CN101985052A - Skin substitute for automatically capturing endothelial progenitor cells and promoting vascularization and construction method thereof - Google Patents

Abstract

The invention relates to the technical fields of tissue engineering and medical wound repair. At present, the traditional skin substitutes have slow vascularization and low survival rate after transplantation, so they cannot be applied clinically. The present invention constructs a new type of skin substitute that can automatically capture endothelial progenitor cells in peripheral blood and accelerate vascularization, transfects the stromal cell-derived factor 9HRE-CMV gene regulated by the hypoxia response element into fibroblasts, and then transforms the epidermal Cells and transgenic fibroblasts were respectively planted on both sides of the dermal scaffold to form a sandwich-type skin substitute. After transplantation, SDF-1α was expressed and secreted in a timely manner, which mediated the migration and homing of EPCs in peripheral blood to the transplantation site of the skin substitute. This promotes vascularization of the skin substitute and improves post-transplant survival. The skin substitute of the present invention can significantly improve the induction of chemotaxis to EPC cells and accelerate the vascularization of the skin substitute, with an average survival rate of 95%, while the average survival rate of traditional skin substitutes without transgenic fibroblasts is only 72%. %.

Description

Automatically capturing endothelial ancestral cell promotes the Graftskin and the construction method thereof of vascularization

Technical field

The present invention relates to organizational project and medical science wound repair technical field, be specifically related to a kind ofly can catch endothelial progenitor cells of peripheral blood automatically, promote the novel skin substitute of vascularization, can obviously improve the transplanting success of Graftskin.

Background technology

Skin tissue engineering is through the development in surplus 20 years, and the development of simple epidermis cell cultivation and transplanting, dermis scaffold and application technology be maturation comparatively all, and successfully is applied to clinical.On this basis, researcher is planted the epidermis cell of In vitro culture in the dermis scaffold surface and has been made up the sandwich Graftskin that contains cuticular cellulose, attempts the epidermal area and the skin corium of disposable repair deficiency.Yet, the sandwich Graftskin to be transplanted in deep burn cut the crust wound surface, anti-infection ability is poor, survival rate is low, and fluctuation is difficult to apply clinically between 0%～50%.One of key reason is that vascularization speed was slow after Graftskin was transplanted.Transplanting in early days, Graftskin blood is for poor, cuticular cellulose nutrition supply deficiency, cause propagation slowly, even death (the Caruso DM that comes off, Schuh WH, Al-Kasspooles MF, et al.Culturedcomposite autografts as coverage for an extensive body surface area burn:casereport and review of the technology.Burns, 1999,25 (8): 771-779.Scuderi N, OnestiMG, Bistoni G, et al.The clinical application of autologous bioengineered skinbased on a hyaluronic acid scaffold.Biomaterials, 2008,29 (11): 1620-9.).Therefore, making up the Graftskin that a kind of vascularization speed is fast, the transplanting survival rate is high is to need a urgent difficult problem that solves in the skin tissue engineering research.

(endothelial progenitor cells EPC) is the crucial participant that new vessels forms to be present in endothelial progenitor cells in the peripheral circulation blood.At (after etc.) under certain pathological state as tissue ischemia, operation on vessels of heart, hypoxic-ischemic tissue local secretion stroma cell derivative factor-1 α (stromal cell-derivedfactor-1 α, SDF-1 α), SDF-1 α is powerful chemoattractant (the Avci-Adali M of EPC, ZiemerG, Wendel HP.Induction of EPC homing on biofunctionalized vascular grafts forrapid in vivo self-endothelization-a review of current strategies.BiotechnolAdv.2010; 28 (1): 119-29), it by with specific receptor CXCR4 (the chemokineCXC receptor type 4 on EPC surface, CXCR4) combination, can prompting mobilization at short notice, EPC enters peripheral blood in the bone marrow, and the migration of the EPC in the mediation peripheral blood, the hypoxic-ischemic tissue local of going back to the nest, thereby participated in (the Madeddu P of endothelialization again of angiogenesis and injured blood vessel, Kraenkel N, Barcelos LS, et al.Phosphoinositide 3-kinase gamma gene knockout impairs postischemicneovascularization and endothelial progenitor cell functions.Arterioscler ThrombVasc Biol, 2008,28 (1): 68-76.).Researcher arrives the SDF-1a gene transfection of plasmid DNA coding in the Mus ischemic limb, observe that EPC increases in the peripheral blood of transfection Mus, the ishemic part capillary density increases, the hemoperfusion amount is obviously recovered (Hiasa K, Ishibashi M, Ohtani K, et al.Gene transfer ofstromal cell-derived factor-1alpha enhances ischemic vasculogenesis andangiogenesis via vascular endothelial growth factor/endothelial nitric oxidesynthase-related pathway:next-generation chemokine therapy for therapeuticneovascularization.Circulation, 2004,25; 109 (20): 2454-61.).And directly isolating EPC kind in the peripheral blood is planted the dermis scaffold surface, behind In vitro culture, transplant, visible vesselsization is obviously accelerated, and confirm that the EPC that transplants has participated in local new vessels formation simultaneously and this two kinds of revascularization process (Kung EF take place the stem cell blood vessel, Wang F, Schechner JS.In vivo perfusion of human skinsubstitutes with microvessels formed by adult circulating endothelial progenitorcells.Dermatol Surg, 2008,34 (2): 137-46.Shepherd BR, Enis DR, Wang F, et al.Vascularization and engraftment of a human skin substitute using circulatingprogenitor cell-derived endothelial cells.FASEB J, 2006,20 (10): 1739-41.).Therefore, the discovery of EPC provides new thinking with the revascularization that is applied as the organizational project organ.

Yet,, make the application of EPC be subjected to great restriction owing to separate, cultivate from the method for body EPC immaturity still.Both enabled the EPC of capacity that cultivates, increases, being inoculated into the dermis scaffold surface cultivates, the EPC poor adhesion, survival rate is low, and easily cause differentiation of stem cells after long-time the cultivation, lose angiogenesis ability (Hofmann NA, Reinisch A, Strunk D.Isolation and large scale expansion of adulthuman endothelial colony forming progenitor cells.J VisExp.2009; 28 (32): 1524-29).Therefore, adopt the method for traditional separation, cultivation and transplanting EPC to promote the vascularization of Graftskin to have various technical bottlenecks.

Summary of the invention

The object of the invention is to make up the Graftskin that a kind of vascularization speed is fast, the transplanting survival rate is high.

The present invention's imagination: if make Graftskin high expressed SDF-1a, can mediate EPC migration, the Graftskin transplantation site of going back to the nest in the peripheral blood rapidly by the SDF-1/CXCR4 axle so, quicken vascularization, improve the transplanting survival rate.Thereby avoid EPC in-vitro separation, multiple deficiency that cultivation, transplanting brought.

Technical scheme of the present invention is by the gene transfection technology, made up a kind of high expressed stroma cell derivative factor-1 α (stromal cell-derived factor-1 α, SDF-1 α) Graftskin, can discharge SDF-1 α after the transplanting, with (the endothelial progenitor cells of endothelial progenitor cells in the peripheral blood, EPC) surface specific receptor CXCR 4 (chemokine CXC receptor type 4, CXCR4) combination, thereby rapid migration, the Graftskin transplantation site of going back to the nest of mediation EPC, quicken vascularization, improve the transplanting survival rate.Specifically be that fibroblast is arrived in stroma cell derivative factor-1 α (SDF-1 α) gene transfection of hypoxia response element regulation and control, then epidermis cell and genetically modified fibroblast are planted dermis scaffold two sides formation sandwich Graftskin respectively, after the transplanting in good time, moderately express, secrete SDF-1 α, promote the vascularization of Graftskin, improve the transplanting survival rate.

The invention provides the construction method that the automatic capturing endothelial ancestral cell of a kind of energy promotes the Graftskin of vascularization, this method comprises the steps:

1. separate, cultivate people's epithelial cells, fibroblast (reference: Liu Dewu, Li Guohui, Zou Ping, Liu Deming. epidermis cell, the compound acellular dermal matrix of fibroblast make up organization engineering skin. Chinese clinical rehabilitation, 2004,8 (8): 1439-1441.);

2. make up the carrier that contains SDF-1 α gene expression, expression vector is pcDNA3.1-SDF-1 α;

3. make up the SDF-1 α expression vector of hypoxia response element control, and the transfection fibroblast;

4. make up and contain the fibroblastic Graftskin of epidermis cell-transgenic.

Construction method concrete steps of the present invention are as follows:

1. separate, cultivate people's epithelial cells, fibroblast:

The utilization discarded prepuce tissues of postoperative of peritomizing adopts enzyme digestion to separate, cultivate epithelial cells, fibroblast.

2. make up and contain SDF-1 α expression carrier

From people's endotheliocyte, clone SDF-1 α gene and order-checking, SDF-1 α gene is inserted the pcDNA3.1 carrier, obtain pcDNA3.1-SDF-1 alpha expression carrier.

3. make up the SDF-1 α expression vector of hypoxia response element control

Adopt Protocols in Molecular Biology, obtain multicopy hypoxia response element promoter (9HRE-CMV), and be connected with SDF-1 α gene segment, insert the pcDNA3.1 carrier, make up the SDF-1 α expression vector of hypoxia response element control: pcDNA3.1-9HRE-CMV-SDF-1 α carrier.

4.pcDNA3.1-9HRE-CMV-SDF-1 α expression vector transfection fibroblast

Utilize the fibroblast of Lipofectamine 2000 mediation pcDNA3.1-9HRE-CMV-SDF-1 α carrier transfection In vitro culture.

5. make up and contain the fibroblastic Graftskin of epidermis cell-transgenic

With the dermis scaffold is carrier, plants the fibroblast of the epidermis cell and the transfection 9HRE-CMV-SDF-1 α gene of In vitro culture respectively in its two sides, forms the sandwich Graftskin behind In vitro culture.

Chang Yong dermis scaffold has acellular dermal, spongy collagem membrane, hyaluronic acid membrane and polylactic acid/polyglycolic acid film etc. clinically.

The present invention also provides the Graftskin that makes up according to said method.

The present invention also provides the application as the wound repair graft materials of the Graftskin that makes up according to said method.

The present invention has carried out obvious improvement to the method for currently used acceleration Graftskin vascularization.Traditional method is to separate cultured and amplified in vitro behind the EPC from peripheral blood, plants the surface of Graftskin again, transplants the back vascularization thereby quicken Graftskin.The present invention has made up the Graftskin of expressing SDF-1 α gene outcome, this Graftskin is transplanted the back and is mediated EPC migration, the Graftskin transplantation site of going back to the nest in the peripheral blood rapidly by the SDF-1/CXCR4 axle, quicken vascularization, avoided the various deficiencies of conventional separation, cultivation, plantation EPC.In addition, this Graftskin with hypoxia response element (HRE) as oxygen condition gene expression gauge tap, regulation and control SDF-1 α expression of gene and closing, promptly Graftskin is not before the vascularization, under anaerobic environment, be subjected to the regulation and control of HRE, SDF-1 α effective expression, and after vascularization, Graftskin is under the normal oxygen condition, the expression of genes of interest is ended at once, has improved the safety of gene therapy.

Prove that through animal implant tests textured novel skin substitute of the present invention is compared with not containing genetically modified Graftskin, can obviously improve the chemotactic of peripheral blood EPC cell is induced that quicken the vascularization of Graftskin, the transplanting survival rate brings up to 95% by 72%.

Description of drawings

Fig. 1 is a pcDNA3.1-9HRE-CMV-SDF-1 α vector construction sketch map.

The specific embodiment

Now in conjunction with the embodiments and accompanying drawing, the present invention is further described, but enforcement of the present invention is not limited in this.

Embodiment 1. preparations are the novel skin substitute of capturing endothelial ancestral cell promotion vascularization automatically

1. separate, cultivate people's epithelial cells, fibroblast:

The utilization discarded prepuce tissues of postoperative of peritomizing adopts enzyme digestion to separate, cultivate epithelial cells, fibroblast.Be prepared into single cell suspension respectively, press 2-3 * 10 ⁵Individual/ml cell density is inoculated in the culture bottle, place 37 ℃ of incubators, epithelial cells is cultivated (DK-SFM with serum-free medium, Gibco, the U.S.), fibroblast is cultivated with perfect form DMEM culture fluid (Gibco, the U.S.), goes down to posterity, increases when 70%-80% merges when cell reaches.

2. make up the carrier that contains SDF-1 α gene expression

The sequence of SDF-1 α gene:

ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAGTAA

Design primer: forward primer 5 '-CGGGATCCATGAACGCCAAGGTCG-3 '

Downstream primer 5 '-GGAATTCGGACCTCTTTCGAAATTTGTTCATT-3 '

MRNA in extraction people's the endotheliocyte, obtain cDNA by the RT-PCR reverse transcription, obtain SDF-1 α by specific primer PCR, test kit reclaims the PCR product, connect pMD-18T (TaKaRa), do conversion, enzyme action is identified after extracting plasmid, choose correct plasmid and send company's order-checking (entrusting the order-checking of Invitrogen company), after order-checking is correct, use BamH I, EcoR I comes digested plasmid pMD-18T-SDF-1 α, obtains having the SDF-1 α gene of sticky end, with the T4 ligase with SDF-1 α gene with used BamH I, pcDNA3.1 (+) the plasmid link that EcoR I enzyme action is good, transformed into escherichia coli, extract plasmid pcDNA3.1-SDF-1 α, send the order-checking of Invitrogen company to identify, obtain pcDNA3.1-SDF-1 alpha expression carrier.

3. make up the SDF-1 α expression vector of hypoxia response element control

Multicopy hypoxia response element promoter (9HRE-CMV) entrusts Invitrogen company to make up, add the KpnI restriction enzyme site with round pcr at the gene 5 ' end, 3 ' end adds BamH I restriction enzyme site, be connected with pcDNA3.1 (+) carrier behind the enzyme action with SDF-1 α gene behind the enzyme action, make up the SDF-1 α expression vector of hypoxia response element control: pcDNA3.1-9HRE-CMV-SDF-1 α carrier (as shown in Figure 1).

4.pcDNA3.1-9HRE-CMV-SDF-1 α expression vector transfection fibroblast

Transfection the previous day, trypsin digestion cell and counting, the cell bed board, make its transfection day density be 90%.The cell bed board contains serum at 0.5ml, does not contain in the culture medium of antibiotic normal growth.For every porocyte, use 50 μ l serum-free mediums to dilute 0.8 μ g-1.0 μ g DNA.For every porocyte, use 50 μ l OPTI-MEM I culture medium to dilute 1 μ l-3 μ l LIPOFECTAMINE, 2000 reagent.(the pcDNA3.1-9HRE-CMV-SDF-1 α of dilution mixed together in 30 minutes, and temperature retention time is long can to reduce activity in 5 minutes in Lipofectamine 2000 dilution back insulations.) DNA (the 2nd step) of mixed diluting and the Lipofectamine 2000 (the 3rd step) of dilution.Room temperature insulation 20 minutes.Directly complex is joined in every hole wave and culture plate, mixing gently.At 37 ℃, 5% CO ₂In insulation 24-48 hour, need not remove complex or change culture medium or after 4-5 hour, change growth medium and also can not reduce transfection activity.In cell, add complex after 24-72 hour, analysis of cells extract or carry out the original position cell dyeing, the examining report gene activity.This depends on cell type and promoter activity.To stably express, the beginning transfection after one day with passage to fresh culture, add the screening antibiotic two days later.Carry out stably express and need a couple of days or several weeks.

5. make up and contain the fibroblastic Graftskin of epidermis cell-transgenic

(PELNAC, the prefecture is a company limited, Japan) is laid on the culture plate with spongy collagem membrane, with the fibroblast of transfection pcDNA3.1-9HRE-CMV-SDF-1 α with 1-3 * 10 ⁵Individual/cm ²Density plant in the collagem membrane surface, in In vitro culture 1-2 days.With collagem membrane upset, the plantation fibroblast faces down subsequently, to the side with 3-5 * 10 ⁵Individual/cm ²Density inoculation epithelial cells, treat cell fusion after, carry out liquid-vapor interface and cultivated for 2 weeks, changed liquid once in per 2～3 days, form and contain the fibroblastic sandwich Graftskin of epidermis cell-transgenic.

The zoografting test of embodiment 2. Graftskins of the present invention

Male Balb/c-nu mice (nude mice, west, Shanghai pul-Bi Kai laboratory animal company limited provides), body weight 18 ± 2 grams, 40, being divided into is four groups.Be respectively and contain the fibroblastic Graftskin of transfection SDF-1 α gene, contain the transfection end and add the fibroblastic Graftskin of pcDNA3.1-9HRE-CMV-SDF-1 α, contain the Graftskin of the normal fibroblast of fibroblastic Graftskin of transfection empty carrier and untransfected gene.

Nude mice is shaved except that the skin of back hair after the ketamine intraperitoneal anesthesia, and excision spinal column inclined to one side veutro portion's holostrome skin and deep fascia reach Musclar layer.The viable skin substitute through In vitro culture 2-3 week described in the embodiment 1 is transplanted in wound surface.All skin shrinks and epidermis is creeped in order to prevent to create, and result's observation is transplanted in influence, adopts cage ring with wound surface and the isolation of peripheral skin.Cover oil-sand on the Graftskin, periodic replacement dressing is also observed the wound healing situation.Draw materials respectively after 1,3,6,9,12,14 day after transplanting and observe vascularization, survival rate and newborn skin histology.

The result shows that novel skin substitute of the present invention can obviously improve induces the chemotactic of EPC cell, quickens the vascularization of Graftskin, and average survival rate reaches 95%, and tradition not contain the average survival rate of the fibroblastic Graftskin of transgenic only be 72%.

Sequence table

SEQUENCE?LISTING

＜110〉Second Military Medical University, PLA

＜120〉automatic capturing endothelial ancestral cell promotes the Graftskin and the construction method thereof of vascularization

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Claims (5)

1. A method for constructing a skin substitute that can automatically capture endothelial progenitor cells to promote vascularization, the method comprising the steps of: A) separating and culturing human keratinocytes and fibroblasts; B) constructing a vector containing SDF-1α gene expression; C) constructing the SDF-1α gene expression vector controlled by the hypoxia response element, and transfecting fibroblasts; D) Construction of skin substitutes containing epidermal cells-transgenic fibroblasts. 2. The method for constructing a skin substitute capable of automatically capturing endothelial progenitor cells to promote vascularization according to claim 1, characterized in that the specific steps of the method are as follows: A) Separation and cultivation of human keratinocytes and fibroblasts: Use the discarded foreskin tissue after circumcision, separate and culture keratinocytes and fibroblasts by enzymatic digestion, prepare single cell suspensions, and inoculate in culture flasks at a cell density of 2-3× ¹⁰⁵ cells/ml , placed in a 37°C incubator, cultured with serum-free medium for keratinocytes, cultured with complete DMEM medium for fibroblasts, and passaged and expanded when the cells reached 70%-80% confluence; B) Construct the expression vector containing SDF-1α gene: The SDF-1α gene was cloned and sequenced from human endothelial cells, and the SDF-1α gene was inserted into the pcDNA3.1 vector to obtain the pcDNA3.1-SDF-1α expression vector. C) Construct the SDF-1α gene expression vector controlled by the hypoxia response element, and transfect fibroblasts: Obtain multiple copies of the hypoxia response element promoter 9HRE-CMV, connect it with the SDF-1α gene fragment, insert it into the pcDNA3.1 vector, and construct the SDF-1α gene expression vector controlled by the hypoxia response element: pcDNA3.1-9HRE-CMV- SDF-1α carrier; Using Lipofectamine 2000 to mediate pcDNA3.1-9HRE-CMV-SDF-1α vector transfection into fibroblasts cultured in vitro; D) Construction of skin substitutes containing epidermal cells-transgenic fibroblasts: Using the dermal scaffold as a carrier, in vitro cultured epidermal cells and fibroblasts transfected with 9HRE-CMV-SDF-1α gene were planted on both sides of the scaffold, and a sandwich skin substitute was formed after in vitro culture. 3. The method for constructing a skin substitute that can automatically capture endothelial progenitor cells to promote vascularization according to claim 2, wherein the dermal scaffold in step D) is decellularized dermis, spongy collagen membrane, hyaluronic acid membrane or polylactic acid/polyglycolic acid membrane. 4. A skin substitute constructed by the method of claim 1, 2 or 3. 5. The application of the skin substitute as claimed in claim 4 in the preparation of graft materials for wound repair.

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