support can prevent that also this artery from peeling off along medial part.By make arterial lumens keep having reduced nearly 30% ISR with support greater than adopting the formed inner chamber of PTCA separately.But although obtain such success, support still can not be eliminated ISR fully.(Suryapranata etc., 1998." for comparing at random of coronary artery bracket art in the patients of acute myocardial infarction through selecting and balloon angioplasty ", Randomized comparison ofcoronary stenting with balloon angioplasty in selectedpatients with acutemyocardial infarction Circulation, 97:2502-2502).

Arteriarctia also can occur in other blood vessels outside the coronary artery, comprises artery under sustainer bone artery, the groin, the dark femoral artery in distally, Yuan Ce popliteal artery, tibial artery, subclavian artery and mesenteric artery etc.The atherosclerosis of peripheral arterial (PAD) illness rate depends on the impaired concrete region of anatomy, and used standard is blocked in diagnosis.Traditionally, the doctor adopts the Charcot's syndrome method of testing to judge whether to have PAD.Yet this method may have been underestimated the actual incidence of disease of this disease among the crowd greatly.The incidence of disease of PAD is different with the age, and the incidence of disease of older individuals PAD increases.Estimate to have every year 55, the 000 routine male sex and 44,000 routine women to be diagnosed as chronic PAD first according to U.S.'s state hospital investigation data of leaving hospital; And have 60, the 000 routine male sex and 50,000 routine women to be diagnosed as acute PAD first.91% acute PAD case all relates to the lower limb position.In PAD patient, the ratio of the CAD that occurs together may surpass 50%.In addition, in PAD patient, the cranial vascular disease incidence of disease increases.

Can adopt percutaneous transluminal balloon angioplasty (PTA) to treat PAD.Adopt PTA and, can reduce the restenosis incidence of disease in conjunction with support.Yet, at the postoperative effect that medical treatment device obtained that adopts such as support, the effect that is obtained not as the revascularization of employing standard (that is, adopt vein or repair and use the bypass material).(" Surgery Principle", Principles of Surgery, volumes such as Schwartz, the 20th chapter, " arterial disease ", Arterial Disease, the 7th edition, McGraw-Hill Health Professions Division, New York 1999).

The preferred method treatment PAD that sets up bypass that adopts walks around the obstructing arterial section with graft in the method and sets up bypass.(" Principles of surgery", Principles of Surgery, volumes such as Schwartz, the 20th chapter, " arterial disease ", Arterial Disease, the 7th edition, McGraw-Hill Health Professions Division, New York 1999).Described graft can comprise: autogenous vein section (for example, saphena) or synthetic graft (for example, the graft for preparing with polyester, polytetrafluoroethylene (PTFE) or expanded polytetrafluoroethyltoe (ePTFE) or other various polymeric materials).Its postoperative vessel open rate depends on multiple different factor, and these factors comprise: the chamber internal diameter of bypass graft, as the synthetic material type of graft, and flow out the position.Yet even if adopted bypass graft, neointimal hyperplasia and thrombosis remain serious problem.For example, arterial bypass is after 3 years under the groin of employing ePTFE bypass graft, and the vessel open rate of burst-popliteal bypasses is 54%, and the bypass of thigh-shin then only is 12%.

Therefore, obviously need improve, with the incidence of disease and the case fatality rate of further minimizing CAD and PAD to support performance, synthetic bypass graft and other long-term blood contact surfaces and/or device performance.For example, can adopt to make the method for blood vessel radial dilatation (outside or positivity is moulded again) compensate the carrying out property growth of atherosclerotic plaque, thereby postpone the development of blood flow limited narrow (flow-limiting stenosis).

For support, the method that has adopted is to apply support with various anticoagulants or anti-restenosis agent, to reduce thrombosis and ISR.For example, as if can suppress ISR with the radioactive substance impregnated stents by migration and the propagation that is suppressed to myofibroblast.(U.S. Patent number 5,059,166,5,199,939 and 5,302,168).But the blood vessel that irradiation is treated can cause the edge ISR problem that the patient is serious.In addition, irradiation is handled inhomogeneous to encroaching on blood vessel.

Perhaps, also adopted chemical agent to apply support, for example heparin, phosphocholine, rapamycin and taxol, all these medicines all show can extenuate thrombosis and/or ISR.As if can obviously reduce the thrombosis of animal model in short-term though heparin and phosphocholine show, this class medicament does not have long-time effect to prevention of restenosis.In addition, heparin may bring out thrombopenia, causes serious thrombus complication, for example apoplexy.Therefore, in the practice mode of this treatment ISR, also infeasible with the support that heparin of enough treating effective dose or phosphocholine load.

The ISR and the thrombosis that have adopted synthetic graft to treat in many ways to reduce postoperative.(Boe etc., 1998." small diameter vascular shifting plant prosthetic: present situation ", Small-Diameter Vascular GraftProstheses:Current Status, Archives Physio.Biochem,106:100-115).For example, it is reported that it is suitable with the ePTFE graft that polyurethane synthetic (for example, porous Merlon urethanes) reduces the effect of ISR.Also adopted radio frequency glow discharge that modification is carried out on the surface of described graft, the dibenzoate graft has been fluoridized so that gather.Also adopted the synthetic graft of biomolecule dipping.Yet none can significantly reduce thrombosis or ISR incidence over a long time these methods.

Endothelial cell (EC) layer is a kind of important component of normal blood vessels wall, and it provides the interface between blood flow and the vascular wall surrounding tissue.Endothelial cell also can participate in physiological activity, comprising: angiogenesis, inflammatory process and prevent thrombosis (Rodgers GM.FASSEB J 1988; 2:116-123).Discovering in recent years, endothelial cell is except forming vascular system, and postnatal EC and endothelial progenitor cells (EPC) also circulate in peripheral blood.(Asahara T etc.Science, 1997; 275:964-967; Yin AH, etc.Blood, 1997; 90:5002-5012; Shi Q, etc.Blood, 1998; 92:362-367; Gehling UM, etc., Blood, 2000; 95:3106-3112; Lin Y etc., J Clin Invest, 2000; 105:71-77).It is believed that EPC can migrate to the impaired endodermis of circulatory system zone, comprises wound and ischemic injury position (Takahashi T etc., Nat Med, 1999; 5:434-438).In normal adult, the EPC concentration in the peripheral blood is 3-10/mm ³(Tkahashi T etc., Nat Med, 1999; 5:434-438; Kalka C, etc., Ann Thorac Surg, 2000; 70:629-834).Nowadays verified, each stage that blood vessel is replied for damage all is subjected to the influence (if not controlling) of endothelial tissue.It is believed that; impaired and settle the blood vessel section of support to rebuild the ability of the various materials that functional endodermis can be by harmful effect that the barrier to the circulating cells factor, anti-tampon are provided and the generation protection lower floor layer of smooth muscle cells that they had rapidly, and help the severe complication that prevents that these are potential.(Van Belle etc., 1997; " the interior dermatoplasty of support ", Stent Endothelialization, Circulation, 95:438-448; Bos etc., 1998." small diameter vascular shifting plant prosthetic: present situation ", Small-Diameter Vascular Graft Prostheses:Current Status, Archives Physio. Biochem.106:100-115).

Behind implant frame,, can promote growth (Van Belle etc., 1997 of endothelial cell at rack surface by local delivery vascular endothelial growth factor (VEGF, a kind of endothelial cell mitogen); " the interior dermatoplasty of support ", Stent Endothelialization, Circulation,95:438-448).Bring out required action effect though can use the saline solution of recombinant protein growth factor VEGF at damaged part, VEGF can send with the passage balloon catheter behind implant frame.But this technology is unsatisfactory, and this is because it is confirmed that the too low effect that can not produce unanimity of the effect of single delivery dosage.Thereby this method can not accurately be reappeared effect at every turn.

Also adopted endothelial cell seeding synthetic material graft, but the clinical effectiveness of plantation endothelial cell is all very poor usually, the tube chamber that is postoperative opens the low (Lio etc. of rate, 1998, " new ideas of capilary transplantation and new material: endothelial cell seeding and the gene therapy of reparation property graft ", New concepts and Materials inMicrovascular Grafting:Prosthetic Graft Endothelial Cell Seeding and GeneTherapy, Microsurgery, 18:263-256), its reason is that cell is failed properly to be attached on the graft and/or because isolated operation causes cell to lose its EC function most probably.

Thereby in the adjusting of sticking, growing and breaking up of vascular injury site endothelial cell, original position endothelial cell growth factor (ECGF) and environmental condition are vital.In view of the above, for ISR and other vascular disease, be necessary to develop the new method and composition of the medical treatment device that is used to apply the graft that comprises support and synthesize, to promote and acceleration functional endothelium of formation on implanted device, thereby on the inner chamber of target vessel section or implantation, form continuous EC individual layer, suppress the new intima hyperplasia thus.

About diseases such as cancers, present most of medicine can produce the whole body systemic reaction to the patient, owing to use the medicine of conventional oral or intravenous form, not only can influence cancer cell, also can influence any fissility cell (dividing cell) in the body.In many cases, because the character of the disease that need treat and the character (for example dissolubility, body internal stability, bioavilability etc.) of medicine, the general administration is ineffective.When the general administration, medicine is through blood circulation transportation and be distributed into the body area that comprises normal structure.At diseased region, originally drug concentration low and invalid, can bring up to toxic level after the frequent drug administration, and in non-lesion region, the existence of medicine then causes bad side effect.In some cases, medicine is easy to be subjected to metabolic degradation after administration.Therefore, often obtain drug effect and time expand by improving drug dose, this just cause the normal structure system burden increase and with the increase of patient's correlative charges.In other cases, because the toxic and side effect of some active drug can not be brought into play its treatment potential fully.

Therefore, done effectiveness and the target that many effort improve drug delivery system.For example, the advantage that adopts the liposome delivery medicine to have be usually their can prolong drug in blood circulation timei, reduce side effect, reduce drug degradation, prolong therapeutic action after each administration, reduce by free drug concentrations in the restriction of blood flow the demand of administration frequency and the amount that reduces required medicine.Yet present available liposome system shows that the effect that delivers drugs into target site in vivo is limited.Referring to Kaye etc., 1979, Poznansky etc., 1984, United States Patent (USP) 5,043,165 and United States Patent (USP) 4,920,016.

For realizing that efficiently the sending of therapeutic compound developed the viral vectors that can mix transgenosis DNA, yet the quantity of clinical practice success is still limited.The successful number that in external and animal model, is obtained, proposed gene transfer technique is combined with cell therapy.Ex vivo gene transfer is gone into might prove in the various cell types than direct body and function carrier transfer to have bigger treatment feasibility.Referring to Kohn etc., 1987, Bilbao etc., 1997 and Giannoukakis etc., 2003.

Recently, developed the localized drug delivery carrier, for example bracket for eluting medicament (drug elutingstent, DES).Referring to United States Patent (USP) 6,273,913, United States Patent (USP) 6,258,121 and United States Patent (USP) 6,231,600.Yet bracket for eluting medicament of the prior art is subjected to the restriction of many factors, for example, and the time of drug type, the medication amount that will discharge and release medicine.The other factors that need consider about bracket for eluting medicament is that medicine and other support apply the interaction between composition (for example polymeric material) and the character (for example integrality after lipophilicity, molecular weight, the sterilization and activity and effectiveness and toxicity) of individual drugs.As for the polymeric material in the bracket for eluting medicament, what must consider is type, polymerization ratio, the ability of drug loading and the biocompatibility of this polymer of polymer, and the compatibility of drug-polymer (for example, the pharmacokinetics of medicine).

In addition, the drug dose in the bracket for eluting medicament is a preloaded, can not realize the drug dose of individual condition and demand is regulated.As for pharmaceutical release time, bracket for eluting medicament begins to discharge medicine after in a single day implanting immediately, and the real-time release that can not realize ideal.

Therefore, press for and develop a kind of effective general and local drug delivery system, to overcome the defective of current available technologies.The present invention just provided a kind of peace and and controlled way carry out the system of part or systemic delivery medicine.

Summary of the invention

An object of the present invention is to provide the method for a kind of curative drug delivery system and treatment patient disease.This treatment or drug delivery system comprise the medical treatment device with coating, this coating is by containing that at least one class can be discerned and form in conjunction with the matrix of the part of target cell, and described target cell is for for example: the mammalian cell of endothelial progenitor cells or hereditary change and the mammalian cell that has been subjected to the hereditary change of single or dual transfection.

Medical treatment device of the present invention can be any implantable patient's device.For example, in one embodiment, described device is the device that can insert intravascular space or hollow organ, repair screen, artificial heart, external and built-in left ventricular assist device (LVAD) and synthetic vessel graft as support, support implant, cardiac valves, conduit, blood vessel, they are used for the treatment of following disease: cancer for example; Vascular disease comprise ISR, atherosclerotic, thrombosis, angiemphraxis; Or these install other any purposes that is covered.

In one embodiment, coating on the described medical treatment device comprises: biocompatible matrix and at least a substrate or part, described substrate or ligand specificity identification and in conjunction with target cell (for example endothelial progenitor cells), with for example prevention or treatment ISR, or the mammalian cell that makes hereditary change adheres to the surface of described device, for example to treat vascular remodeling and cancer.

In addition, the coating of described medical treatment device can randomly comprise and at least aly can regulate the engineered gene expression of hereditary change cell and the reactive compound of secretion.The example of the activator of energy activated compounds includes but not limited to: chemical molecular and peptide, for example growth factor.Comprise in the embodiment of at least a compound in coating, stimulus, anakmetomeres or compound can be used for the irritation cell expression and/or secrete at least a treatment of diseases thing for the treatment of.

In one embodiment, coating on the medical treatment device comprises the biocompatible matrix that has outer surface, described outer surface be used to adhere at least a part (for example, the combination of antibody, antibody fragment or antibody and antibody fragment) for the treatment of effective dose or at least a can be in conjunction with the molecule of the engineered mark of genetically altered cell surface.The identification of antibody of the present invention or antibody fragment and in conjunction with the antigen on cell membrane or target cell surface or specificity through genetically engineered cell surface marker, thereby make the surface of cell fixation at described device.In one embodiment, described coating can randomly comprise at least a compound that the stimulating endothelial CFU-GM is fixed that is used for of effective dose, if target cell is the circulation CFU-GM, then can promote the formation of maturation, functionalization endothelium, if perhaps target is the hereditary change cell on the surfaces of medical devices, then can stimulate the required gene outcome of cellular expression justacrine of combination.

Medical treatment device of the present invention can be any device that is used to implant the organ that comprises cavity or body part, also can be but is not limited to: support, stent graft, synthetic blood vessel graft, cardiac valves, conduit, blood vessel is repaired screen, pacemaker, the pacemaker lead, defibrillator, in hole, the ovum garden patent (PFO) every closing device, blood vessel clip, the vascular aneurysms dead lock, the haemodialysis graft, hemodialysis catheter, the chamber current divider, aortic blood tuberculation graft device or assembly, venous valve, suture, the vascular anastomosis folder, remain-type vein or arterial duct, vagina vasorum and medicine delivery port.According to the difference of device, described device can be made from a variety of materials.For example, support of the present invention can be made with stainless steel, Nitinol (NiTi) or evanohm and Biodegradable material.Synthetic blood vessel graft can be made with crosslinked PVA hydrogel, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethyltoe (ePTFE), porous type high density polyethylene (HDPE) (HDPE), polyurethane and poly terephthalic acid vinyl acetate or Biodegradable material.

Form described medical treatment device biological compatibility of coating matrix including but not limited to synthetic material, for example, polyurethane, block polyurethane-urea/heparin, poly--L-lactic acid, cellulose esters, polyethylene glycol, polyvinyl acetate, glucan or gelatin; And/or naturally occurring material, the basement membrane composition of collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin and so on for example, heparin, fibrin, cellulose and amorphous carbon or fullerene (fullerene).

In an embodiment of the invention, contain the biocompatible matrix that comprises fullerene in the described medical treatment device.In this embodiment, the carbon number of fullerene can be about C ₂₀-C ₁₅₀, more specifically, described fullerene is C ₆₀Or C ₇₀Fullerene of the present invention also can be arranged in nanotube (nanotube) on the surface of medical treatment device.

In an embodiment of the invention, described part is put on the surface that medical treatment device contacts with blood, described part can specific recognition and in conjunction with the lip-deep epi-position of required component or target cell in the circulation blood.In one embodiment, described part be specifically designed as by on the cell membrane of only discerning the hereditary change cell through genetically engineered labelled molecule, and can only discern and in conjunction with the mammalian cell of hereditary change.The combination of target cell with this cell fixation the device the surface on.

In one embodiment, according to the part of selecting through genetically engineered cell membrane labelled molecule to be used on the surfaces of medical devices in conjunction with genetically-altered cells.That is to say that described part only combines with the cell membrane labelled molecule or the antigen of being expressed by the extrachromosomal genetic element that offers this cell, thereby makes the part on the described surfaces of medical devices only discern genetically modified cell.In this way, have only genetically modified cell just to can be incorporated into the surface of medical treatment device.For example, if described mammalian cell is an endothelial cell, described part can be at least a antibody, antibody fragment or their combination; Produce described antibody specifically at lip-deep specific target epi-position of target cell or labelled molecule.In this one side of the present invention, described antibody can be monoclone antibody, polyclonal antibody, chimeric antibody or humanized antibody, it is by the only identification and combine the endothelial cell of this hereditary change with the surface markers intermolecular reaction of the endothelial cell of hereditary change, and regulates this cell thus so that it adheres to surfaces of medical devices.Antibody of the present invention or antibody fragment covalently or non-covalently can be connected in the surface of matrix, or pass through the covalently bound outermost layer that arrives the matrix of coated medical devices of linkers.In this embodiment, for example, monoclone antibody also can comprise Fab or F (ab ') ₂Fragment.Antibody fragment of the present invention comprises the fragment of any size, for example, has kept antibody recognition and in conjunction with the big molecule and the little molecule of target antigen characteristic.

In another embodiment, the mammiferous antigen that antibody of the present invention or antibody fragment capable are discerned specifically and combination is treated, and their specificity does not depend on cell-line.In one embodiment, for example, in the treatment ISR when cell not genetically modified and contain the specific cell membrane marker and divide the period of the day from 11 p.m. to 1 a.m, described antibody or fragment can be selected and specifically in conjunction with the surface antigen of circulation endothelium progenitor cell, for example CD133, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146), CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit part), Tie-2, MHC (for example, H-2K ^kAnd HAD-DR).

In another embodiment, the coating of described medical treatment device comprises the above-mentioned biocompatible matrix of one deck at least, and this matrix contains and is useful on the outer surface that adheres at least a natural or synthesized micromolecule for the treatment of effective dose.Described little molecular energy identification for example, the endothelial progenitor cells in the treatment of restenosis, and with its interaction, thereby and this cell fixation is formed endodermis at apparatus surface.Described little molecule can be used for and this medical treatment device therapeutic alliance various diseases, and can be derived from various sources (cell component for example, as fatty acid, protein, nucleic acid, carbohydrate etc.), and can produce result or the effect identical with the lip-deep AI of endothelial progenitor cells with antibody.Of the present invention this on the one hand, but the inclusion compound also of the coating on the medical treatment device for example is connected in the above-mentioned growth factor of this paper on the coating that comprises antibody or antibody fragment.

In one embodiment, the compound in the coating of the present invention (for example being used for treatment of restenosis) comprises stimulating or to promote growth of progenitor cells and differentiation to become any compound of ripe functional endothelial cell.In another embodiment, described compound is used to stimulate the genetically modified required gene outcome of cellular expression justacrine.For example, the compound that is used for the present invention can be growth factor, for example vascular endothelial growth factor (VEGF), basic fibroblast growth factor, blood platelet growth factor, transforminggrowthfactor-, acid fibroblast growth factor, osteonectin, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2), IDGF (insulin-like growth factor), granulocyte macrophage colony stimulating factor, platelet derived growth factor AA, platelet derived growth factor BB, platelet derived growth factor AB and the endothelium PAS albumen 1 of inducing.

In another embodiment, for example when using the mammalian cell of hereditary change, can be used for irritation cell and express activator or the compound of justacrine through genetically engineered gene outcome, include but not limited to: oestrogenic hormone, tetracycline and other antibiotic, Tamoxifen etc., can offer the patient through various methods of administration, for example pass through percutaneous drug delivery with paster and subcutaneous form.

The present invention also is provided for treating the method for various diseases, and described disease is for for example: vascular disease, cancer, vascular remodeling, serious coronary heart disease, atherosclerotic, ISR, thrombosis, aneurysm and angiemphraxis.In one embodiment, provide a kind of be used to keep or seal insert vascular wall medical treatment device (for example, support or synthetic blood vessel graft, cardiac valves, abdominal aortic aneurysm device and their parts) and set up environment in the blood vessel stable state, thus prevent method as the excessive endometrial hyperplasia in the ISR.In the atherosclerotic the inventive method of treatment, described artery can be coronary artery or peripheral arterial (for example femoral artery).Also available these technology and medical treatment device treatment vein.

For the treatment of ISR, the present invention also provides a kind of engineered method that is used to induce healing reaction.In one embodiment, a kind of method that is used for the endodermis that rapid induction formation is merged on the luminal surface of the implanted device of grafting vessel target region is provided, and wherein said endothelial cell can be expressed nitricoxide synthase and other anti-inflammatory and inflammation regulatory factor.The present invention also provides a kind of medical treatment device, it has higher biocompatibility than prior-art devices, and can be by reducing or suppressing smooth muscle cell migration, smooth muscle cell differentiation and along the collagen deposition of the surface of internal cavity of medical treatment device implant site, the excessive endometrial hyperplasia and the ISR that reduce or suppresses to organize.

In one embodiment, the method that is used for coated medical devices may further comprise the steps: will be at least one deck biocompatible matrix put on surfaces of medical devices, wherein said biocompatible matrix comprises at least a component that is selected from down group: polyurethane, block polyurethane-urea/heparin, poly--L-lactic acid, cellulose esters, polyethylene glycol, gather ethyl acetate, glucan, gelatin, collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose and carbon and fullerene; And simultaneously or apply in succession: at least a antibody, antibody fragment or their combination of treatment effective dose, and the compound of growth of at least a stimulating endothelial cell and differentiation to described biocompatible matrix.

The present invention also provides a kind of method for the treatment of mammal blood vessel disease, described method comprises: medical treatment device is implanted in mammiferous blood vessel or the pipe inner chamber, wherein said medical treatment device is coated with (a) biocompatible matrix, (b) at least a antibody, antibody fragment or their combination of treatment effective dose, and (c) at least a compound; Wherein said antibody or antibody fragment capable identification and in conjunction with the lip-deep antigen of endothelial progenitor cells, thus endothelial progenitor cells is fixed on the stromal surface, and described compound then is used to stimulate fixing endothelial progenitor cells to form endothelium on surfaces of medical devices.

Treatment/the drug delivery system of treatment patient disease also is provided in one embodiment.Described treatment or drug delivery system contain the mammalian cell of hereditary change and are used to implant patient's medical treatment device, and the mammalian cell of wherein said hereditary change comprises coding through the genetically engineered cell surface marker and the exogenous nucleic acid of at least a therapeutic gene product.In one embodiment, with the suitable described genetic engineering modified cell of the transfection carrier in-vitro transfection that comprises exogenous genetic material, for described cell provides required gene.In this embodiment, described cell can be any mammalian cell, no matter from body, allochthonous or xenogenesis, for example endothelial cell, fibroblast, sarcoblast etc.In this embodiment, described medical treatment device applies with biocompatible matrix, described matrix comprises part, and described part passes through combination through genetically engineered cell membrane labelled molecule or the antigen on the cell surface, and only is incorporated into the mammalian cell of hereditary change.

In treatment of the present invention and/or drug delivery system, described genetically-altered cells has exogenous genetic material and has imported the required gene of at least a Codocyte surface markers molecule or antigen and the gene of at least a coding therapeutic gene product.This system option ground comprises signal system, for example can stimulate the mammalian cell expression of hereditary change and/or secrete the reactive compound or the molecule of required gene outcome and/or marker gene.

Therefore, in one embodiment, but with import in the mammal described exogenous genetic material through engineered be the cell membrane mark of part on the codified specificity combining device.For example, when described device was used to insert intravascular space, this exogenous genetic material should be encoded except offering patient's the cell membrane mark that does not all have in any cyclicity cell in auterblood through genetically engineered cell.

A kind of medical treatment device and method through applying that is used for the treatment of various diseases also is provided, and described disease is: vascular disease for example include but not limited to atherosclerotic, cancer and rheumatoid arthritis.Medical treatment device of the present invention comprises and is used in vivo that specificity is caught and the fixing coating of the mammalian cell of hereditary change that the mammalian cell of described hereditary change is to import simultaneously or subsequently when described medical treatment device through applying is implanted the patient.

Also provide and be used for fixing expression and/or secrete at least a material of specific disease or the fixing hereditary change cell of therapeutic agent of being used for the treatment of.In this one side of the present invention, for example in the treatment cancer, make described cell (for example, endothelial cell) that hereditary change take place by in described cell, importing exogenous genetic material.In one embodiment, in described genetic material transfered cell nuclear, and it is DNA (a for example exchromosomal DNA).Described exchromosomal DNA can be carrier, for example adenovirus vector, plasmid (as the gymnoplasm grain), linearity or short dna etc.In one embodiment, described DNA comprises regulation and control/expression cassette that required mark and/or therapeutic gene are expressed in control.In one embodiment, described regulation and control box can comprise the controlling element that is used for constructive expression's therapeutic gene, and maybe can comprise can be by needs of patients control or the element of expressing.

In one embodiment, the described medical treatment device that is used to implant the patient comprises coating; Described coating comprises can identification and in conjunction with the part of target cell by matrix load at least a.At cell is in the embodiment of genetically-altered cells, and described part is only discerned and in conjunction with through engineered and add the specific cell membrane marker molecule or the antigen of cell.Therefore in this embodiment, part is only discerned the mammalian cell of the hereditary change that has imported the patient, the mammalian cell of this hereditary change is incorporated on the described medical treatment device, expresses justacrine labelled molecule or antigen and at least a therapeutic gene product.

In another embodiment, described treatment or drug delivery system also can comprise activating molecules (activatingmolecula), and this activating molecules is used to stimulate the mammalian cell expression of described hereditary change and/or secretes required therapeutic gene product.In this one side of the present invention, can will supply with the patient such as the compound of chemical irritant or peptide by several different methods, these methods comprise: oral route, warm paster (thermal patch), intravenous, intracutaneous injection etc.In this embodiment, the mammalian cell of described hereditary change can be from body or external source, for example mature endothelial cell, fibroblast, myocyte, epithelial cell etc., the exogenous nucleic acid that they comprise can be exchromosomal DNA.In one embodiment, described DNA provides with carrier format, for example adenovirus vector, naked plasmid dna, linear DNA etc.In one embodiment, described exchromosomal DNA comprises the regulation and control box, and promptly the gene of Codocyte membranous antigen and at least a coding are used for the treatment of the gene of the peptide of disease.In aspect of this embodiment, described cell membrane specific gene coding, for example BMP or prostatic cell memebrane protein.

In one embodiment, described extrachromosomal genetic element comprises the gene of the treatment/pharmaceutical product of encoding, and these products are for example to be used for the vascular endothelial growth factor and the angiogenesis factor of vascular remodeling, or is used for the treatment of the anti-angiogenesis of cancer.

In another embodiment, provide a kind of method that is used for the treatment of disease.Described method comprises:

The mammalian cell of hereditary change is offered the patient; Described cell comprises coding through genetically engineered cell membrane labelled molecule and at least a therapeutic gene product exogenous nucleic acid;

The medical treatment device that will comprise coating is implanted the patient; The matrix of at least a part that described coating has comprised load, wherein this part can discern and in conjunction with on the mammalian cell of described hereditary change through genetically engineered cell membrane labelled molecule, the mammalian cell of wherein said hereditary change is incorporated into medical treatment device, and expresses and secrete described therapeutic gene product.In an embodiment of the invention, described therapeutic genes and gene outcome comprise for example vascular endothelial growth factor, angiogenesis factor, anti-angiogenesis and fibroblast growth factor.

The present invention also provides the method for treatment patient disease, and described method comprises: the mammalian cell of hereditary change is offered the patient; Medical treatment device is implanted the patient; Described medical treatment device comprises coating, described coating comprises the matrix that load has at least a part, described part can specific recognition and in conjunction with at least a labelled molecule, the acceptor on the mammalian cell of this hereditary change for example, the mammalian cell of wherein said hereditary change is incorporated on the described medical treatment device, comprises the exogenous nucleic acid that is used to express and secrete therapeutic gene product.

In another embodiment, provide a kind of method that is used in vivo cell being raised the surface that contacts blood.Described method comprises provides the surface that is arranged in object blood flow contact blood, and the surface of described contact blood is through setting up the surface that the cyclicity target cell in the object blood flow can be raised described contact blood; And the surface of described target cell being raised described contact blood.In this embodiment, the surface of described contact blood comprises the surface of internal cavity of the medical treatment device of implanting described object.In this embodiment of the present invention, the target cell of being raised on the surface (for example support or graft) of contact blood can recover the normal endothelial tissue with surface self endothelialization (self-endothelialize) of described device and at the damaged blood vessels position.The surface of described contact blood can be the biodegradable framework maybe can be coated with biodegradable biocompatible materials.In this one side of the present invention, described biodegradable framework will be degraded when implantable intravascular in position, and the newborn endothelium that forms on described device inner chamber has been rebuild the coherent blood vessel by the injury, thereby form functional new vessels.

In another embodiment, the present invention includes a kind of dummy, described dummy comprises: (a) have outer surface and the carrier film that contacts the blood surface; (b) be coated on the lip-deep ground floor cross-linked polymer compound of contact blood of described carrier film; (c) be coated on the second layer on the ground floor, the described second layer contains at least a part that in vivo target cell is had compatibility.

In another embodiment, a kind of method that is used for generating in vivo self endothelialization graft is provided, described method comprises: (a) provide the function of setting up to be the framework as blood vessel graft, described framework has surface of internal cavity and outer surface, and described surface of internal cavity comprises specificity and is used for part in conjunction with endothelial progenitor cells; (b) described framework is implanted in the blood vessel of object; (c) the cyclicity endothelial progenitor cells is raised on the described surface of internal cavity of described framework to form newborn endothelium.

In another embodiment, provide a kind of method that is used for original position generation self endothelialization graft, described method comprises: the reparation construction that the surface with contact blood circulation (a) is provided; (b) described reparation construction is implanted object; (c) from blood, raise cyclicity cell (for example, the mammalian cell of endothelial progenitor cells and hereditary change) and make on its surface that is attached to described reparation construction, thereby form newborn endothelium thereon.

In another embodiment, provide the method for original position generation self endothelialization graft, described method comprises: (a) provide the function of setting up to be the biodegradable framework as interim blood vessel graft, described framework has surface of internal cavity and outer surface; (b) with in the described biodegradable framework implantable intravascular; (c) raise circulating cells (for example mammalian cell of endothelial progenitor cells and hereditary change) and make it to be attached on the surface of internal cavity of described dummy (for example graft, support or biodegradable framework), thereby form newborn endothelium; (d) make vascular tissue wrap up the outer surface of described framework to form outside hemostasis blood vessel structure; (e) in vivo under the condition, can make described newborn endothelial tissue and outside blood vessel structure form in the time period of functional new vessels, described biodegradable framework is degraded.

In one embodiment, provide a kind of biodegradable framework that original position forms the endothelialization blood vessel graft that is used for, described framework comprises: the porous biological degradable carrier film that (a) has surface of internal cavity and outer surface; (b) described surface of internal cavity comprises the ground floor of being made up of at least a polymerizable compound that is coated on the described carrier film, wherein said compound can form covalent bond through the crosslinking agent self-crosslinking, described covalent bond can be subjected to enzymatic lysis or non-enzymatic hydrolysis under the condition and in vivo (c) to have the part of specificity affinity in the body in conjunction with the mammalian cell of hereditary change.

In another embodiment, provide a kind of method that original position produces self endothelialization graft that is used for, described method comprises: (a) provide to have the circulate reparation construction on surface of blood of contact patient; (b) described reparation construction is implanted object or patient; (c) mammalian cell with hereditary change gives described patient; (d) from blood, raise cyclicity cell (for example mammalian cell of hereditary change) and make on its surface that is attached to described reparation construction, thereby on the surface of described reparation construction, form one deck genetically-altered cells.

In another embodiment, a kind of method that promotes vascular remodeling is provided, for example the girth that increases artery by outside or positivity reconstruct to be partly or entirely compensating because of forming that atherosclerotic plaque causes or the injured back of artery is occupied because of the inner chamber that the inner membrance paraplasm causes, thus prevent or suppress damaged blood vessels inwardly or negativity reconstruct.In this embodiment, for example providing aforesaid is covered and can be in conjunction with the support of genetic engineering modified cell by matrix and part, to be used to catch genetically modified autogenous cell (for example endothelial progenitor cells), these cells can be secreted at least a potent anticoagulant and vasodilator, for example: prostacyclin, as prostaglandin 12, PG12; CGRP is as α-CGRP etc.Can comprise nitric oxide (nitric oxide synthase gene), matrix metalloproteinase, acetylcholine, adenosine, serotonin, P material (substance P), adrenomedulin etc. through engineered other product that produces by cell.Can adopt its product as vasodilator and/or anticoagulant or have vasodilator and/or the gene of anticoagulant characteristic, for example can cause the vasodilator of vascular smooth muscle relaxation.Can offer endothelial progenitor cells or endothelial cell by will the encode gene (for example prostacyclin synthase gene) of vasodilator of gene transfer technique, described gene transfer technique can be the viral gene that for example adopts cistron (cistronic) gene constructs and shifts, with regard to prostacyclin, for example cistron cyclooxygenase-1/ prostacyclin synthase gene constructs can provide continuous local prostaglandin to send.In this embodiment, prostaglandin local delivery system for example can be used for treatment, cerebral infarction and coronary vessels diseases.Also the positivity reconstruct of blood vessel can be generated (being ripe blood vessel, the formation of for example be grown up arteriole and artery) to form the methods of treatment of attached blood vessel as regulating artery.

In another embodiment, the cell that the bicistronic mRNA carrier transfection of available code vasodilation compound and unique cell surface marker (for example MHC-I of brachymemma) is suitable, as fibroblast, endothelial cell or endothelial progenitor cells, described cell surface marker can be by part (for example being fixed in the antibody on the dummy in the blood vessel) identification.For example, the support that part (as antibody) can be applied is implanted in the patient's coronary artery, then genetically-altered cells (for example endothelial cell of hereditary change) is implanted among the patient who needs the treatment vascular disease.In this embodiment that uses genetically-altered cells and other embodiment, can adopt the technique for gene engineering of standard for example to utilize plasmid vector before implanting cell foreign gene to be sent into cell, described plasmid vector can be for example bicistronic mRNA pMACSK ^K.II plasmid vector (Miltenyi Biotec, Germany), it comprises multiple clone site and interested gene can be inserted wherein selected marker as used mammal cell line, described interested gene be for example prostacyclin synthase and marker gene (as the MHC I type molecule of brachymemma, H-2K).

In another embodiment, the foreign gene delivery system that is used for transfection mammalian cell in being used for the treatment of can comprise, for example comprises the MHC I type antigen of brachymemma and the slow virus carrier of vasodilator transgenosis (prostacyclin synthase and/or the α-CGRP gene that for example are used for the treatment of vascular disease).In this embodiment, describedly want transfected mammalian cell to can be endothelial cell or endothelial progenitor cells from body, and the ligands specific of the MHC I type antigen of available this brachymemma of described reparation device (anti--H-2K for example ^kAntibody) apply.

The accompanying drawing summary

Figure 1A passes through the corsslinking molecular covalent coupling in the schematic diagram of matrix for antibody.Figure 1B is C ₆₀The O molecule is anchored on the schematic diagram on the matrix.Fig. 1 C is coated with the schematic diagram of the support of matrix for the present invention.

Fig. 2 A is the phase contrast microscope photo that sticks to the endothelial progenitor cells on the slide that is applied by fibronectin, contains from the enriched medium isolated cells on the described slide.Fig. 2 B is the phase contrast microscope photo attached to the endothelial progenitor cells on the slide of fibronectin coating, contains the magnetic bead isolated cells of useful anti-CD34 antibody sandwich on the described slide.Fig. 2 D and 2F are the microphotos of having hatched 7 days and having carried out the endothelial progenitor cells of nuclear staining with PI.By these figure as seen, as respectively the antibody fluorescence of Tie-2 (Fig. 2 E and 2G) and VEGFR-2 (Fig. 2 C) antibody response being shown, shown in cellular expression the mark of mature endothelial cell.

Fig. 3 A and 3B are 2% Ago-Gel sxemiquantitative RT-PCR photos of the ethidium bromide staining of endothelial nitric oxide synthase (eNOS) and GAPD (GAPDH).After being incubated at last 3 day of slide (Fig. 3 B) and 7 days (Fig. 3 A) of fibronectin coating, endothelial progenitor cells begins to express eNOS mRNA.

Fig. 4 A-4E sticks to the HUVEC cell and cultivates and dye with iodate third ingot: CMDx and anti-CD34 antibody (4A); Gelatin and anti-CD34 antibody (4B); Blank stainless steel disk (4C); The photo of HUVEC on (4E) stainless steel disk that (4D) that CMDx applies and gelatin apply.

Fig. 5 A-5C is the microphoto of the contrast that applies of the CMDx of the no antibody of hatching with human blood leukocyte's component.Described cell is with the anti--KDR antibody staining of iodate third ingot and FITC mark.Fig. 5 D-5F is incorporated into the microphoto of the contrast stainless steel disk that its surperficial gelatin applies with what human blood leukocyte's component was hatched by no antibody.Described cell is with the anti--KDR antibody staining of iodate third ingot and FITC mark.

Fig. 6 A-6C is the microphoto that is combined with the stainless steel disk that the CMDx matrix of anti-CD34 antibody applies on the surface of hatching with HUVEC.Described cell is with the anti--KDR antibody staining of iodate third ingot and FITC mark.Fig. 6 D-6F is the microphoto that is combined with antibody stainless steel disk with the gelatin coating surface that HUVECS is hatched.Described cell is with the anti--KDR antibody staining of iodate third ingot and FITC mark.

Fig. 7 is the microphoto that the surface of hatching altogether 24 hours with CFU-GM is combined with the stainless steel disk that the CMDx of antibody applies.Described cell is with the anti--KDR antibody staining of iodate third ingot and FITC mark.

Fig. 8 A and 8B are the microphotos that the surface of hatching altogether 7 days with CFU-GM is combined with the stainless steel disk that the CMDx matrix of anti-CD34 antibody applies.Described cell is with the anti--KDR antibody staining of iodate third ingot and FITC mark.

Fig. 9 A and 9B are the microphotos that the surface of hatching altogether 7 days with CFU-GM is combined with the stainless steel disk that the CMDx matrix of anti-CD34 antibody applies.Described cell is with the anti-Tie-2 antibody staining of iodate third ingot and FITC mark.

Figure 10 A-10C is the phase contrast microscope photo of hatching the stainless steel disk that the CMDx in 3 weeks applies in the endothelial cell growth medium with CFU-GM, has shown ripe endothelial cell.

Figure 11 is the schematic diagram that is combined with the rack surface that the functional fullerene of the usefulness of the present invention of CFU-GM applies.

Figure 12 A-12D is the photo that has or do not have the sample that the fullerene of anti-CD34 antibody applies.Described sample and human leukocyte component are hatched altogether, and with anti-VEGFR-2 antibody staining of iodate third ingot and FITC mark.

Figure 13 A-13D is the low power and the high magnification micrographs of coronary artery extract histology transverse section, sample (Figure 13 B and 13D) 4 weeks that described extract has been implanted exposed stainless steel stent (Figure 13 A and 13C) and applied with fullerene.The section hematoxylin eosin staining.

Figure 14 A-14G is an ESEM microphoto of implanting male yorker 1 and 48 hours after-poppet extracts.The extract of (Figure 14 A) and the glucan that glucan applies/(14B) support that anti-CD34 antibody applies is for implanting back 1 hour.Figure 14 C and 14D are depicted as the control sample extract, and Figure 14 E-G is for implanting the support that back 48 hours glucan/anti-CD34 antibody applies.Figure 14 H-14M is the cross section histology microphoto of coronary artery extract of taking from 4 weeks of implantation of male yorker: uncoated (blank stainless steel) (14H and 14I), glucan apply (14L and the 14M) that contrast (14J and 14K) and glucan/anti-CD34 antibody applies.

Figure 15 A, 15B and 15C are respectively the fluorescence micrograph of 48 hours 18mm of the implantation support that long glucan with the no antibody in surface-blood plasma applies, glucan-blood plasma coating/support that anti-CD34 antibody applies.

Figure 16 A and 16B are the microphotos of the sample of iodate third ingot and antiagglutinin/FITC coupling.

Detailed Description Of The Invention

The invention provides a kind of implantable medical device (for example support or graft) of coating and be used for coating The method and composition of this medical treatment device, and the method for the treatment of vascular diseases with the medical treatment device of described coating. This Invention also provides a kind of method that is used for the treatment of disease (for example ISR and cancer), and described method comprises having The medical treatment device of coating is implanted the patient who needs treatment, and moves for described patient provides through genetically engineered lactation Thing cell, described cell can be incorporated in vivo the surface of described medical treatment device and can produce engineered with required Therapeutic agent (for example gene outcome). Figure 1A-1C is the schematic diagram of medical device surface coatings of the present invention. The medical treatment dress The coating that is set up comprises: biocompatible matrix, it can promote (for example to form the fused cell layer at apparatus surface The mammalian cell of hereditary change is such as endothelial cell or fibroblast) in the patient, to regulate or to produce required Result for the treatment of, for example produce can prevent ISR and/or thrombotic anti-angiogenesis or anticoagulant or Generation can suppress the product of neointimal hyperplasia. In one embodiment, the coating on the described prosthetic device comprises Matrix contain synthetic or naturally occurring material, comprise in the described material and can promote (for example heredity of cyclicity cell The mammalian cell that changes is such as endothelial cell, CFU-GM or stem cell) treatment that adheres to medical treatment device is effective At least a antibody of amount and can stimulating endothelial cell growth and differentiation at least a compound (for example grow because of Son). After implanting described device, the cell transformation that is attached to described apparatus surface becomes function ripe and that merge The sexual cell layer for example forms endothelium at described medical treatment device surface of internal cavity. The endothelial cell that exists at medical treatment device Fused layer for example can reduce implant site ISR and thrombosis.

As used herein, " medical treatment device " refers to temporarily or forever introduce in the mammal to prevent or to treat certain The device of one medical science symptom. These devices comprise by subcutaneous, be placed in organ, tissue through what skin or operation were introduced Or any device in the inner chamber of organ (for example artery, vein, ventricle or atrium). Described medical treatment device can comprise: The support of support, stent graft, coating (for example is coated with polytetrafluoroethylene (PTFE) (PTFE), intumescent polytetrafluoroethyl-ne Alkene (ePTFE) or other natural or synthetic coating) or synthetic blood vessel graft, artificial heart valve, Artificial heart and can connect the organ of reparation and the fixture of systemic vascular, vein valve, abdominal aorta moving Arteries and veins knurl (AAA) graft, inferior caval vein screen sheet, permanent infusion administering catheter, spiral shell volume embolus (embolic The embolism materials (for example, crosslinked PVA hydrogel) that coil), when blood vessel embolism forms, uses, blood vessel suture, Vascular anastomosis fixture, saturating myocardial vascular form support and/or other various conduits again.

The coating of the medical treatment device that forms with the compositions and methods of the invention can stimulate described apparatus surface in vivo The mammalian cell layer that upper generation is merged. For example, make it at described dress when the ligand binding endothelial cell that provides When forming functional endodermis on the blood contact surface of putting, namely formed endothelial cell on this surfaces of medical devices Layer, thus prevented ISR, and it is anti-to regulate the local chronic inflammation that is caused by the implantation of described medical treatment device Should with the thrombosis complication.

The matrix that covers medical treatment device can be made up of synthetic material, and for example, the polymerism gel foam is as by poly-second The hydrogel that enol (PVA), polyurethane, Poly-L-lactide, cellulose esters or polyethylene glycol are made. An enforcement In the mode, can comprise very hydrophilic compound in the synthetic material of preparation matrix, for example dextran compound. In another embodiment, described matrix is made up of naturally occurring material, for example collagen, fibrin, elasticity egg In vain, tropoelastin and/or amorphous carbon. Described matrix also can comprise multilayer, and for example ground floor can be by synthetic Material or naturally occurring material form, and the second layer can contain for example part (such as antibody). Each layer can be in succession Arrange in turn, ground floor directly contacts second with medical treatment device (support or synthetic material graft) surface Layer has a surface directly to contact with ground floor, and another surface then contacts with intravascular space.

Described matrix also can comprise at least a growth factor, cell factor, vasodilator, anticoagulant etc. But the growth factor of stimulating endothelial cell propagation and differentiation is for for example: VEGF (VEGF) and same The growth factor (PIGF) that worker's type, basic fibroblast growth factor (bFGF), blood platelet are induced, conversion Grouth factor beta 1 (TGF. β 1), acid fibroblast growth factor (FGF), osteonectin, Angiogenesis Element 1, ANG2, IGF (ILGF), platelet derived growth factor AA (PDGF-AA), PDGF-BB (PDGF-BB), platelet derived growth factor AB (PDGF-AB), granulocyte macrophage colony stimulating factor (GM-CSF) etc., or its functional fragment, They can be used among the present invention. Vasodilator comprises: prostacyclin, α-CGRP etc.

In another embodiment, described matrix can comprise fullerene, and the carbon number of described fullerene is about C₂₀-C ₁₅₀ Described fullerene also can be arranged in nanotube, wherein can mix molecule or protein. Described fullerene Matrix also can be applicable to stainless steel, PTFE or ePTFE surfaces of medical devices, then this layer is carried out functionalization And in its surface coating antibody and growth factor. Perhaps, can for example apply earlier on the stainless steel medical treatment device PTFE or ePTFE layer, and then apply second layer fullerene, add antibody and growth factor.

This matrix can mode non-covalent or covalency be attached on the medical treatment device. Various antibody and growth factor can By different base or with basic difunctional cross-linking reagent, covalent attachment is on this matrix. Can adopt standard technique will grow because of Son adds in this matrix with antibody or after the antibody combination.

As used herein, term " antibody " refers to a class monoclonal, polyclone, humanization or mosaic antibody Or their combination, wherein, described monoclonal, polyclone, humanization or chimeric antibody and a kind of antigen or The functional equivalents of this kind antigen combines. The term antibody fragment comprise with antibody have same effect and Any antibody fragment of effect is (such as Fab, F (ab ')₂Deng), and can be any size, i.e. big molecule or little branch Son. (include most separately antibody of antibody molecule, be equivalent to 6.022 * 10²³Individual molecule/mole antibody).

For example, in one embodiment, available biocompatible matrix drug delivery medical device or synthetic graft, This matrix comprises can regulate circulating cells (for example mammal therapeutic cells and the endothelial progenitor cells of hereditary change) Adhere to antibody, antibody fragment or their combination of described surfaces of medical devices. For example, antibody of the present invention is known Not and the cell membrane labelled molecule of binding specificity, for example the mammalian cell by hereditary change in the circulating produces Endothelial progenitor cells surface antigen and/or membrane molecule, thereby so that described cell be fixed on the surface of device Form functional cell layer (for example functional endothelial tissue). In one embodiment, described antibody comprise can with The monoclonal antibody of following substance reaction (identification and in conjunction with): the mammalian cell surface molecule of hereditary change, in Skin CFU-GM surface antigen or CFU-GM or stem cell surface antigen, for example vascular endothelial growth factor receptor-1,-2 and-3 (isoforms of VEGFR-1, VEGFR-2 and VEGFR-3 and VEGFR receptor family), Tie-1, Tie2, CD34, Thy-1, Thy-2, Muc-18 (CD146), CD30, stem cell antigen-1 (Sca-1), stem cell factor (SCF or c-Kit part), CD133 antigen, VE-cadherin, P1H12, TEK, CD31, Ang-1, Ang-2 Or the antigen of expressing at described cell surface. In one embodiment, can use and a kind of antigen reactive list The antibody of one type. Perhaps, the multiple different antibody for different endothelial progenitor cells surface antigens can be blended in Together and add in the matrix. In another embodiment, adopt the mixture of monoclonal antibody, passed through target Specificity cell surface antigen improves the endothelium formation rate. In this embodiment, for example use capable of being combined is anti--CD34 and anti--CD133 maybe can share these and any or several antigen group listed above in being attached to medical treatment Stromal surface on the device (for example support or graft). Also available antibodies, antibody fragment and/or their combination Apply described medical treatment device.

As used herein, " antibody for the treatment of effective dose " refers to promote that cell is attached to the antibody of medical treatment device Consumption, wherein said cell for example are: the mammalian cell of natural or hereditary change comprises endothelial cell, ancestral Cell or stem cell. Implement antibody amount required for the present invention according to the character of used antibody and difference. For example, used The amount of antibody depend on antibody and with it the reaction antigen between binding constant and/or affinity. This area general Logical technical staff knows the treatment effective dose that how to confirm is used for the antibody of concrete antigen.

As used herein, term " compound " refer to stimulate hereditary change mammalian cell expression and/or Any material of secretion therapeutic gene product.

As used herein, term " growth factor " but refer to irritation cell (for example hereditary change or unaltered in Chrotoplast, stem cell or CFU-GM) grow and be divided into peptide, protein, the sugar of ripe functional endothelial cell Albumen, lipoprotein or their fragment or variant or synthetic property molecule. Ripe endothelial cell can be expressed an oxidation Thereby the nitrogen synthase is released into tissue with nitric oxide. Following table 1 has been listed some that can be used for applying described medical treatment device Growth factor.

Table 1

Growth factor	Endothelial cell specific
Growth factor	Endothelial cell specific	( aFGF ) ( bFGF ) 3 ( FGF-3 ) 4 ( FGF-4 ) 5 ( FGF-5 ) 6 ( FGF-6 ) 7 ( FGF-7 ) 8 ( FGF-8 ) 9 ( FGF-9 ) 1 2 / ( HGF/SF ) ( PDE-CGF ) -α ( TGF-α ) -β ( TGF-β ) -α ( TNF-α ) 121 ( VEGF121 ) 145 ( VEGF145 ) 165 ( VEGF165 )	Not having to have or not have has

VEGF 189 (VEGF189) VEGF 206 (VEGF206) vascular endothelial growth factor B (VEGF-B) vascular endothelial growth factor C (VEGF-C) vascular endothelial growth factor D (VEGF-D) VEGF E (VEGF-E) VEGF F (VEGF-F) placenta growth factor Ang-1 angiopoietin-2 thrombospondin (TSP) proliferin liver joins albumen-A1 (Ephrin-A1, B61) CD62L chicken chemotactic and angiogenesis factor (cCAF) leptin (Leptin) heparin affinity is regulated peptide (HARP) heparin granulocyte colony stimulating factor IGF interleukin 8 thyroxine sphingosine 1-phosphate

Having or not have to have or not does not have

As used herein, term " VEGF " refer to any isoform of listed VEGF in the table 1, Unless can differentiate it with this sequence number or letter abbreviations with the worker specifically.

As used herein, term " growth factor for the treatment of effective dose " refers to stimulate or inducing specific cell mass (example Such as natural or modified endothelial cell, CFU-GM or stem cell) growth and differentiation, thus form the maturation that merges Growth with functional cell layer (for example endothelial cell forms functional endothelial tissue at the medical treatment device surface of internal cavity) Factor consumption. Implement growth factor consumption required for the present invention with the character of used growth factor and growth factor and Binding kinetics between its receptor in target cell and difference. For example, but 100 μ g VEGF have shown that stimulating endothelial is thin Born of the same parents adhere to medical treatment device and form the endothelium fused layer. Those of ordinary skill in the art knows how to confirm and is used for thorn Swash the treatment effective dose of the growth factor of cell (for example, endothelial cell) growth and differentiation.

As used herein, " endometrial hyperplasia " refers in the vascular wall smooth muscle cell proliferation and/or apposition not Good increasing. As used herein, " ISR " refers to that the recurrent of intravascular space is narrow. Blood vessel can be because of ISR Block. Behind PTCA or PTA, from media and adventitia and the common smooth muscle cell that is not present in the inner membrance Breed, move to inner membrance justacrine protein, in inner membrance, form gathering of smooth muscle cell and stromatin. This gathers and has caused the Oligemia that arterial lumens is narrow, flow to the stenosis far-end. As used herein, " suppress again Narrow " refer to the inhibition of smooth muscle cell migration and propagation is also prevented the protein secretion simultaneously, thus prevent again narrow Narrow and cause complication.

The object of available medical treatment device of the present invention, method and composition treatment is mammal, comprising: the people, Dog, cat, pig, horse, rodent and monkey.

It is interior or external that methods for the treatment of of the present invention can be applicable to body.

Term " endothelial progenitor cells " refers to derived from bone marrow, blood or local organization and is in any stage of development Endothelial cell (the functional endothelial cell from CFU-GM or stem cell to maturation), they are non-malignant cells.

The medical treatment device that can be described coating provides genetically modified mammalian cell, for example can be from exsomatizing Separate the endothelial cell of the differentiation of the hereditary change that obtains in artery or the vein (for example human umbilical vein), they are at body The required nucleic acid construct thing of external application has carried out hereditary change, and endothelial progenitor cells is then separable from peripheral blood or marrow. In one embodiment, can be by endothelial cell and the medical treatment device that has applied matrix be hatched jointly, and make institute Stating endothelial cell can be combined with described medical treatment device, and wherein said matrix has been mixed antibody and optional at least a life The long factor or other can be attached to the part of endothelial cell. In another embodiment, described endothelial cell can be and turns to The endothelial cell of changing. The endothelial cell of transfection can comprise the carrier of expressing growth factor or other peptide or protein, this A little growth factors, peptide or protein can directly or indirectly suppress thrombosis, ISR or other any treatment results.

In another embodiment, available mammalian expression vector transfection endothelial cell or other type-stable Mammalian cell (for example fibroblast), described carrier comprise coding and are suitable for the protein of special-purpose or peptide Any clone gene. For example, can make up described carrier and make it contain expression cassette, it is little that described expression cassette comprises coding blood The gene of plate derivative growth factor (PDGF), fibroblast growth factor (FGF) or nitricoxide synthase (NOS), And available conventional method makes up described expression cassette, can be provided by the commercially available source of goods. (referring to for example, available from Stratagene, San Diego, the mammalian expression vector of CA and transfection reagent box). For example, according to the side of Rosengart etc. Method, the adenovirus expression carrier of use VEGF expression cDNA is with VEGF (VEGF) gene transfection The pig endothelial progenitor cells of purifying (" is used for the treatment of the gland that gives VEGF expression 121 cDNA in the direct cardiac muscle of coronary heart disease The assessments in six months that the I phase of the angiogenesis gene therapy of viral vectors tests ", Six-month assessment of A phase I trial of angiogenic gene therapy for the treatment of coronary Artery disease using direct intramyocardial administration of an adenovirus Vector expressing the VEGF121 cDNA; Ann.Surg.230 (4): 466-470,1999, receive Enter this paper as a reference). In this embodiment, the source of described mammalian cell can be from body, of the same race Allosome or xenogenesis. In case transfection contain the foreign DNA of required gene or rna expression box and so that described cell Heredity changes, and the tissue culture technique of available standards is cultivated described cell. The available standards technology is in liquid nitrogen The cell sample of the required gene of frozen expression justacrine. Before use, the tissue culture technique of available standards makes freezing Cell regrow. When implanting described device, the mammalian cell of hereditary change is given in the implant site part Give or in intravenous or artery, give the patient, preferably after the medical treatment device of implanting described coating, give. Transform Cell also can comprise mark or the reporter gene that gives to be used for accurately detecting and differentiating before this cell of patient described cell.

Can implement the method that the present invention treats vascular diseases at any artery or vein. Comprise in the scope of the invention The atherosclerotic of any artery, described artery comprise artery under coronary artery, the groin, sustainer common iliac artery, Subclavian artery, mesenteric artery and the arteria renalis. The present invention also comprises the angiemphraxis of other type, for example between wall What aneurysm caused blocks.

The mammiferous method that treatment suffers from vascular diseases comprises: the medical treatment device that applies is implanted described patient Organ or blood vessel, for example, in the situation of the support that in revascularization, implant to apply. In case enter in the body, Endothelial progenitor cells is trapped in by identification with in conjunction with cellular antigens (for example mammalian cell of genetic modification) On the surface of the support of described coating, or the combination by independent antibody or antibody and other part is trapped in and deposits Be the CFU-GM surface on the described device coating. In case CFU-GM is attached to matrix, the growth on the described coating because of Son just promotes the endothelial progenitor cells growth of new combination with differentiation and in becoming that the surface of internal cavity formation of described support is merged Ripe functional endothelial tissue. Perhaps, before implanting described medical treatment device, can be natural or genetic modification at external use Mammalian cell (for example endothelial cell) applies described medical treatment device, these cells can be separate from blood samples of patients, The CFU-GM of marrow or blood vessel, stem cell or mature endothelial cell. In both cases, described medical treatment device inner chamber The functional cell that exists on the surface can produce required or through engineered function, for example suppresses or prevents inner membrance Hyperplasia and thrombosis.

Endothelial cell

In some embodiments, according to Jaffe etc. (J.Clin.Invest., 52:2745-2757,1973, Include this paper in as a reference and be used for test at this) method, can obtain people's umbilical cord from people's umbilical vein Venous endothelial cell (HUVEC). In brief, process the cell of peeling off from vascular wall with clostridiopetidase A, put into In the tissue culture flasks with the gelatin coating, do not have in the hyclone that contains 10% low endotoxin, 90 mcg/ml The M199 of the pork liver element of anticorrisive agent, 20 mcg/ml endothelial cell growth replenishers (ECGS) and glutamine Cultivate in the culture medium.

Can according to the people's such as Asahara method (be used for the separation of the endothelial progenitor cells of inferring of Angiogenesis, Isolation of putative progenitor endothelial cells for angiogenesis, Science.275:964-967 1997, includes this paper in as a reference) from human peripheral, separate and obtain the endothelium ancestral Cell (EPC). The magnetic bead that is coated with the antibody of anti-CD34 is hatched with the human peripheral liquid that separates through component. Through after hatching, the cell of elution of bound is incubated at EBM-2 culture medium (Clonetics, San Diego, CA) with it In. Perhaps, can adopt the enriched medium partition method to separate these cells. In brief, obtain from the volunteer Peripheric venous blood adopts density－gradient centrifuga－tion method to separate and obtains monocyte, is seeded in these cells coated Have on the cultivation slide of fibronectin (Becton Dickinson), additional 5% hyclone, people VEGF-A, HFGF-2, human epidermal growth factor, insulin-like growth factor-i and anti-bad The EC basal medium-2 (EBM-2) of hematic acid is middle the cultivation (Clonetics). Make endothelial progenitor cells growth 7 days, Per 48 hours replaced mediums. By anti-CD45, CD34, CD31, VEGFR-2, Tie-2 and E-Selectin Fluorescence antibody carry out cell and identify.

In another embodiment, can adopt conventional method, with any table that contains any following clone gene Reach box mammalian cell carried out transfection, described clone gene can contain be coded in the circulating cells uncommon Specific marker molecule (for example PSA or osteocyte antigen), but and also expression of peptides and/ Or protein, for example platelet derived growth factor (PDGF), fibroblast growth factor (FGF) or Nitric oxide synthase (NOS) etc. (referring to, for example available from Stratagene, San Diego, the mammal of CA Expression vector and transfection reagent box). For example, according to the method for Rosengart etc., adopt VEGF expression cDNA Adenovirus expression carrier, (" be used for the pig endothelial progenitor cells of VEGF (VEGF) transfection purifying The angiogenesis gene that gives the adenovirus vector of VEGF expression 121 cDNA in the direct cardiac muscle for the treatment of coronary heart disease is treated The assessments in six months that the I phase of method tests ", Six-month assessment of a phase I trial of Angiogenic gene therapy for the treatment of coronary artery disease using Direct intramyocardial administration of an adenovirus vector expressing The VEGF121 cDNA; Ann.Surg.230 (4): 466-470,1999, include this paper in as a reference).

Antibody

Can prepare the Dan Ke that adopts in the method for the invention according to the standard technique of Kohler and Milstein Grand antibody (" secretion pre-determines the continuous breeding method of the fused cell of specific antibody ", Continuous Cultures of fused cells secreting antibody of predefined specificity, Nature, 265:495-497,1975, include this paper in as a reference), perhaps can directly obtain from commercially available source The monoclonal antibody that must be used for the inventive method. Endothelial cell can be used as the anti-endothelial cell table of the original generation of immunity The monoclonal antibody of face antigen.

HUVEC or purified endothelial progenitor cells can be injected into mouse or rat and prepare anti-endothelial cell Monoclonal antibody. Through after the sufficiently long time, put to death mouse, get its splenocyte. By splenocyte is melted Close in myeloma cell or lymphoma cell and make the immortality cell, merge and usually containing the nonionic surface Carry out in the activating agent (for example polyethylene glycol). Make gained comprise that the cell of amalgamation hybridoma is in selective cultivation Growth in the base (for example HAT-culture medium), and use the limiting dilution condition to make the cell of survival at this culture medium Middle growth. Growth of Cells has required in suitable container (for example microtiter well) in the screening supernatant Specificity, the monoclonal antibody that can react with endothelial cell antigen.

The existing multiple technology that can be used for improving the monoclonal antibody productive rate, for example: hybridoma is injected into connects Be subjected to gather in the crops then ascites in the mammalian hosts abdominal cavity of these cells. When from ascites, not collecting capacity During monoclonal antibody, can from this host's blood, gather in the crops antibody. Existing multiplely can be used for separating and the purifying monoclonal resists Thereby body makes the method that does not contain other oroteins or pollutant in the monoclonal antibody.

Scope of the present invention also comprises the useful binding fragment of antibody (for example anti-endothelial cell monoclonal antibody), for example the Fab of the monoclonal antibody of these anti-endothelial cells, F (ab ')₂ Can obtain described antibody by routine techniques Fragment. For example, can prepare useful knot by carrying out the peptide enzymic digestion with papain or pepsin antagonist Close fragment. These fragments can be used separately, perhaps unite with antibody and the fragment thereof of its origin antibody or other type Use.

Antibody of the present invention can be mouse IgG antibody-like; Yet this does not become restriction. Specificity Antibody, for example no matter above-mentioned antibody is mouse, mammal with those antibody suitable with its function Source property (comprising the humanized) or other sources, or their combination all are included in the category of the present invention, Also comprise other antibody-like that contains isotype, such as IgM, IgA, IgE etc. These antibody capable specific recognition And with high-affinity in conjunction with the target antigen on the target cell membrane, no matter be that this target antigen is on natural molecule or through losing Pass on the engineered antigen. With regard to antibody, term " suitable on the function " refers to two kinds of different antibody separately Same antigen site on the conjugated antigen, in other words, described antibody competition is in conjunction with identical antigen. Described antigen Can be positioned on the identical or different molecule.

In one embodiment, can adopt can with surface endothelial cell antigens (for example, CD34) reaction Dan Ke Grand antibody and/or its fragment. Proved that the anti-CD34 monoclonal antibody that is attached on the solid phase carrier can catch people's periphery Endothelial progenitor cells in the blood. After catching, these CFU-GMs can be divided into endothelial cell. (Asahara etc., 1997, " being used for the separation of the endothelial progenitor cells of inferring of Angiogenesis ", Isolation of putative Progenitor endothelial cells for angiogenesis, Science 275:964-967. ) assorted The anti-CD34 monoclonal antibody of handing over knurl to produce can be collected center (American Type available from U.S. typical organization Tissue Collection, Rockville, MD). In another embodiment, energy and endothelial cell have been used The monoclonal antibody of surface antigen (for example VEGFR-1 and VEGFR-2, CD133 or Tie-2) reaction. Using heredity In the embodiment of the cell that changes, available and mentioned above similar mode adopts standard technique to produce anti-institute State the antibody through genetically engineered gene outcome, after applying matrix, it is applied to described medical treatment device and blood On the surface of liquid contact.

Also can use can with separate from and accept the homozoic anti-endothelial cell phase reaction that this medical treatment device is implanted Polyclonal antibody.

Support

Term " support " in this article refers to when insertion or implantable intravascular inner chamber, and it is transversal to enlarge intravascular space Any medical treatment device of face. Term " support " comprising: the stainless steel that has applied with method of the present invention or other Alloy commercial stents processed; Support through covering for example covers with PTFE or ePTFE. In one embodiment, Comprise dermal delivery treatment coronary occlusion or sealing spleen, arteria carotis, bone section He Tui popliteal section blood vessel otch or moving The support of arteries and veins knurl. In another embodiment, described stent delivery is entered the vein blood vessel. Described support can be by polymerization Thing or hardware form, and are coated with the matrix that contains antibody and compound (for example growth factor) on it. For example, can Adopt the deformable wire support, as authorize disclosed support in the United States Patent (USP) 4,886,062 of Wiktor, should Patent is included this paper in as a reference in full with it. Also can use the elastic polymeric material Self-expanded stent (self-expanding stent), for example name is called " inner chamber medicament elution prosthetic ", Intraluminal United States Patent (USP) 5,871,535 and the disclosed international patent application of Drug Eluting Prosthesis The support that discloses among the WO91/12779 is included these patents in this paper as a reference in full with it. Can use United States Patent (USP) 6,432, other support that discloses in 132 and 6,821,292 is included these patents in this in full with it Literary composition as a reference. Also can use following material to make support: stainless steel, polymer, Ni-Ti, tantalum, gold, platinum-Iridium, cobalt-base alloys or Elgiloy or MP35N and other iron-bearing materials. To prop up to be placed on the conduit and pass by body cavity Deliver to therapentic part, at this position support is discharged from conduit, so that the expansion of described support is for directly contacting blood vessel Inwall. In another embodiment, described support comprises Biodegradable scaffold (H. Tamai, coronary artery The support handbook, Handbook_of_Coronary_stents, 297 pages, the 3rd edition, PW Serruys and MJB Kutryk, Martin Dunitz (2000). Skilled in the art will recognize that can be with antibody of the present invention, growth The factor and matrix are used with other Self-expanded stent design thing (for example elastic metal rack design thing).

Synthetic graft

Term " synthetic graft " is meant that with biocompatibility feature anyone produces and repairs complex.In one embodiment, described synthetic graft can be used PETG (Dacron ^, PET) or polytetrafluoroethylene (PTFE) (Teflon ^, ePTFE) preparation.In another embodiment, described synthetic graft is made of the biocompatible foam body of polyurethane, crosslinked PVA hydrogel and/or hydrogel.In another embodiment, He Cheng graft is by netted Merlon urethanes (polycarbonate urethane) internal layer and the outer formation of netted PETG.Those skilled in the art will know that and covering component of the present invention (for example antibody, growth factor and matrix) can be used with the synthetic graft of any biocompatibility.(Bos etc., 1998, small-bore blood vessel repair materials: present situation, Small-Diameter Vascular Prostheses:CurrentStatus, Archives Physio Biochem.106:100-115 includes this paper in as a reference).Synthetic graft can be used for, for example end-the end of blood vessel, end to side, side-end, side-side or tube chamber are interior and anastomosis of blood vessel, or are used for the shunting of diseased vessel section, for example as the abdominal aortic aneurysm device.

Matrix

(A) Synthetic material---the described matrix that is used to cover support or synthetic graft can be selected from synthetic material, for example: biocompatible foam body or hydrophily glucan, for example Sensor Chip CM 5 of polyurethane, block polyurethane-urea/heparin, poly--L-lactic acid, cellulose esters, polyethylene glycol, crosslinked PVA hydrogel, hydrogel.

(B) Naturally occurring material---described matrix can be selected from naturally occurring material, for example: collagen, fibronectin, vitronectin, elastin laminin, laminin, heparin, fibrin, cellulose and carbon.For the basic demand of matrix is to have enough elasticity and pliability not rupture on the contact surface of support or synthetic graft to guarantee it.

(C) Fullerene---described matrix also can comprise fullerene (term " fullerene " comprises multiple fullerene molecule).Fullerene is carbon-cage (carbon-cage) molecule.The excursion of the carbon in the fullerene (C) molecular number is C ₂₀-C ₁₅₀Fullerene is according to method well known to those skilled in the art, produces by at high temperature making carbon or carbon containing class substance reaction; For example, by carbon being carried out laser vaporization, in electric arc, heat carbon or generating the hydrocarbons that burns in the flame at carbon black.(authorize the United States Patent (USP) 5,292,813 of Patel etc.; Authorize the United States Patent (USP) 5,558,903 of Bhushan etc., its announcement is included this paper in as a reference in full with it).In all cases, the deposit or the cigarette ash of carbon containing have all been produced.Can from this cigarette ash, obtain different fullerenes by extracting with The suitable solvent (for example toluene).Separate fullerene with known method, especially by high performance liquid chromatography (HPLC).Can synthesize or from Dynamic Enterprises, Ltd. (Berkshire, England or SouthernChemical Group, LLC, Tucker, Georgia) or Bucky USA (Houston Texas) buy fullerene.

Available diverse ways with the fullerene deposition from the teeth outwards, described method comprises: distillation, laser vaporization, sputter, ion beam, spraying, dip-coating, roller coating or brushing are (as United States Patent (USP) 5,558, disclose in 903, include this paper in full in as a reference with it), or the derivatization by rack surface.

A key character of fullerene is that they can form " activated carbon ".The electronic structure of fullerene is overlapping π rail system, thereby a plurality of bonding electrons is present in the surface around molecule jointly.(Chemical andEngineering News, Apr.8, include this paper in as a reference by 1991, the 59 pages).As the form with active carbon, fullerene demonstrates the basic Van der Waals force to weak interaction.The absorption property on fullerene surface makes it have the extra modification of guiding specific cell membrane interaction.For example, can be adsorbed onto the fullerene surface with having the specific molecular of selective binding in the chemical property of the cell membrane of particular cell types or specific cell membrane component (for example agglutinin or antibody).Can operate (for example to produce the different cell types of selective binding the adhesion of fullerene surface different molecular, endothelial progenitor cells, endothelial cell, fibroblast, former generation explant or the T-cell subsets) the surface, for example authorize the United States Patent (USP) 5 of Richmond etc., 310,669, its announcement is included this paper in as a reference in full with it; Stephen R.Wilson, the biological property of fullerene, Biological Aspects ofFullerenes, Fullerenes:Chemistry, Physics and Technology, volumes such as Kadish, John Wiley ﹠amp; Sons, NY 2000, include this paper in as a reference.

Fullerene also can form the nanotube that can mix other atom or molecule.(its announcement is included this paper in as a reference for Liu etc., Science 280:1253-1256 (1998)).Synthetic and the preparation method of CNT is well-known in the art.(authorize the United States Patent (USP) 5,753,088 of Olk etc. and authorize the United States Patent (USP) 5,641,466 of Ebbsen etc., both announcements are all included this paper in as a reference in full with it).Also can the inboard of CNT will be mixed such as protein-based molecule.For example, after cutting the nanotube two ends, available enzyme (Zn for example ₂Cd ₂-metallothionein, cromoci and C3 and beta-lactamase) filled with nanotubes.(Davis etc., Inorganica Chim.Acta 272:261 (1998); Full Sci.Tech.5 (4) such as Cook: 695 (1997), both all include this paper in as a reference).

Also can use three-dimensional fullerene structure body.Include this paper United States Patent (USP) 5 of authorizing Mirkin etc. as a reference in full in, disclosed the 3-dimensional multi-layered fullerene structure body that on substrate surface, forms by the following method in 338,571: (i) fullerene is carried out chemical modification and form material (bond-forming species) to produce key; (ii) chemical treatment is carried out on the surface of base material and is formed material so that key to be provided, described material be enough to solution in have the fullerene covalent bond that key forms material; And (iii) modified fullerene solution is contacted with treated substrate surface, be covalently bonded in the Fullerene layer of treated substrate surface with formation.

(D) Matrix is put on medical treatment device

Described matrix should be able to be securely attached to the medical treatment device surface of (comprising support or synthetic graft).In one embodiment, this is to realize by applying continuous thin-layer matrix.Perhaps, can be only with on the outer surface that antibody and growth factor are applied in intravascular space directly contacts.Apply dissimilar matrix pantostrats serially.After being applied over matrix on the support, can be with described antibody covalently or non-covalently in conjunction with being coated on the matrix.

In order to cover medical treatment device, support for example, available moderately viscous matrix liquid solution impregnation or spray described support.After having applied each layer, under applying, make the support drying before one deck.In one embodiment, the gross thickness of paint sample matrix coating is no more than 100 microns.

In one embodiment, the surface of described medical treatment device at first through functionalization, has been added the rack surface of hypothallus for for example then.Antibody and other coating component (for example growth factor) be coupled to stromal surface thereafter.At this on the one hand, the technology that matrix is applied over such as rack surface can produce functional chemical group.For example, described chemical group can be amino, it can be with the functional group reactions of polymer with fixing as discerning and the matrix intermediate layer of the carrier of the part (for example antibody, peptide and/or growth factor) of acquisition target cell.

In another embodiment, can prepare suitable matrix coating solution in the following way: under aseptic condition, with 480 milligrams of (mg) pharmaceutical carriers, for example gather-D, the L-lactide is (as R203 available from Boehringer Inc., Ingelheim Germany) is dissolved in 3 milliliters of (ml) chloroforms.Yet, can use blood-and biodegradability (or the abiotic degradability) matrix of tissue-compatibility (biocompatibility) and solubilized, dispersion or emulsification in principle, as long as can be on medical treatment device after using dry quickly for from adhering to lacquer sample or coating sample coating.

For example, those of ordinary skill in the art knows usable fibers albumen coating support.Fibrin is condensed authorizing to have disclosed by fibrinogen is contacted with fibrin ferment in the United States Patent (USP) 4,548,736 of Muller etc. (it discloses in full and includes this paper in as a reference).Preferably, fibrin in the fibre-bearing albumen support of the present invention contains the XIII factor, and in condensing, there is calcium, (as authorize in the United States Patent (USP) 3,523,807 of Gerendas disclose, its announcement is included this paper in as a reference in full, or such as disclosed european patent application 0366564 announcement, its announcement is included this paper in as a reference in full), to improve the mechanical property and the biological stability of implanted device.In this embodiment,, be used to prepare fibrinous fibrinogen of the present invention and fibrin ferment and derive from and the animal or human that will implant animal or human's identical type of this support for fear of any interspecies immunity reaction (for example anti-ox of people).The fibrin product of producd fibers extract of protein fine film form in the following manner: the fibrinogen and the fibrin ferment that mix are poured into film, from described film, remove moisture by the semipermeable membrane diafiltration then.(its announcement is included this paper in as a reference in full) disclosed base material (preferably have highly porous or fibrin ferment or fibrinogen are had high-affinity) contacted with thrombin solution with fibrinogen solution in european patent application 0366564.The result is by fibrinogenic polymerization, forms the fibrin layer on the surface of medical treatment device.The multi-layer fiber albumen that applies with this method can provide the fibrin layer of any desired thickness.Perhaps, can fibrin be condensed, be ground into powder then, this powder and mixed being incorporated in of water are embossed into required form (United States Patent (USP) 3,523,807) in the heated mold.Can produce the fibrin that is shaped and the stability that obtains to improve by fibrin is contacted with fixative (for example glutaraldehyde or formaldehyde).Can be used for preparation and form fibrinous these methods being used for the present invention with well known by persons skilled in the art with other method.

If apply described synthetic graft with collagen, preparation collagen also makes the method that forms coating on synthetic graft device know, as authorizes the United States Patent (USP) 5,851 of Weadock etc., disclose in 230, its announcement is included this paper in as a reference in full.This patent has been described the method that is used for applying with collagen synthetic graft.Be used for the method that collagen adheres to porous graft base material generally included the collagen dispersion liquid is applied over base material, make it dry and repeat this process.The preparation of collagen dispersion is normally by being mixed into insoluble collagen (about 1-2 weight %) dispersion liquid of acid pH (pH is 2-4).Usually described dispersion liquid is gone in the graft inner chamber with injector to inject, and rub to press with hand and make the collagen slurries cover whole inner surface area.Remove excessive collagen slurries from one of two openends of described graft.Repetitive coatings and drying steps are for several times to provide enough processing.

In another embodiment, apply support or synthetic graft with amorphous carbon.At United States Patent (USP) 5,198, in 263 (its announcement is included this paper in as a reference in full) described a kind of be used for through fluorinated gas or contain other halogen gas in the presence of, the method for producing height ratio low temperature depositing amorphous c film.The deposition of carrying out according to the method for this invention can be lower than under 100 ℃ the temperature (comprising ambient room temperature), and, chemical vapor deposition process auxiliary with radio frequency, plasma carries out.The amorphous c film of using method generation of the present invention can adhere to the base material of numerous species well, and described base material for example comprises: glass, metal, semiconductor and plastics.

Fullerene molecule is combined with the reactive amino group site of amido polymer with to form fullerene-support, and containing amino polymer can be by United States Patent (USP) 5,292, and 813 (its announcement is included this paper in as a reference in full) are finished.The chemical modification of carrying out with this mode can make that fullerene directly mixes in the support.In another embodiment, can as described above fullerene be deposited on the surface of support or synthetic graft.(referring to, authorize the WO 99/32184 of Leone etc., its announcement is included this paper in as a reference in full).Fullerene (C for example ₆₀) also can be attached to (Yamago etc. on the stainless steel surfaces by epoxy bond, " organic property fullerene forms the chemically derived of reaction by oxidation, reduction and C-O and C-C bonding ", Chemical Derivatization of Organofullerenes through Oxidation, Reduction and C-O and C-C Bond FormingReactions, J.Org.Chem., 584796-4798 (1998), its announcement is included this paper in as a reference in full).This adheres to and is by finishing with the covalent bond of oxygen.Described compound and coupling scheme can (BuckyUSA, Houston Texas) buy from BuckyUSA..

(E) Part (for example antibody, peptide and/or growth factor) is added matrix---can promote the antibody that endothelial progenitor cells adheres to and can promote the growth factor of cell growth and differentiation covalently or non-covalently to be incorporated in the matrix.Can be in the following way with part coating (for example antibody, antibody fragment, hormone, peptide, growth factor and/or like that) doped matrix layer: part is mixed with matrix coating solution, then described solution is applied on the surface of device.In some embodiments, with antibody, fragment or their combination and/or growth factor outermost surface attached to the matrix that is applied over the device surface of internal cavity, thereby make part (for example antibody) give prominence on this surface and can contact, and keep their affinity target cell with blood circulation.In these embodiments, can adopt standard technique that described part (for example antibody) is applied on the stromal surface.

In one embodiment, described antibody adding is contained in the solution of matrix.For example, with the concentration of 500-800mg/dl, the Fab fragment and the solution that contains the human fibrinogen of anti-CD34 monoclone antibody are hatched jointly.The concentration that should understand anti-CD34Fab fragment can be different, and those of ordinary skill in the art need not just can determine optium concentration through undue experimentation.Support is added in the Fab/ fibrin mixture, add the fibrin ferment (concentration is at least 1000U/ml) that concentrates again and come activated fiber albumen.The polymer fiber mixed liquid of protein that gained is contained the Fab fragment directly adds matrix, is pressed into film (being thinner than 100pm) on the surface of support or synthetic graft.In fact, before applying support or synthetic graft, in this way can antibody or antibody fragment doped matrix solution with any kind in.

For example, in another embodiment, the whole antibody and the growth factor that have or do not have antibody fragment are that covalent coupling is in matrix.In one embodiment, by heterogeneous or homogeneity bifunctional linker molecule described antibody and one or more growth factors are covalently attached to matrix.As used herein, term " connect (tethered) " is meant by linkers the antibody covalent coupling in matrix.Adopt using of linkers related to the present invention to be usually directed to before matrix is attached to support, with the linkers covalent coupling in described matrix.After matrix, linkers provides many functional activity groups that can be used for one or more antibody of covalent coupling for matrix at covalent coupling.In an embodiment of this embodiment, Figure 1A provides the example by the corsslinking molecular coupling.Endothelial cell 1.01 is incorporated into antibody 1.03 by surface antigen 1.02.Described antibody is connected in matrix 1.05-1.06 by corsslinking molecular 1.04.Matrix 1.05-1.06 is attached to support 1.07.Described linkers can directly be connected on the matrix and (that is, pass through carboxyl), or the coupling chemical reaction by knowing, for example esterification, amidation and acylation.Described linkers can be two or the triamido functional compound, and they are connected with matrix by direct formation amido link, and provide can with the amido functional group of antibody response.For example, described linkers can be the polyamines functional polymer, as polymine (PEI), polyene propyl amides (PALLA) or polyethylene glycol (PEG).Can (Birmingham Alabama) have bought the scheme of multiple polyethyleneglycol derivative (for example, mPEG-succinyl imidodicarbonic diamide base propionic ester or mPEG-N-N-Hydroxysuccinimide) and covalent coupling by Shearwater Corporation.(also can referring to, Weiner etc., the polyethylene glycol sept is for the influence by the immobilized antibody capture antigen, Influence of a poly-ethyleneglycol spacer on antigen capture byimmobilized antibody, J.Biochem.Biophys.Methods 45:211-219 (2000) includes this paper in as a reference).Be understood that the selection of concrete coupling agent can be depending on the type of used antibody, and this type of selection need not can make through too much examination.Also can use these mixture of polymers.These molecules contain multiple drapability amido functional group, and these functional groups can be used for the surface and fix one or more antibody, peptide, protein, hormone and other coating composition.

In one embodiment, antibody can be attached to the C that directly is deposited on the rack surface ₆₀Fullerene layer.Crosslinking agent can be covalently attached to fullerene.Make antibody be attached to crosslinking agent then, and then adhere to support.Figure 1B provides by fullerene C ₆₀The example of coupling.Endothelial cell 2.01 is incorporated into antibody 2.03 by cellular antigens 2.02, and then covalently or non-covalently is incorporated into matrix 2.04.Matrix 2.04 is passed through C ₆₀2.05 covalent coupling is in support 2.06.

Little molecule of the present invention can comprise synthetic or naturally occurring molecule or the peptide that is used to replace antibody, antibody fragment, growth factor etc.For example, agglutinin is a kind of naturally occurring non-immunogenicity sugar binding peptide.(Schatz etc. 2000 for the agglutinin antigen of endothelial cell specific (Ulex Europaeus Uea 1), people's endometrium endothelial cell: tissue factor separates with 1 type blood plasminogen activator inhibitor, characterized and inflammation mediated expression, Human Endometrial Endothelial cells:Isolation, Characterization, and Inflammatory-Mediated Expression of Tissue Factor andType 1 Plasminogen Activator Inhibitor., Biol Reprod 62:691-697), for example alternative cell surface that is incorporated into endothelial progenitor cells.

Createed synthetic " the little molecule " of the different cell surfaces of energy target, protein, glycoprotein, polysaccharide and acceptor.The surface molecular that these molecular energy selective binding are specific, and can the specific cell type of target, for example endothelial progenitor cells.Synthetic little molecule can be discerned endothelial cell surface mark, for example VEGF.SU11248 (SugenInc.) (Mendel etc., 2003, " with vascular endothelial growth factor receptor and platelet derived growth factor receptor is a kind of novel tyrosine kinase inhibitor SU11248 antitumor activity in vivo of target: the mensuration of pharmacokinetics/pharmacodynamics correlation ", In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growthfactor and platelet-derived growth factor receptors:determination of apharmacokinetic/pharmacodynamic relationship, Clin Cancer Res.Jan; 9 (1): 327-37), PTK787/ZK222584 (Drevs J. etc., 2003, " receptor tyrosine kinase: some new main targets of anticancer disease treatment ", Receptor tyrosine kinases:the maintargets for new anticancer therapy, Curr Drug Targets.Feb; 4 (2): 113-21) and SU6668 (Laird, AD etc., 2002, " SU6668 is to tumor vessel system rapid apoptosis and the tumour decline of inhibitory action in causing the mouse body of Flk-1/KDR and PDGFR-β in vivo ", SU6668 inhibitsFlk-1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumorvasculature and tumor regression in mice.FASEB J.May; 16 (7): 681-90) be little molecule in conjunction with VEGFR-2.

Another subclass synthesized micromolecule of target endothelial cell surface is α (v) β (a 3) integrin inhibitors.SM256 and SD983 (Kerr JS. etc., 1999, " new small molecule material α v integrates plain antagonist: compare with the anticancer disease effect of known various angiogenesis hormone class mortifiers ", Novel small molecule alpha vintergrin antagonists:comparative anti-cancer efficacy with knownangiogenesis inhibitors.Anticancer Res Mar-Apr; 19 (2A): 959-68) being can target and be incorporated into α on the endothelial cell surface (the v) synthesized micromolecule of β (3).

The invention provides a kind of drug delivery system, it comprises: the medical treatment device of coating, for example support, stent graft, cardiac valves, conduit, blood vessel are repaired screen, artificial heart, external and built-in left ventricular assist device (LVADs) and synthetic blood vessel graft, they can be used for treating disease, these diseases comprise: tumour and vascular disease, for example: ISR, atherosclerotic, thrombosis, angiemphraxis etc.In one embodiment, the coating on the medical treatment device of the present invention comprises: the matrix of biocompatibility; At least a antibody, antibody fragment or their combination; And/or at least a compound, as part or therapeutic agent (as estradiol, angiogenesis factor, FGF etc.).

In one embodiment, transgenic cell is mixed with at least a metastatic gene, and described metastatic gene can import described cell by the genetic method based on virus or non-virus.At least a medicine of described metastatic gene codified, but continuous expression or expression under the inducing that stimulates.In one embodiment, described medicine can be hormone, peptide, protein etc.Also have at least a antigen on the surface of described transgenic cell, described antigen can be coated on the antibody of surfaces of medical devices and discern and combination.

As used herein, " antibody " is meant antibody or antibody fragment, or the combination of antibody and fragment, and they can be monoclone antibody, polyclonal antibody, chimeric antibody or humanized antibody.Antibody fragment of the present invention comprises the antibody fragment (for example big molecule and little molecule) of any size, and these fragments keep the identification identical with this antibody and in conjunction with the characteristic (Figure 1A, 1B and 11) of target antigen.

As used herein, " part " is meant can be in conjunction with the molecule of another kind of molecule (for example acceptor on the mammalian cell).For example, part can be antibody, antibody fragment (Figure 1A, 1B, 11 and 17), cell adhesion molecule or basement membrane composition, and they can be discerned and in conjunction with specificity epitope on the target cell membrane or structure.In the embodiment of the mammalian cell that adopts hereditary change, be used for that part on the medical treatment device coating can be discerned through special the selection and in conjunction with by the gene outcome that foreign DNA produced that imports in the transgenic cell.

As used herein, " protein " is meant the amino acid polymer of any length.Described polymer can be straight or branched, can comprise modified amino acid, also can between be inserted with non-amino acid.Described polymer can be naturally occurring peptide, protein, or its modification and synthesized form, comprises it: bioactive fragment, derivative, congener, analogies and do not have function or dominant negative mutant.

Described medical treatment device can be the organ that is used for the implantation belt chamber or any device of body area, and it can include but not limited to: support, stent graft, synthetic blood vessel graft, cardiac valves, conduit, blood vessel is repaired screen, pacemaker, the pacemaker lead, defibrillator, in hole, the ovum garden patent (PFO) every closing device, blood vessel clip, the vascular aneurysms dead lock, the haemodialysis graft, hemodialysis catheter, the chamber current divider, aortic blood tuberculation graft device or assembly, venous valve, suture, the vascular anastomosis folder, remain-type vein or arterial duct, vagina vasorum and medicine delivery port.According to the difference of device, described device can be made from a variety of materials.For example, support of the present invention can be made with stainless steel, Nitinol (NiTi) or evanohm.Synthetic blood vessel graft can be made with crosslinked PVA hydrogel, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethyltoe (ePTFE), porous high density polyethylene (HDPE) (HDPE), polyurethane and poly terephthalic acid vinyl acetate.

Forming apparatus of the present invention biological compatibility of coating matrix comprises: synthetic material, for example polyurethane, block polyurethane-urea/heparin, poly--L-lactic acid, cellulose esters, polyethylene glycol, poly-ethyl acetate, glucan and gelatin; Naturally occurring material, basement membrane components for example is as collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose; And amorphous carbon or fullerene etc.

In one embodiment, described medical treatment device comprises the biocompatible matrix that contains fullerene.In this embodiment, the carbon number of described fullerene can be about C ₂₀-C ₁₅₀, more particularly, described fullerene is C ₆₀-C ₇₀Fullerene of the present invention also can be arranged as nanotube on surfaces of medical devices.

The antibody that offers described medical treatment device coating comprise at least a can identification and in conjunction with the antibody of transgenic cell surface antigen, this antigen can be expressed and can be regulated the adhesion of described cell to surfaces of medical devices by foreign gene or metastatic gene.Described antibody can be covalently or non-covalently attached in stromal surface, or is covalently attached to the outermost layer of the matrix that covers medical treatment device by linkers.Of the present invention this on the one hand, for example, described monoclone antibody also can comprise Fab or F (ab ') ₂Fragment.

Described antibody can be discerned and the subject mammiferous antigen of combination butt joint specifically, and their specificity does not depend on cell-line.In one embodiment, described antibody has specificity to people's endothelial progenitor cells surface antigen, and described surface antigen is for for example: CD133, CD14, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146), CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit part), Tie-2, HAD-DR and other antigen (anti--H-2K for example ^kAntibody).

In another embodiment, the coating of described medical treatment device comprises the aforesaid biocompatible matrix of one deck at least, and this matrix comprises the micromolecular outer surface that is used to adhere at least a natural or synthetic source for the treatment of effective dose.Described little molecular energy identification transgenic cell surface antigen and with its interaction, thereby transgenic cell is fixed on the surface of device, and induce the expression of metastatic gene.Described little molecule can for example from cellular component, as fatty acid, protein, nucleic acid, carbohydrate etc., and can interact with the transgenic cell surface receptor from various sources.In this embodiment of the present invention, the coating on the medical treatment device also can comprise compound, for example the part that combines with the coating that contains antibody.

Genetic method based on virus and non-virus all can be used for importing metastatic gene to produce transgenic cell.Transgenic cell of the present invention can be expressed the medicine of justacrine by the of short duration or stable metastatic gene coding that mixes.Also can be in conjunction with other metastatic gene to give its survival, selectivity and/or growth vigor.Can to various cells modify with produce non-regeneration or again propagation transgenic cell, these cells are for for example: endothelial cell or granulocyte, comprise neutrophil cell, eosinophile granulocyte, basophilic granulocyte, monocyte and lymphocyte or somatic cell, or the combination of these cells.Can be at the culture in vitro transgenic cell, it is collected and preserves.Can generate the transgenic cell that can produce various medicines by mixing different metastatic genes, to be used for different therapeutic purposes.Can pass through whole body or local approach, give transgenic cell with single or mixed cellularity group.At individual symptom, the transgenic cell that can give different amounts is to secrete the medicine of different amounts.In one embodiment, the renewable endothelial progenitor cells of described transgenic cell.In another embodiment, can use based on the mode of sending of conduit or with two air bag plavini (dual balloon inflation method) topical administrations and give the transgenosis endothelial progenitor cells.

In one embodiment, transgenic cell also comprises other metastatic gene of expressing exogenous cell surface antigen, and these antigens can be coated on ground identification of described antibody specificity and the combination in the medical treatment device matrix.The expression of metastatic gene and the secretion of product can be to be recurred or activates inducibility promotor and temporary transient the generation at exogenous stimulation.

Therapeutic compound by metastatic gene coding of the present invention can be any molecule with required physiological action, it can be but is not limited to: defined protein-based, comprise that growth factor, chemotactic factor (CF) and cell factor, part and acceptor and other functional protein and nonprotein can secrete compound.In one embodiment, therapeutic compound is the protein that is selected from down group: endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), the anti-inflammatory factor and inflammation regulatory factor.

Medicine, for example in the embodiment of the mammalian cell that adopts hereditary change, be to stimulate the compound that metastatic gene is expressed and the target product is secreted, can be part or other composition in the described medical treatment device coating, they can and activate the downstream signal pipeline of extrachromosomal nucleic acid (for example import target cell in DNA construction) in conjunction with the transgenic cell surface antigen.In another embodiment, the metastatic gene of the mammalian cell of hereditary change is expressed and can be activated by for example part or medicine, and described part or medicine can be taken in by described transgenic cell and be expressed by inducibility promotor stimulated gene.In one embodiment, described part or medicine are the general administrations.In another embodiment, described part or medicine are to be coated in the matrix of implanted device and topical.

The invention provides the method that is used for the treatment of multiple disease, described disease can be but is not limited to: tumour, vascular disease and healing reaction.Described method relatively and the progress of prior art be as required various medicines are carried out target site send with aequum.

The invention provides a kind of method for the treatment of tumour and transfer thereof.In this embodiment, described metastatic gene codified (1) anti-angiogenesis, for example interferon (IFN), thrombospondin (TSP), angiostatin, Endostatin, oncostatin M (OSM) and Rho, they can suppress to generate as the new vessels of carrying out property of tumour growth precondition; Or (2) tumor suppressor protein, for example kinases (CDK) of the antibody of p53, Rb, E1, BRCA1, cell growth activator (for example growth factor) or dominant negative mutant or cyclin dependence or cyclin, E2F, NFKB; Or the combination of these genes.In one embodiment, described metastatic gene can comprise the gene of the following material of encoding: for example, and prostacyclin and/or cyclooxygenase, α-CGRP, matrix metalloprotease and/or endothelial nitric oxide synthase.

As used herein, phrase " anti-angiogenesis " is meant the molecule that can suppress angiogenesis or angiogenic growth.

The present invention also provides the method that is used for the treatment of vascular disease.In one embodiment, a kind of method for the treatment of ischemic disease is provided, metastatic gene is used to the angiogenesis factor of encoding in the method, for example the antibody or the dominant negative mutant of the multiple-effect factor (pleiotrophin), angiogenesis factor, angiogenin, the plain stimulating factor of integration and/or anti-angiogenesis.

As used herein, phrase " angiogenesis factor " is meant the molecule that can stimulate angiogenesis or angiogenic growth.

In another embodiment, described invention is used for the treatment of atherosclerotic, ISR, thrombosis, aneurysm or angiemphraxis.In this embodiment of the present invention, metastatic gene is used for eNOS or the VEGF that coding (a) promotes that endothelium is rebuild; Or (b) anti-inflammatory or inflammation regulatory factor, for example IFN-β, IFN-α, TGF-β or interleukin 10 (IL-10); Or (c) be used to suppress smooth muscle cell growth, migration or the differentiation inhibitors of endometrial hyperplasia; Or the combination of these genes.

The present invention also provides the engineered method that is used to induce healing reaction.In one embodiment, provide a kind of implanted device surface of internal cavity rapid induction in being placed in implantable intravascular target lesion to merge the method that endodermis forms, transgenic cell wherein is the endothelial progenitor cells of expressing eNOS, VEGF or the anti-inflammatory factor or inflammation regulatory factor.In this embodiment, compare with prior-art devices, the biocompatibility of medical treatment device of the present invention increases, can be deposited on the implant site of medical treatment device along surface of internal cavity by reducing or suppressing smooth muscle cell migration, smooth muscle cell differentiation and collagen, and reduce or suppress excessive endometrial hyperplasia and ISR based on tissue.

In one embodiment, the method that is used for coated medical devices may further comprise the steps: one deck biocompatible matrix is applied over the surface of medical treatment device at least, wherein said biocompatible matrix can comprise at least a component that is selected from down group: polyurethane, block polyurethane-urea/heparin, poly--L-lactic acid, cellulose esters, polyethylene glycol, poly-ethyl acetate, polysaccharide (glucan for example, gelatin), collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose and carbon and fullerene, and simultaneously or on described biocompatible matrix, apply at least a antibody and the optional a kind of compound that can induce metastatic gene to express successively.

The present invention also provides a kind of method that is used for the treatment of disease, and described disease is for for example, mammiferous tumour, vascular disease and wound healing.The present invention includes medical treatment device is implanted in mammiferous blood vessel or the pipe, wherein said medical treatment device is coated with: (a) biocompatible matrix; (b) at least a antibody; (c) a kind of composition randomly; Transgenic cell is imported the described mammal that needs described treatment, and randomly give compound, wherein be coated on the antibody recognition in the medical treatment device matrix and be combined in the antigen of expressing on the transgenic cell surface, thereby described transgenic cell is fixed on the surface of matrix, and by the described cellular expression that be fixed of at least a medicine by being activated by compound of metastatic gene coding, described compound be a medicine and at the therapeutic gene product of specifying the site to secrete for example.

The present invention also provides a kind of method that is used for the treatment of mammal medium vessels disease, and described method comprises: medical treatment device is implanted described mammiferous blood vessel or pipe, and wherein said medical treatment device is coated with: (a) biocompatible matrix; (b) at least a antibody; (c) a kind of composition randomly; Transgenic cell is imported the described mammal that needs described treatment, and randomly give compound, wherein be coated in the medical treatment device matrix antibody recognition and in conjunction with the antigen of only on transgenic cell film surface, expressing, thereby described transgenic cell is fixed on the surface of matrix.Described transgenosis (hereditary change) cell also can comprise the genetic material of at least a therapeutic gene product of encoding, and described gene outcome can be continuous expression or expresses when signal (compound that for example comprises hormone and peptide) activates.

Transgenic cell of the present invention can comprise at least a effable metastatic gene, this gene can be used for coding (but being not limited to): (1) growth factor, comprise its family member: platelet derived growth factor (PDGF) for example, TGF (TGF), endothelial growth factors (EGF), fibroblast growth factor (FGF), IDGF (insulin-like growth factor) (IGF), vascular endothelial growth factor (VEGF), heparin-bounding growth factor, the growth factor that liver cancer is derived (HDGF), hepatocyte growth factor/dispersion factor (HGF), placenta growth factor (PIGF), platelet-derived endothelial cell growth factor (ECGF) (PD-ECGF), stem cell factor (SCF), and their other albumen form; (2) chemotactic factor (CF): for example CXC family, CC family, C family, and their other albumen form; (3) cell factor: for example adam protein (ADAM), annexin V, B7﹠amp; The CD28/CTLA-4 receptor family, bone morphogenetic protein (BMP), caspase, CD44, CD44H, endothelin-1 (ET-1), eph, hematopoietin (Epo), intercellular adhesion molecule-3/CD50 (ICAM-3), macrophage stimulating protein (MSP), matrix metalloproteinase (MMP), neurotrophic factor, endothelial nitric oxide synthase (eNOS), NKG2D, PECAM-1 (PECAM-1/CD31), the multiple-effect factor/midkine (PTN/MK), TfR (sTfR), protection peptide (hedgehog peptide), STAT, stem cell labeling, Th1/Th2, TPO (Tpo), tnf family cytokines, VCAM-1/CD16, the non-specific inhibiting factor β of monoclonal (MNSF β), 6Ckine (SLC), B-lymphocyte chemotactic inducer (BCA-1/BLC), leukaemia inhibitory factor, the peptide of the neutrophil activation of monocyte derived (GRO), and their other albumen form; (4) participate in other functional protein of signal conduction adjusting, Cycle Regulation, cell division and/or cell differentiation, they are for for example: part, acceptor, phosphorylase, kinases, transcription factor, and their other albumen form.

In one embodiment, be used for anti-angiogenesis of the present invention for for example: interferon (IFNs), thrombospondin (TSP), angiostatin and Endostatin, oncostatin M (OSM), integrate the antibody of the plain retarding agent of arranging, metal protease inhibitors, endothelial cell phosphorylation inhibitor, the dominant acceptor that is used for blood vessel generation derivant, blood vessel generation derivant, the protein by the alternate manner effect, and their other albumen form.Other angiogenesis factor comprises angiogenesis factor, angiogenin, the plain stimulating factor (for example Del-1) of integration, and their other albumen form.

Be used for other growth factor of the present invention for for example: the multiple-effect factor, midkine, VEGF family (comprises VEGF-2, VEGF-C and VEGF-D), FGF family (comprises FGF-1, FGF-2, FGF-5 and FGF-18), the growth factor that liver cancer is derived (HDGF), hepatocyte growth factor/dispersion factor (HGF), endothelial growth factors (EGF) family member (comprises transforming growth factor, EGF and TGF-α-HII I and platelet derived growth factor (PDGF) (comprise AA, AB and BB hypotype).

Experimental example

The present invention will be described in following tentative detailed description part.These following parts are in order to understand the present invention, rather than and should not be construed as the scope of the invention that limits by any way by described in the claims behind the embodiment.

Embodiment 1

The phenotype of endothelial progenitor cells

Separate endothelial progenitor cells (EPC) by the following method: by CD34+ magnetic bead partition method (Dynal Biotech) or enriched medium partition method (Asahara T as describing recently, Murohara T, Sullivan A etc., " feeder vessels generates the separating method of the endothelial progenitor cells of inferring of usefulness ", I solation of putativeprogenitor endothelial cells for angiogenesis, Science 1997; 275:964-7).In brief, obtain peripheric venous blood from healthy male volunteers, and by density gradient centrifugation acquisition monocyte component, then with this cell inoculation (Becton Dickinson) on the cultivation slide that is coated with people's fibronectin, medium be EC basal medium-2 (EBM-2) (Clonetics), wherein be supplemented with 5% hyclone, people VEGF-A, human fibroblastic growth factor-2, people's endothelial growth factors, insulin-like growth factor-i and ascorbic acid.Make EPC growth 7 days, per 48 hours replacing medium.The results are shown among Fig. 2 A and the 2B of these tests.Fig. 2 A and 2B have shown that anti-CD34 isolated cells seems more seemingly spindle sample, and this just shows that these cells just are being divided into endothelial cell.

Measure the phenotype of EC by immunohistochemical method.In brief, EPC (Sigma) is fixed 10 minutes with 2% paraformaldehyde (PFA) phosphate buffered solution (PBS) (Sigma), with PBS washing 3 times, and with the dyeing of different EC specific marker: rabbit human VEGFR-3 resistant-2 (Alpha Diagnostics Intl.Inc.), mouse-anti-human T ie-2 (Clone Ab33, Upstate Biotechnology), mouse-anti people CD34 (Becton Dickinson), EC-agglutinin (Ulex Europaeus Ueal) are (Sigma) and mouse-anti people 8 factors (Sigma).Make two of cells contacting fluorescein isothiocynate (FITC) combination resist the existence of verifying antibody.Iodate third ingot (PI) is used as the nuclear mark.These tests the results are shown in Fig. 2 C-2G.Fig. 2 C shows has VEGFR-2 to express after 24 hours in the culture, this has just confirmed that described cell is an endothelial cell.Fig. 2 D and 2F are depicted as 7 days and hatch the nuclear staining of back in conjunction with cell, and Fig. 2 E and 2G are depicted as the cell of using the anti-Tie-2 antibody staining of FITC coupling in the same visual field.

ENOS mRNA is carried out reverse transcriptase-polymerase chain reaction (rt-PCR) measure the ability that EPC expresses endothelial nitric oxide synthase (eNOS, a kind of EC function sign).EPC was grown 7 days in the EBM-2 medium, use the total RNA kit of GenElute mammal (Sigma) to separate total RNA then, and quantitative by absorbance under 260nm.With 1 μ g random primer the total RNA of 20 μ L is carried out reverse transcription with Omniscript RT kit (Qiagen).For each RT product, end reaction volume (2-10 μ L) to equal portions in two parallel PCR reactions increases, used eNOS Auele Specific Primer (299bp product, justice 5 '-TTCCGGGGATTCTGGCAGGAG-3 ' is arranged, SEQ IDNO:1, antisense 5 '-GCCATGGTAACATCGCCGCAG-3 ', SEQ ID NO:2) or GAPDH Auele Specific Primer (343bp product, justice 5 '-CTCTAAGGCTGTGGGCAAGGTCAT-3 ' is arranged, SEQ ID NO:3, antisense 5 '-GAGATCCACCACCCTGTTGCTGTA-3 ', SEQ ID NO:4) and Taq polymerase (Pharmacia BiotechAmersham).PCR circulation is as follows: 94 ℃ 5 minutes, 65 ℃ 45 seconds, 72 ℃ 30 seconds (to eNOS is 35 circulations, then is 25 circulations to GAPDH).Analyze the rt-PCR product by 2% agarose gel electrophoresis, with the ethidium bromide colour developing, and the optical density standard measure.This test the results are shown in Fig. 3 A and 3B.By Fig. 3 A and 3B as seen, cell has been expressed nitricoxide synthase (eNOS) after hatching 3 days under the condition that has or do not exist oxygen in medium.Cultivate and still continue to express eNOS mRNA after 7 days.The expression of eNOS mRNA shows that described cell has been divided into ripe endothelial cell in 3 days, and begins to exercise the function that is similar to the endothelial cell that breaks up fully.

Embodiment 2

Catch endothelial cell with the stainless steel disk that anti-CD34 (antibody) applies: in process of the test, (American type culture collection, American Type CultureCollection) is incubated in the endothelial cell growth medium with Human umbilical vein endothelial cells (HUVEC).Cell is hatched with sample or blank stainless steel (SST) sample with CMDX and gelatin coating that the surface contains or do not contain binding antibody.After hatching, remove medium, and with PBS washing sample 2 times.Fixed cell is 10 minutes in 2% paraformaldehyde (PFA), and with PBS washing 3 times, each 10 minutes to guarantee to remove all fixatives.At room temperature, each sample was hatched 30 minutes with lock solution, to seal all non-specific binding.With PBS washing sample 1 time, and 1: 100 dilution of contact VEGFR-2 antibody is cultivated and is spent the night.Use the PBS washing sample then 3 times, one anti-to guarantee to remove all.Add FITC coupling two anti-of doing 1: 100 dilution with lock solution in each sample, incubated at room is 45 minutes on Belly Dancer device.After hatching, use PBS washing sample 3 times, with the PBS washing that contains 0.1%Tween 20 once, wash with PBS again.With iodate third ingot (PI) fixed sample, and under Laser Scanning Confocal Microscope, observe.

Fig. 4 A-4E has applied CMDX and anti-CD34 antibody (Fig. 4 A), gelatin and anti-CD34 antibody (Fig. 4 B) as mentioned above, blank SST (Fig. 4 C), CMDX coating but has not had antibody (Fig. 4 D) and gelatin coating but do not have the SST sample microphoto of antibody (Fig. 4 E).Accompanying drawing is depicted as and demonstrates the sample that only applies antibody by PI dyeing and comprise many cells that adhere to sample surfaces.Blank SST contrast dish shows that few cell adhesion is in its surface.

Fig. 5 A-5C has applied CMDX and has not had the microphoto that antibody is incorporated into its surperficial control sample.Fig. 5 A shows by the visible few cell adhesion of PI dyeing in the surface of sample.Fig. 5 B shows that the cell that adheres to is the VEGFR-2 positive, and this shows that they are endothelial cells, and Fig. 5 C is depicted as the combination of the nuclear and the positive green fluorescence of VEGFR-2 of dyeing.Fig. 5 D-F has applied gelatin and the microphoto that do not have the control sample of antibody in its surface.Fig. 5 D shows acellular existence, and this is because this sample does not have the green fluorescence (referring to Fig. 5 E and 5F) that PI dyeing is not also launched.

Fig. 6 A-6C is the microphoto that has applied CMDX and had the SST sample of the anti-CD34 antibody that is incorporated into its surface.These accompanying drawing show samples comprise many adherent cells, as green fluorescence shows they set up approaching fusion individual layer (Fig. 6 A), be VEGFR-2 positive cell (Fig. 6 B and 6C).Similarly, the microphoto of the sample of Fig. 6 D-6F gelatin that is the coating that is combined with anti-CD34 antibody of its surface.These accompanying drawings are many red staining nuclears and the green fluorescence by producing from VEGFR-2/FITC antibody also, has shown that HUVEC adheres to sample surfaces (Fig. 6 E and 6F).

Embodiment 3

The VEGFR-2 of endothelial progenitor cells dyes with Tie-2: separate the CFU-GM that obtains in the human blood as described in example 1 above, incubated in vitro 24 hours, 7 days and 3 weeks in medium.After hatching, remove growth medium, use PBS washing sample 2 times.With 2% paraformaldehyde (PFA) fixed cell 10 minutes, with PBS washing 3 times, each 10 minutes, guarantee to remove all fixatives then.Each sample was hatched at room temperature 30 minutes with sheep (for VEGFR-2) or horse (the being used for Tie-2) confining liquid of 440 μ l, to seal all non-specific binding.With PBS washing sample 1 time, add VEGFR-2 or the Tie-2 antibody of doing dilution in 1: 100 with confining liquid then, hatch sample and spend the night.Then with PBS washing 3 times, anti-all by flush away to guarantee all one.In each sample, add two anti-(200 μ l), and on Belly Dancer device, at room temperature hatched 45 minutes with the FITC coupling of horse or 1: 100 dilution of sheep confining liquid do.After hatching, use PBS washing sample 3 times, with the PBS washing that contains 0.1%Tween 20 once, again with washing among the PBS.With iodate third ingot (PI) fixed sample, and under Laser Scanning Confocal Microscope, observe.

Fig. 7 be contain on its surface CD34 antibody coating the microphoto of sample of CMXD, this sample was hatched 24 hours with cell, showed that CFU-GM is trapped in the surface of sample, this point can be bright by the authentication of the red staining that exists on the sample surfaces.This accompanying drawing has shown that also about 75% cell is the VEGFR-2 positive, and form is circular.

Fig. 8 A and 8B are the sample of hatching with cell 7 days.Shown in Fig. 8 A, the nuclear of red staining shows that cell is present on the sample, and these cells are the VEGFR-2 positive (Fig. 8 B, 100%), and by the endothelialization more structurally of these cells shown in the spindle of cell.Fig. 9 A and 9B be comprise in its surface CD34 antibody coating the sample microphoto of CMXD, this sample was hatched 7 days with cell, after hatching, made this sample contact Tie-2 antibody.Shown in Fig. 9 A, the nuclear of red staining has shown has many cell adhesions in sample surfaces.As the green fluorescence of cell emission as seen, the cell that adheres to sample also is the Tie-2 positive (100%) (Fig. 9 B).Generally speaking, shown in the VEGFR-2 and Tie-2 acceptor that exists in sample surfaces and adherent cell surface by many cell adhesions: after cell hatched 7 days with sample, the sample that has applied CD34 antibody can be trapped in endothelial cell their surface.In addition, on sample surfaces, exist 100% endothelial cell to show that non-endothelial cell may come off in the time of 7 days, or all adherent cells have all begun to express endothelial cell marker in the time of the 7th day.

Figure 10 A-10C is the phase contrast microscope photo that is grown in the endothelial progenitor cells in 3 weeks in the endothelial cell growth medium.Figure 10 A showed cell has been broken up becomes ripe endothelial cell, and this point can be proved by the intravascular space of the two-dimentional tubular structure at arrow place.Figure 10 B shows the three-dimensional cell stack that has multilayer, and promptly a cell is positioned on another cell, and this begins to form the report that is positioned at the layer on another layer with regard to the endothelial cell that has confirmed growth in time expand.Figure 10 C is depicted as the CFU-GM of cultivating for 3 weeks behind kind of the plate, and these cells have the outward appearance of endothelial cell, and as CD34/FITC antibody green fluorescence of their surface existence proves, this accompanying drawing has confirmed that these cells are endothelial cell.

The leucocyte that above-mentioned data show is separated from human blood contains the positive CFU-GM of CD34, and these cells can be grown for mature endothelial cell and are easy to express surface endothelial cell antigens.(VEGFR-2 and Tie-2).These data also show can adopt at the antibody of CFU-GM or stem cell surface antigen with these cell captures on the surface of the medical treatment device that the present invention applies.

Embodiment 4

Applied fullerene and applied fullerene and had anti-CD34 antibody

And/or endothelial cell growth factor (ECGF) (Ang-2, body of stainless steel VEGF)

Adopt following method to come with the stainless steel stent and the disk that produce functional Fullerene layer with binding antibody and/or growth factor (that is, VEGF or Ang-2):

In the first step, with the surface of 0.5M HCl activation SST support or disk, described HCl has also removed lip-deep any passivation pollutant simultaneously.From activating bath, take out metal sample, use distilled water flushing, the dry and oven dry under 75 ℃ of methyl alcohol.Then described support be impregnated in and contain oxidation fullerene (C ₆₀In-O) the toluene derivative solution maximum 24 hours.Described oxidation fullerene can be incorporated into support by the Fe-O on the support, Cr-O and Ni-O.Support is taken out from the bath of deriving,, placed Soxhlet Extractor then 16 hours, remove the C of any physical adsorption with fresh toluene with the toluene flushing ₆₀Take out support, and oven dry is spent the night under 105 ℃.This reaction has produced support of deriving fully or the disk with fullerene individual layer.

In step 2, make decanedioic acid and thionyl chloride or sulfur oxychloride (SOCl ₂) reaction formation sebacoyl chloride, to form the dialdehyde molecular solution.Gained sebacoyl chloride and the LiAI[t-O butyl of making as follows] ₃H and diethylene glycol dimethyl ether reaction are to obtain 1, the 10-decanedioyl:

In step 3, on available from the support of step 1 or disk, form N-methylpyrrole alkane derivatives.As follows, come fullerene molecule is further derived by following reaction: under the nitrogen, in the toluene solution that refluxes, with 1 in the fullerene of equimolar amounts and sarcosine and the step 2,10-decanedioyl product reaction 48 hours is to obtain fullerene stainless steel stent or the disk that the N-crassitude is derived.

Stainless steel stent that washing is derived or disk are used for standard method binding antibody and/or (VEGF or Ang-2) to remove any chemical residue.As the CFU-GM in the separation human blood as described in the embodiment 1, make the fullerene disk of its contact with anti-CD34 antibody coating.After hatching, remove growth medium, use PBS washing sample 2 times.With 2% paraformaldehyde (PFA) fixed cell 10 minutes, with PBS washing 3 times, washed 10 minutes, then to guarantee to remove all fixatives at every turn.Each sample was at room temperature hatched 30 minutes with lock solution, to seal all non-specific binding.With PBS washing sample 1 time, the VEGFR-2 antibody incubation that adds dilution in 1: 100 spends the night.Use the PBS washing sample then 3 times, one anti-to guarantee to remove all.Add FITC coupling two anti-of doing dilution in 1: 100 with confining liquid in each sample, incubated at room is 45 minutes on Belly Dancer device.After hatching, use PBS washing sample 3 times, with the PBS washing that contains 0.1%Tween 20 once, wash with PBS again.With iodate third ingot (PI) fixed sample, and under Laser Scanning Confocal Microscope, observe.Figure 11 shows that the present invention combine CFU-GM coating the schematic diagram of rack surface of functional fullerene.Figure 12 A-12B respectively for the coating of PI dyeing (12A) and anti-VEGFR-2/FITC-coupling antibody staining fullerene do not have the microphoto of the control sample of antibody.Figure 12 C and 12D are the microphotos that has applied the sample of fullerene/anti-CD34 antibody coating.As shown in the figure, the sample that is coated with anti-CD34 antibody contains the VEGFR-2 positive cells that adhere to the surface more.

As described in embodiment 5, will contain or not contain antibody coating the sample of fullerene implant yorker.Take out support and make histologic analysis,, fix up to processing with 10% formalin buffer then with 10% formalin buffer flushed zone support section 30 seconds.From each support, cut following 5 sections; Close on support 1mm place, support and close on terminal 1mm place, support middle section, rack far end 1mm place and support end 1mm place.Use Su Mujing ﹠amp; Yihong (HE) and three look elastin laminin stained.Figure 13 A-13D is the support cross section microphoto of implanting for 4 weeks that takes out from coronary artery.The support that data show has applied fullerene (Figure 13 B and 13D) has suppressed the neointimal hyperplasia (blank support, Figure 13 A and 13C) at support position of living in compared with the control.

Embodiment 5

Pig air bag damage research: the structural transplantation that will apply antibody is gone into yorker childhood of heavy 25-30kg.Take care of animal according to " management of laboratory animal and guide for use " (Guide for the Care and Use of LaboratoryAnimals, NIH publication number 80-23 revises in 1985).Behind the overnight fasting, make animal calmness (20mg/kg) with ketalar.Use pentothal (12mg/kg) induced anesthesia then, animal is carried out intubate, and connect the snorkel give oxygen and nitrogen mixture (1: the 2[volume/volume]).Isoflurane with 0.5-2.5 volume % is kept anesthesia.Intramuscular injection 1,000mg procaine penicillin-G and tardocillin-G (streptomycin) mixture is to provide the prevention of antibiosis disposition.

Under aseptic condition, carry out the left neck artery otomy, the 8F guide sheath is placed left neck artery.Give all animal 100IU heparin/kg body weight.In the whole surgery process, regularly append 2, the heparin medicine group (boluse) of 500IU is to remain on ACT more than 300 seconds.The 6F guiding tube is imported by carotid sheath, be advanced to coronary ostium.After giving 200 μ g nitroglycerine in the coronary artery, carry out angiography, with quantitative coronary angioradiographic system analysis image.The 3F Embolectomy catheters inserted near-end coronarius and be advanced into and select to be used for implant frame and the exposed section of endothelium to far-end.The R support through applying that adds anti-CD34 antibody is inserted by guiding tube, and in the exposed section of coronary artery, launch.With blank stainless steel stent or applied matrix but the support that do not contain antibody in contrast.Be that 1.1 ratio is implanted LADCA (LAD) or arteria coronaria dextra (RCA) or circumflex branch of coronary artery (Cx) with support with support to artery.With the size and the position of angiography evaluation support, take out guide sheath, closed two-layer skin.In process of the test, give the ASA of animal 300mg.

Behind implant frame, put to death animal 1,3,7,14 and 28 day the time.Calm and the anesthesia with animal earlier as mentioned above.Take out the coronary artery of implant frame, have the unsupported blood vessel of 1cm at its near-end and far-end.Handle the blood vessel of implant frame with three kinds of methods: histology, SABC method or ESEM method.

Detect for SABC, softly wash the support 30 seconds of incision, place 10% formalin/PBS solution until processing with 10% formalin.Specify with the PBS solution flushing that contains 2% paraformaldehyde (PFA) and to be used for the support 30 seconds that SABC detects, placed 2%PFA solution then 15 minutes, and detecting until the SABC of using rabbit human VEGFR-3 resistant-2 or mouse-anti-human T ie-2 antibody with PBS washing and storage.

Prepare to be used for the support of SEM by the following method:, in containing the 0.1M sodium cacodylate buffer liquid of 2.5% glutaraldehyde and 2%PFA, fixedly spend the night then with 10% formalin buffer flushing 30 seconds.Use dimethyl arsenic acid buffer liquid washing sample three times then, and its cleaning is spent the night.Finish fixedly post processing by following steps: the 0.1M dimethyl arsenic acid buffer liquid with 1% osmium tetroxide (Sigma) is handled, and uses ethanol dehydration (30% ethanol, 50%, 70%, 85%, 95%, 100%, 100%) then, uses CO subsequently ₂Carry out critical point drying.After the drying, sample is carried out metal spraying, and under SEM, observe.(" coronary artery to normal pig adopts the Palmatz-Schatz support that applies through heparin to reduce or remit thrombotic support mould bases ", Reduction in thromboticevents with heparin-coated Palmaz-Schatz stent in normal porcine coronaryarteries, Circulation 93:423-430 includes this paper in as a reference).

Be used for the support section 30 seconds of histologic analysis with 10% formalin buffer flushing, fix until processing with 10% formalin buffer then.Cut following 5 sections from each support; Close on support 1mm place, support and close on terminal 1mm place, support middle section, rack far end 1mm place and support end 1mm place.Use Su Mujing ﹠amp; Yihong (HE) and three look elastin laminin stained.

Figure 14 A-14G is for implanting back 1 hour (Figure 14 A and 14B) and 48 hours (Figure 14 C-14G) observed taking-up support under ESEM.Microphoto has clearly shown the support (14B with glucan/anti-CD34 antibody applies, 14E-G) caught endothelial progenitor cells, this point can by with the contrast that applies with glucan by contrast, section in 48 hours than high power (400X) down cell be spindle outward appearance (14A, 14C and 14D).

The transverse section of porcine coronary separator also shows compared with the control (blank stainless steel 14H and 14I; 14J and 14K with the glucan coating), apply (14L, 14M) with glucan-anti-CD34 antibody and make neointimal hyperplasia (thickness of arterial smooth muscle layer) be subjected to significant inhibition.The stent graft that applies with fullerene has suppressed neointimal hyperplasia (shown in Figure 13 B-13D) better than the blank stainless steel stent.

Figure 15 A and 15B are respectively long the applying but the support separator of the no antibody in surface and with copolymerization Jiao microphoto of the anti-CD34 antibody support of glucan-blood plasma coating with glucan-blood plasma through the 18mm that takes out after 48 hours.Described support was once implanted the coronary artery of male York children pig.The support that takes out is carried out SABC processing and VEGFR-2 dyeing, resist with two of FITC coupling then and handle and under Laser Scanning Confocal Microscope, study.Figure 15 B has shown that with 15C lacking endothelium fully with the support that does not contain antibody compares, and the support that contains antibody is covered by endothelial cell, and this green fluorescence by this section is confirmed (Figure 15 A).

Embodiment 6

Endothelial growth factors is mixed the matrix of antibody that put on fixing of support: following the step that the antibody that will resist the endothelial progenitor cells surface antigen is fixed in the biocompatible matrix that puts on endovascular stent described, endothelial growth factors be adsorbed in subsequently support with promote circulation endothelium progenitor cell adhere to and when contacting with blood maturation be functional endothelium.

Apposition: adopted method known to those skilled in the art, handled stainless steel stent to import amino functional at rack surface with plasma deposition technique.Employing is called the standard method of water-soluble carbodiimide coupling chemical method, glucan (CMDX) carboxyl by activation under aqueous conditions is incorporated into the amino-functional layer that is deposited on the support with carboxyl group functional glucan layer, forms amido link at the amino that is positioned under the described aqueous conditions on the plasma deposition layer between plasmasphere and functionality CDMX.

Antibody immobilization: by in the acidic buffer solution of moisture water-soluble carbodiimide chemical reagent, hatching, with antibody (for example mouse monoclonal anti-human CD34) covalent coupling of anti-endothelial progenitor cells surface antigen in the support that applies with CDMX.

The absorption of growth factor: after monoclonal anti-human CD34 is fixed on the CMDX matrix that puts on support, with described device be incubated in contain suitable concentration endothelial growth factors (for example, angiogenin 2) in the aqueous solution, make growth factor be absorbed into CMDX matrix.With the treated device of normal saline solution flushing, and be stored in the sodium azide preservative agent.

Employing standard angiogram technology, when said apparatus being implanted porcine coronary and contacting human blood, produced and strengthen circulation endothelium progenitor cell and catch and be attached to treated or apply rack surface and promote that cell maturation is the effect of functional endothelial tissue.The quick foundation of functional endothelial tissue can reduce device thrombosis that causes and the degree of regulating neointimal hyperplasia.

Embodiment 7

Endothelial growth factors and antibody are fixed on the support: the following antibody and the growth factor that will resist the endothelial progenitor cells cell surface antigen described is fixed on the biocompatible matrix that puts on endovascular stent, thereby promotes adhering to maturation of circulation endothelium progenitor cell to be functional endothelial tissue step when contacting with blood.

Apposition: apposition has used method known to those skilled in the art, handles stainless steel stent to import amino functional on rack surface with plasma deposition technique.Employing is called the standard method of water-soluble carbodiimide coupling chemical method, under aqueous conditions, carboxyl group functional glucan layer is incorporated into the amino-functional layer that is deposited on the support, between plasmasphere and functionality CDMX, forms amido link at the amino that is positioned under the described aqueous conditions on the plasma deposition layer by activated dextran (CMDX) carboxyl.

The immobilization of antibody and growth factor: by under acid condition, with etc. the antibody (for example mouse monoclonal anti-human CD34) of anti-endothelial progenitor cells surface antigen of molar concentration and endothelial growth factors (for example, angiopoietin-2) hatches in the water-soluble carbodiimide reagent solution, make itself and the support covalent coupling that applies with CDMX.With the treated device of normal saline solution flushing, and be stored in the sodium azide preservative agent.

Embodiment 8

The little molecular functionization of support: obtain endothelial progenitor cells as separation as described in the embodiment 1.The slide that cell inoculation is covered in fibronectin also grew 7 days it in the EBM-2 medium.Fixed cell is also used the endothelial cell specific agglutinin dyeing (Ulex Europaeus Ueal) of iodate third ingot (PI) and FITC coupling.The results are shown among Figure 16 A and the 16B of these tests.These accompanying drawings have shown that endothelial progenitor cells is incorporated into the slide that fibronectin applies, and these cells are expressed the part that this agglutinin is arranged on their surface.

Embodiment 9

Bicistronic mRNA carrier transfection pig endothelial progenitor cells (EPC) with the coding vasodilation compound and the cell surface marker (MHC-I of brachymemma) of uniqueness.MHC-I can be fixed in the identification of the specific antibody on the dummy in the blood vessel.The support that antibody applies is implanted in the porcine coronary, and the EPC with hereditary change implants pig then.Because antibody-AI, EPC are trapped on the support of coating, and form endothelial cell monolayer on stent strut.Captive cell can be secreted vasodilator, the increase far-end CBF of expressing and trigger positivity and mould.

The selection of plasmid: Miltenyi Biotec (Germany) has developed and has contained pMASCSK ^kThe MACSelect K System of plasmid vector.Described pMACSK.II plasmid is a kind of bicistronic mRNA carrier (5229bp) that contains multiple clone site (MCS), and the clone has the cDNA of coding prostacyclin synthase and the gene of coding mouse MHC I type molecule H-2K therein.Develop this system and be in order to select to be used for cells transfected, with the MHC molecule of brachymemma as selected marker.The expression of natural H-2K is limited to (for example, AKRiJA or CBNJ) in some rare mouse strain, therefore at H-2K ^kIrrelevant reaction should can not take place with other surface antigen in the monoclone antibody of surface protein (Miltenyi Biotec) basically.

The assessment with The cross reactivity of whole blood: in order to ensure anti-H-2K ^kAntibody not with the pig whole blood in cellular component generation cross reaction, make the anti-H-2K antibody response of whole blood and FITC coupling, and carry out whole blood facs analysis (BeckmanCoulter Cytomics FC 500).With expressing H-2K ^k(whole blood of ATCC) " reinforcement " (" spiked ") is as positive control for American type culture collection, American Type Culture Collection for the mouse spleen fibroblast strain AKRIJASp of surface antigen.

Fibroblastic cultivation: the AKR/JA.Sp fibroblast is incubated in the T-75 plastic bottle of uncoated (Sarstedt, Montreal), condition of culture is: 37 ℃, 5%CO ₂, medium is Da Erbaike (family name) improvement Iger (family name) medium (DMEM) that contains 4mM L-glutamine, 4500mg/L glucose, 1mM Sodium Pyruvate, 1500mg/L sodium bicarbonate and 10% hyclone.Carry out cell separation with trypsase/EDTA (Invitrogen).By immunohistochemical analysis, use fluorescently-labeled H-2K ^kAntibody has confirmed H-2K ^kExpression.In brief, with 0.5 * 10 ⁶Individual cell/cm ²Cell inoculation is gone in the band chamber slide (chamber slide) of 2 hole uncoated.In the time of the 1st, 2,3 and 4 day, with 2% paraformaldehyde fixed culture, and with the H-2K antibody (Miltenyi Biotec, Germany) of FITC coupling and nuclear mark iodate third ingot (PI) (Vectashield Mounting Medium, Vector Laboratories) dyeing.Adopt Laser Scanning Confocal Microscope (Nikon Eclipse E800-BioradRadiance 2100) to analyze with quantitative.Human fibroblasts is used as negative control.

The analysis of adherent cell: H-2K not ^kThe reservation of surface protein is the feature of the AKRIJA.Sp cell of non-cohesive form, is used to confirm to adopt when blood exists the feasibility of this system.As mentioned above with cell culture in the T-75 of uncoated bottle.Made the cell detachment of adhesion at the 4th day with trypsase/EDTA, and with the H-2K of FITC coupling ^kAntibody and facs analysis (Beckman Coulter Cytomics FC500) are measured and are expressed H-2K ^kThe cell quantity of surface protein.With the mouse IGg2 α isotype of FITC mark as negative control.

The structure of plasmid: utilize will the encode cDNA of prostacyclin synthase of BamHl in the multiple clone site and the restricted sequence of HindIII to be cloned into bicistronic mRNA carrier pMACS K ^k.II (Miltenyi Biotec, Germany).Used the cDNA of 1153 base-pairs that contain prostacyclin synthase gene and pVAX-1 that are arranged in the plasmid construction thing.In the presence of selective agent ampicillin (50ng/ml), carry out the colibacillary conversion of HG70.

Be used for open biosystem (Open Biosystems, the classification number #MHS 1768-9144117 of the full-length cDNA of people α-CGRP available from plasmid vector pPCR-Script Amp SK (+); Huntsville AL).Then this fragment and BamHl/EcoRl are connected into bicistronic mRNA plasmid vector pMACS K.II.Transform the JM109 Escherichia coli to obtain a large amount of described plasmids.

The transfection of EPC: use Ficoll gradient centrifugation enrichment pig monocyte from the pig whole blood, and separate EPC with above-mentioned enriched medium.Cultivate after 7 days, with the method (Amaxa Nucleofector, Germany) of nuclear perforation, with the bicistronic mRNA plamid vector transfection EPC that contains metastatic gene, described metastatic gene comprises α-CGRP or prostacyclin synthase.Utilize reporter gene and endothelial nitric oxide synthase (eNOS) in the pVAXt plasmid, obtained＞70% EPC electroporation transfection efficient (data not shown goes out).To successful transfection and express H-2K ^kThe EPC of surface protein carries out purifying, and removes kit (MACS Dead cell removal kit), MACSelect K with the MACS dead cell ^kMicroballon (MACSelect K ^kMicroBead) separate with MS splitter (MS Separation Column) (Miltenyi Biotec).MACSelect K ^kMicroballon is biodegradable, and it disappeared with cell culture in 24 hours.The mensuration that vasodilator is expressed:

The mensuration of prostacyclin synthase activity: after 2 days, keep cultivating EPC through transfection in transfection.Change medium, and assess the activity of prostacyclin synthase according to manufacturer's explanation with the level of prostacyclin synthase metabolite 6-ketone-PGF1 (6-keto-PGFIcu) in radio immunoassay (Amersham Corp.) the mensuration medium.

The mensuration of α-CGRP activity: measure the expression of α-CGRP in the transfectional cell with SABC method color reagent box (Bachem USA).Under-10 ℃ with methyl alcohol fixing cultivate 3 days through EPC5 minute of transfection.Wash described cell and air-dry.Fixing cell is hatched 7 minutes with deactivation endogenous peroxide activity in the PBS of 0.5% hydrogen peroxide solution.Cell is hatched in the serum confining liquid 20 minutes with the sealing non-specific binding.Anti-(rabbit monoclonal is Bachem) with three kinds of dilution factors: handled cell 2 hours in 1: 100,1: 200 and 1: 500 with anti-α-CGRP one then.Wash slide then, make it contact biotinylated two and resist 30 minutes.Wash cell then and use HRP-streptavidin compound to handle 30 minutes.With after the PBS washing, made cells contacting substrate-chromogen mixture 3 minutes.Add deionized water with cessation reaction.With MayerShi haematoxylin redyeing slide 3 minutes.Wash slide with running water then, place PBS, use the distilled water rinsing until becoming blue.With 95% and 100% ethanol and dimethylbenzene slide is dewatered again.Check in covered on the slide and under light microscopic.

Support with the antibody coating: foregoing with glucan and anti-H-2K ^kAntibody applies stainless steel stent (9mm is long).

Cells in vivo is caught: all tests all in the children pig of male York (＞carry out in 30kg).Obtain arterial passageway by the arteriotomy of on left neck artery, carrying out.After in coronary artery, giving 200pg nitroglycerine, obtain coronary angiography, and carry out online quantitative coronary angiogram and measure.With 1.1: 1 supports to blood vessel than support is circled round or the proximal section of arteria coronaria dextra is launched at LAD at random.After implanting, just give 200pg nitroglycerine in the intravascular.Carry out intravascular ultrasound (IVUS) then measuring external caliber, with the distally of stent prop up with remote edge as far-end and near-end reference.Utilize prototype series connection balloon catheter (CordisCorporation) to finish through the giving of transfectional cell, described cell is with the bicistronic mRNA carrier transfection of coding prostacyclin synthase or α-CGRP.This conduit constitutes by being positioned at two of apparatus adjacent far-end air bags of highly fitting, by same air-filled pore this two air bags that expand.In case after expanding, separate the long blood vessel section of 1.0cm between two air bags, to produce a regional perfusion chamber.Provide the far-end blood flow by central chamber, and be fed into solution or the chamber by two separation the whole chamber of solution suction.Filling cavity end at the far-end air bag near, discharge side then ends at a near opening of proximal balloon.The series connection balloon catheter is reached the position of implant frame forward, and air bag is expand into 25psi (1.7 atmospheric pressure).Send salt solution by fill orifice, in the section that separates, do not contain blood.The artery section of implant frame is carried out random packet to accept perfusion of saline or cell is sent.In 10 minutes, be total up to the 2ml cell suspension of 3 * 10 EPC with 200pL/ minute irrigation rate, hatched then 10 minutes.Closed then position of implementing arteriotomy is restored animal.Handled the back letting animals feed 28 days at cell.Coprocessing 34 animals (10 is saline control, and 14 is prostacyclin synthase, and 14 is α-CGRP).Delivery of cells after 1 hour is killed two animals of every group.The section that separates implant frame, and prepare to be used for the artery of SEM by following steps: fix 30 seconds at 10% formalin buffer PBS, further in the 0.1M sodium cacodylate buffer liquid (Sigma) that contains 2.5% glutaraldehyde (BDH Inc.) and 2%PFA, fixedly spend the night then through the implant frame of flushing.Finish fixedly post processing by following steps: the 0.1M arsenate buffer solution with 1% osmium tetroxide (Sigma) is handled, and with the dehydration of ethanol gradient, uses CO subsequently then ₂Carry out critical point drying.After the drying, sample is carried out metal spraying and whether has the cell that is incorporated into stent strut in the following observation of ESEM (SEM).Behind implant frame the 5th day put to death two animals of prostacyclin synthase group and two animals of α-CGRP group.The artery section that contains implant frame that takes out is placed 10% formalin/PBS solution, until carrying out the normal structure chemical analysis.Cut 5 sections from each support; Close on support 1mm place, support and close on terminal 1mm place, support middle section, rack far end 1mm place and support end 1mm place.Use Su Mujing ﹠amp; Yihong (HE) and three look elastin laminins dye to section.Measure inflammation [section's Milunovich index, Kornowski Score (0-3)] index with the rejection sign of assessment to the input cell.After carrying out exponential process (about 28 days), anesthetized animal also carries out coronary angiography by the arteria coronaria dextra angiotomy.Carry out the quantitative coronary angiography, utilize IVUS to carry out the blood vessel inquiry, with the variation of standard clinical algorithm record external caliber.

Embodiment 10

Be used for the mammalian cells in vitro transfection of vascular remodeling: to endothelial progenitor cells, described plasmid comprises coding and is responsible for the protein of adenosine generation and the gene of prostate specific epicyte protein the method for employing electroporation with the bicistronic mRNA plasmid transfection.Two kinds of genes are subjected to them to initiate self the control of son separately, thereby make gene be able to the constructive expression.

Make up following carrier with being similar to above-mentioned method, described carrier comprises the gene of coding prostate specific membrane albumen and its natural promoter, and the gene of coding VEGF, and these two kinds of genes are arranged in series in same expression vector.This plasmid construction thing can be used for the cell that is used for the patient described in the transfection embodiment 9, mammalian cell.

＜110〉Orbus Medical Technologies Inc

(ORBUS?MEDICAL?TECHNOLOGIES，INC.)

＜120〉have the medical treatment device and the using method thereof of the coating of cell that can be capturing genetically-altered

<130>ORB-101(PCT)

<140>TBA

<141>2005-04-29

<150>US?60/566,829

<151>20040430

<150>US?10/835,767

<151>2004-04-30

<160>4

<170>PatentIn?version?3.3

<210>1

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>primer?bind

<222>(1)..(21)

＜223〉the nitricoxide synthase sequence in endothelial cell source has an adopted PCR primer

<400>1

ttccggggat?tctggcagga?g 21

<210>2

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>primer_bind

<222>(1)..(21)

＜223〉the antisense PCR primer of the nitricoxide synthase sequence in endothelial cell source

<400>2

gccatggtaa?catcgccgca?g 21

<210>3

<211>24

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>primer_bind

<222>(1)..(24)

＜223〉glyceraldehyde phosphate dehydrogenase gene has an adopted PCR primer

<400>3

ctctaaggct?gtgggcaagg?tcat 24

<210>4

<211>24

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>4

gagatccacc?accctgttgc?tgta 24

Claims

1. therapy system for the treatment of patient disease, described system comprises:

The mammalian cell of hereditary change, described cell comprise coding through the genetically engineered cell surface marker molecule and the exogenous nucleic acid of at least a therapeutic gene product;

Be used to implant described patient's medical treatment device, described device comprises coating; Described coating comprises the matrix that load has at least a part, wherein said part identification and in conjunction with the described cell membrane labelled molecule of the mammalian cell of described hereditary change,

Wherein, the mammalian cell of described hereditary change is incorporated into described medical treatment device and expresses and secrete described at least a therapeutic gene product.

2. therapy system as claimed in claim 1, it also comprises and is used to the medicine or the compound that stimulate the mammalian cell expression of described hereditary change and/or secrete described therapeutic gene product.

3. therapy system as claimed in claim 1 is characterized in that, the mammalian cell of described hereditary change is from body, allogeneic or heterogenous cell.

4. therapy system as claimed in claim 3 is characterized in that, described is endothelial progenitor cells or mature endothelial cell from body, allogeneic or heterogenous cell.

5. therapy system as claimed in claim 1 is characterized in that, the described exogenous nucleic acid that is present in the mammalian cell of described hereditary change comprises DNA or RNA molecule, and described molecule comprises at least a gene of at least a therapeutic gene product of encoding.

6. therapy system as claimed in claim 1 is characterized in that described exogenous nucleic acid is an exchromosomal DNA.

7. therapy system as claimed in claim 5 is characterized in that described dna molecular comprises plasmid.

8. therapy system as claimed in claim 6 is characterized in that, described exchromosomal DNA comprises that regulation and control box, cell membrane specific gene and at least a coding are used for the treatment of the gene of the peptide of disease.

9. therapy system as claimed in claim 8 is characterized in that, described cell membrane specific gene is encoded into bone protein, prostatic cell memebrane protein or H-2K ^kSurface protein.

10. therapy system as claimed in claim 5, it is characterized in that described at least a gene code is selected from down the therapeutic gene product of group: prostacyclin, CGRP, vascular endothelial growth factor, angiogenesis factor, anti-angiogenesis or fibroblast growth factor.

11. therapy system as claimed in claim 1 is characterized in that, described part is combination, peptide or the little molecule of antibody, antibody fragment, antibody and antibody fragment.

12. a method for the treatment of patient disease, described method comprises:

The mammalian cell of hereditary change is offered described patient; Described cell comprises coding through the genetically engineered cell surface marker and the exogenous nucleic acid of at least a therapeutic gene product;

The medical treatment device that will comprise coating is implanted described patient; Described coating comprises the matrix that load has at least a part, wherein said part identification and in conjunction with the described cell membrane mark of the mammalian cell of described hereditary change,

13. method as claimed in claim 12, it also comprises and will be used to stimulate mammalian cell expression and/or the medicine of secretion specific gene product or the step that compound gives described patient of described hereditary change.

14. system as claimed in claim 12 is characterized in that, the mammalian cell of described hereditary change is from body, allogeneic or heterogenous cell.

15. one kind is used for the treatment of method for cancer, described method comprises:

16. method as claimed in claim 14, it also comprises mammalian cell expression that stimulates described hereditary change and/or the step of secreting described therapeutic gene product.

17. method as claimed in claim 14 is characterized in that, the mammalian cell of described hereditary change is from body, allogeneic or heterogenous cell.

18. method as claimed in claim 15, it is characterized in that the step of the mammalian cell expression of the described hereditary change of described stimulation and/or secretion therapeutic gene product comprises making with the interactional compound of the mammalian cell of described hereditary change and is released into blood flow.

19. method as claimed in claim 16 is characterized in that, described is mature endothelial cell from body, allogeneic or heterogenous cell.

20. method as claimed in claim 14 is characterized in that, described genetically-altered cells comprises exogenous DNA that gives or RNA molecule, and described molecule comprises at least a gene of at least a therapeutic gene product of encoding.

21. therapy system as claimed in claim 5 is characterized in that, described dna molecular is a plasmid.

22. therapy system as claimed in claim 6 is characterized in that, described exchromosomal DNA comprises that regulation and control box, cell membrane specific gene and at least a coding are used for the treatment of the gene of the peptide of disease.

23. therapy system as claimed in claim 8 is characterized in that, described cell membrane specific gene is encoded into bone protein, prostatic cell memebrane protein or H-2K ^kSurface protein.

24. therapy system as claimed in claim 5, it is characterized in that described at least a gene code is selected from down the therapeutic gene product of group: prostacyclin, CGRP, vascular endothelial growth factor, angiogenesis factor, anti-angiogenesis or fibroblast growth factor.

25. method as claimed in claim 15, it is characterized in that described therapeutic gene product is selected from down group: prostacyclin, CGRP, vascular endothelial growth factor, angiogenesis factor, anti-angiogenesis or fibroblast growth factor.

26. a drug delivery system, described system comprises:

The mammalian cell of hereditary change, described cell comprise coding through the genetically engineered cell surface marker and the exogenous nucleic acid of at least a therapeutic gene product;

Be used to implant described patient's medical treatment device, described device comprises coating; Described coating comprises the matrix that load has at least a part, wherein said part identification and in conjunction with the described cell membrane mark of the mammalian cell of described hereditary change,

27. drug delivery system as claimed in claim 25, it also comprises and is used to the activating molecules or the inducible promoter that stimulate the mammalian cell expression of described hereditary change and/or secrete described therapeutic gene product.

28. therapy system as claimed in claim 25 is characterized in that, the mammalian cell of described hereditary change is from body, allogeneic or heterogenous cell.

29. drug delivery system as claimed in claim 27 is characterized in that, described is mature endothelial cell from body, allogeneic or heterogenous cell.

30. drug delivery system as claimed in claim 25, it is characterized in that, the described exogenous nucleic acid that is present in the mammalian cell of described hereditary change comprises foreign DNA or RNA carrier, and described foreign DNA or RNA carrier comprise at least a gene of at least a therapeutic gene product of encoding.

31. drug delivery system as claimed in claim 25 is characterized in that, described exogenous nucleic acid is an exchromosomal DNA.

32. drug delivery system as claimed in claim 25 is characterized in that, described dna vector comprises plasmid.

33. drug delivery system as claimed in claim 30 is characterized in that, described exchromosomal DNA comprises that regulation and control box, cell membrane specific gene and at least a coding are used for the treatment of the gene of the peptide of disease.

34. drug delivery system as claimed in claim 32, it is characterized in that described cell membrane specific gene coding prostacyclin, CGRP, vascular endothelial growth factor, angiogenesis factor, anti-angiogenesis and fibroblast growth factor.

35. drug delivery system as claimed in claim 29, it is characterized in that described at least a gene code is selected from down the therapeutic gene product of group: prostacyclin, CGRP, vascular endothelial growth factor, angiogenesis factor, anti-angiogenesis or fibroblast growth factor.