CN104083762A - Reagent for blocking interaction between Netrin-1 and CD146 and tumor treatment application thereof - Google Patents
Reagent for blocking interaction between Netrin-1 and CD146 and tumor treatment application thereof Download PDFInfo
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Abstract
本发明公开了阻断Netrin-1与CD146之间的相互作用的试剂在制备用于抑制新生血管形成的药物中的用途以及在制备用于治疗肿瘤的药物中的用途。本发明还公开了通过阻断Netrin-1与CD146之间的相互作用来抑制新生血管形成和治疗肿瘤的方法。The invention discloses the use of a reagent for blocking the interaction between Netrin-1 and CD146 in the preparation of a drug for inhibiting neovascularization and the use in the preparation of a drug for treating tumors. The invention also discloses a method for inhibiting the formation of new blood vessels and treating tumors by blocking the interaction between Netrin-1 and CD146.
Description
Technical field
The present invention relates to block interactional reagent between Netrin-1 and CD146 in purposes and the purposes in the medicine for the preparation for the treatment of tumor for the preparation of suppressing in medicine that new vessels forms.The invention still further relates to by the interaction between blocking-up Netrin-1 and CD146 and suppress the method that new vessels formed and treated tumor.
Background technology
Cancer is the No.1 killer of current harm humans life and health.The number of the infected of the annual malignant tumor of China approximately 3,120,000.The whole nation is per minute has 6 people to be diagnosed as cancer, dies from cancer person and accounts for the more than 20% of total toll, occupies first (according to statistics in 2012) of urbanite's cause of death.
At present, in tumor three large conventional (radiotherapy, chemotherapy and operation) treatment, Drug therapy is an importance.The factor that affects tumor pharmacother effect is many-sided, comprises specificity, means of transportation, permeability and the induced tumor drug resistance of medicine.The specificity of its Chinese medicine and the drug resistance of induced tumor are the key issues that tumor therapeutics is needed solution badly.Tumor vasculature targeting has become a kind of important means in oncotherapy with advantages such as its tumour-specific, broad spectrum activity, no or low drug resistance.Its theoretical foundation is mainly the concept of the tumor-blood-vessel growth that the seventies Folkman proposes in last century, and new vessels divides supply for tumor provides nutrition and oxygen, and then promotes growth and the transfer of tumor.
Studies have found that, neurite-outgrowth guiding factor Netrin-1 can promote growth and the migration of vascular endothelial cell, and then promotes angiogenesis.In growth course, neurocyte, epithelial cell, the various kinds of cell secretion Netrin-1 such as lymphocyte, promote growth and the molding of blood vessel system.Under pathological conditions, tumor cell can synthesize and secrete a large amount of Netrin-1, promotes tumor-blood-vessel growth.Yet Netrin-1 promotes the mechanism of angiogenesis it be unclear that, and its receptor and downstream signal are unknown, therefore there is no at present the anti-tumor angiogenesis drug for Netrin-1.
In research in the past, found immunoglobulin superfamily adhesion molecule CD146 up-regulated expression on tumor vascular endothelial cell, and promoted tumor-blood-vessel growth.The monoclonal antibody AA98 of targeting CD146 can effectively suppress leiomyosarcoma, the angiogenesis in cancer of pancreas and hepatocarcinoma, the nutrient supply that cuts off tumor, thereby the growth of inhibition tumor, migration and deterioration.
Summary of the invention
The inventor proposes the functional part that Netrin-1 is CD146 first, and CD146 is the receptor of Netrin-1 on vascular endothelial cell.Netrin-1 activates downstream signal path by being attached on the CD146 of Surface of Vascular Endothelial Cells, promotes growth and the migration of endotheliocyte.The monoclonal antibody AA98 of anti-CD146 can hinder the combination of Netrin-1 and CD146, therefore can suppress downstream signal and the angiogenesis that Netrin-1 causes in vivo with in external level.
The application in antineoplaston is based on following important scientific discovery as potential treatment target spot to using Netrin-1: (1) Netrin-1 and CD146 direct interaction are the functional parts of CD146; (2) the monoclonal antibody AA98 of anti-CD146 can block the interaction between Netrin-1 and CD146, and then blocking-up Netrin-1 stimulates the activation of the downstream signal (p38 and ERK/NF-κ B) causing; (3) anti-CD146 monoclonal antibody AA98 can suppress the endothelial cell proliferation that Netrin-1 induction causes, migration and angiogenesis; (4), in mice arterial ring and Matrigel plug model, AA98 suppresses the angiogenesis that Netrin-1 induces in level in vivo; (5) in research report in the past, verified anti-CD146 antibody A A98 can pass through to suppress tumor-blood-vessel growth, thereby suppresses leiomyosarcoma, the growth of cancer of pancreas and hepatocarcinoma and deterioration.
In sum, Netrin-1, as the functional part of CD146, can activate the downstream signal of CD146, and then promotes angiogenesis.The CD146 that studies confirm that in past is the target spot molecule in oncotherapy, and the monoclonal antibody AA98 of anti-CD146 can effectively suppress tumor-blood-vessel growth.Therefore, we propose targeting Netrin-1 molecule can effectively suppress angiogenesis, and then the nutrient supply of blocking-up tumor, suppresses tumor growth.
Concrete, the invention provides the following:
1. the purposes of the interactional reagent between blocking-up Netrin-1 and CD146 in the medicine for the preparation for the treatment of tumor.
2. according to the purposes described in 1, the interactional reagent between wherein said obstruction Netrin-1 and CD146 is antibody.
3. according to the purposes described in 2, wherein said antibody is the monoclonal antibody for CD146.
4. according to the purposes described in 3, the wherein said monoclonal antibody for CD146 is AA98.
5. according to the purposes described in 4, wherein said tumor is selected from leiomyosarcoma, cancer of pancreas and hepatocarcinoma.
6. the interactional reagent between blocking-up Netrin-1 and CD146 is in the purposes for the preparation of suppressing in medicine that new vessels forms.
7. according to the purposes described in 6, the interactional reagent between wherein said obstruction Netrin-1 and CD146 is antibody.
8. according to the purposes described in 7, wherein said antibody is the monoclonal antibody for CD146.
9. according to the purposes described in 8, the wherein said monoclonal antibody for CD146 is AA98.
10. treat the method for tumor, described method comprises the interaction between blocking-up Netrin-1 and CD146.
11. according to the method described in 10, wherein by using for the monoclonal antibody of CD146, blocks the interaction between Netrin-1 and CD146.
12. according to the method described in 11, and the wherein said monoclonal antibody for CD146 is AA98.
13. according to the method described in 12, and wherein said tumor is selected from leiomyosarcoma, cancer of pancreas and hepatocarcinoma.
14. suppress the method that new vessels forms, and described method comprises the interaction between blocking-up Netrin-1 and CD146.
15. according to the method described in 14, wherein by using for the monoclonal antibody of CD146, blocks the interaction between Netrin-1 and CD146.
16. according to the method described in 15, and the wherein said monoclonal antibody for CD146 is AA98.
Accompanying drawing explanation
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 .Netrin-1 and CD146 interact at molecular level.A, in human embryo kidney epithelial cell (HEK293), transfection plasmid utilizes the method for co-immunoprecipitation to find that Netrin-1 and CD146 interact; B, external pull-down experiment finds that Netrin-1 and CD146 extracellular region exist direct interaction; C, external pull-down experiment finds that anti-CD146 monoclonal antibody AA98 can block the interaction of Netrin-1 and CD146; D, utilizes interactional affinity constant between surface plasma resonance (SPR) measuring Netrin-1 and CD146.
Fig. 2, level in vitro, Netrin-1 promotes angiogenesis to depend on the CD146 of Surface of Vascular Endothelial Cells to be combined.A-C, utilizes the specific siRNA of CD146 to strike after the CD146 in low Human umbilical vein endothelial cells (HUVEC), and Netrin-1 promotes endothelial cell proliferation, and the function of migration and angiogenesis is suppressed; D-F, the monoclonal antibody AA98 of anti-CD146 can suppress the HUVEC propagation of Netrin-1 induction, migration and angiogenesis.
Fig. 3, Netrin-1 activation downstream angiogenesis signal depends on the CD146 of Surface of Vascular Endothelial Cells is combined.A, the CD146 that utilizes the specific siRNA of CD146 to strike in low HUVEC can suppress the downstream signal that Netrin-1 activates; B, the monoclonal antibody AA98 of anti-CD146 can suppress the signal activation of Netrin-1 induction.
Fig. 4, in mice angiogenesis model, Netrin-1 promotion angiogenesis depends on the combination with CD146.A-B, in arterial ring model, is used CD146 endothelium specificity knock-out mice or AA98 can suppress the endotheliocyte that Netrin-1 causes and sprouts; C-D, in Matrigel plug model, is used CD146 endothelium specificity knock-out mice or AA98 can suppress the angiogenesis of Netrin-1 induction.
The specific embodiment
The antibody A A98 using in this description (Yan, the A novel anti-CD146monoclonal antibody such as X, AA98, inhibits angiogenesis and tumor growth.Blood.2003; 102 (1): 184-191.), AA1 (autonomous production of this laboratory, preservation mechanism: Chinese common micro-organisms preservation center; Preserving number: CGMCC No.2310; Preservation date: 2007.12.28) etc. can obtain according to the description of Chinese Patent Application No. 99107586.2 (CN1234405), Chinese Patent Application No. 200810057260.7 (CN101245101) respectively.
Embodiment 1: cell and external level, Netrin-1 and CD146 interact
CD146 plays a crucial role in angiogenesis especially pathological angiogenesis generates as endothelial cell adhesion molecule.The Netrin-1 promotion angiogenesis that is also in the news, but its receptor unclear.In order to study relation between the two, we utilize the methods such as co-immunoprecipitation to confirm that Netrin-1 and CD146 exist direct interaction in cell and external level.The monoclonal antibody of anti-CD146 can be blocked the interaction between Netrin-1 and CD146.
Main material: human embryo kidney epithelial cell (HEK293) (ATCC, CRL-1573).
Main agents: cell pyrolysis liquid, PBS, HEPES.Anti-CD146 monoclonal antibody AA1 (this laboratory autonomous production of Mus source.Preservation mechanism: Chinese common micro-organisms preservation center; Preserving number: CGMCC No.2310; Preservation date: 2007.12.28).Mus source anti-CD146 monoclonal antibody AA98, anti-Netrin-1 mouse monoclonal antibody is (purchased from Enzo Life Science company, article No. ALX-804-838), recombinant human Netrin-1 albumen is (purchased from Enzo Life Science company, article No. ALX-522-100), Fc-CD146 albumen is (purchased from Sino Biological Inc., article No. 10115-H02H), Fc albumen is (purchased from Sino Biological Inc., article No. 10702-HNAH), His-CD146 albumen (purchased from Sino Biological Inc., article No. 10115-H08H).CD146 specific siRNA and contrast siRNA (synthesized by Invitrogen company, CD146 specific siRNA sequence is as follows).
Forward: 5 '-CCAGCUCCGCGUCUACAAAdTdT-3 '
Reverse: 5 '-UUUGUAGACGCGGAGCUGGdTdT-3 '
Main method: co-immunoprecipitation, external pull-down and surface plasma resonance experiment, concrete grammar is as follows:
Co-immunoprecipitation:
1) the HEK293 cell through transient transfection Netrin-1-pcDNA3.1/myc-his (-) b plasmid and CD146-p3xFLAG-cmv-14 plasmid (10 μ g) is inoculated in 100mm culture dish, cell is scraped gently to 4 ℃ of centrifugal 5 minutes centrifugal being collected in Ep pipe after cell density reaches 90%.
The relevant information of CD146-p3xFLAG-cmv-14 plasmid is shown in document Zheng, C. wait Endothelial CD146is required in vitro tumor-induced angiogenesis:the role of a disulfide bond in signaling and dimerization.The international journal of biochemistry & cell biology41,2163-2172, doi:10.1016/j.biocel.2009.03.014 (2009).
Netrin-1-pEGFP-N1 plasmid is built by this laboratory.Use pEGFP-N1 carrier.The gene order following (Gene ID:9423) of people source Netrin-1:
ATGATGCGCGCAGTGTGGGAGGCGCTGGCGGCGCTGGCGGCGGTGGCGTGCCTGGTGGGCGCGGTGCGCGGCGGGCCCGGGCTCAGCATGTTCGCGGGCCAGGCGGCGCAGCCCGATCCCTGCTCGGACGAGAACGGCCACCCGCGCCGCTGCATCCCGGACTTTGTCAATGCGGCCTTCGGCAAGGACGTGCGCGTGTCCAGCACCTGCGGCCGGCCCCCGGCGCGCTACTGCGTGGTGAGCGAGCGCGGCGAGGAGCGGCTGCGCTCGTGCCACCTCTGCAACGCGTCCGACCCCAAGAAGGCGCACCCGCCCGCCTTCCTCACCGACCTCAACAACCCGCACAACCTGACGTGCTGGCAGTCCGAGAACTACCTGCAGTTCCCGCACAACGTCACGCTCACACTGTCCCTCGGCAAGAAGTTCGAAGTGACCTACGTGAGCCTGCAGTTCTGCTCGCCGCGGCCCGAGTCCATGGCCATCTACAAGTCCATGGACTACGGGCGCACGTGGGTGCCCTTCCAGTTCTACTCCACGCAGTGCCGCAAGATGTACAACCGGCCGCACCGCGCGCCCATCACCAAGCAGAACGAGCAGGAGGCCGTGTGCACCGACTCGCACACCGACATGCGCCCGCTCTCGGGCGGCCTCATCGCCTTCAGCACGCTGGACGGGCGGCCCTCGGCGCACGACTTCGACAACTCGCCCGTGCTGCAGGACTGGGTCACGGCCACAGACATCCGCGTGGCCTTCAGCCGCCTGCACACGTTCGGCGACGAGAACGAGGACGACTCGGAGCTGGCGCGCGACTCGTACTTCTACGCGGTGTCCGACCTGCAGGTGGGCGGCCGGTGCAAGTGCAACGGCCACGCGGCCCGCTGCGTGCGCGACCGCGACGACAGCCTGGTGTGCGACTGCAGGCACAACACGGCCGGCCCGGAGTGCGACCGCTGCAAGCCCTTCCACTACGACCGGCCCTGGCAGCGCGCCACAGCCCGCGAAGCCAACGAGTGCGTGGCCTGTAACTGCAACCTGCATGCCCGGCGCTGCCGCTTCAACATGGAGCTCTACAAGCTTTCGGGGCGCAAGAGCGGAGGTGTCTGCCTCAACTGTCGCCACAACACCGCCGGCCGCCACTGCCATTACTGCAAGGAGGGCTACTACCGCGACATGGGCAAGCCCATCACCCACCGGAAGGCCTGCAAAGCCTGTGATTGCCACCCTGTGGGTGCTGCTGGCAAAACCTGCAACCAAACCACCGGCCAGTGTCCCTGCAAGGACGGCGTGACGGGTATCACCTGCAACCGCTGCGCCAAAGGCTACCAGCAGAGCCGCTCTCCCATCGCCCCCTGCATAAAGATCCCTGTAGCGCCGCCGACGACTGCAGCCAGCAGCGTGGAGGAGCCTGAAGACTGCGATTCCTACTGCAAGGCCTCCAAGGGGAAGCTGAAGATTAACATGAAAAAGTACTGCAAGAAGGACTATGCCGTCCAGATCCACATCCTGAAGGCGGACAAGGCGGGGGACTGGTGGAAGTTCACGGTGAACATCATCTCCGTGTATAAGCAGGGCACGAGCCGCATCCGCCGCGGTGACCAGAGCCTGTGGATCCGCTCGCGGGACATCGCCTGCAAGTGTCCCAAAATCAAGCCCCTCAAGAAGTACCTGCTGCTGGGCAACGCGGAGGACTCTCCGGACCAGAGCGGCATCGTGGCCGATAAAAGCAGCCTGGTGATCCAGTGGCGGGACACGTGGGCGCGGCGGCTGCGCAAGTTCCAGCAGCGTGAGAAGAAGGGCAAGTGCAAGAAGGCCTAG
Insertion point: forward is restricted enzyme Bgl II (AGATCT) restriction enzyme site, is reversed EcoR I (GAATTC) restriction enzyme site.
2) add 600 μ l lysate RIPA Buffer (50mM Tris-HCl, pH7.4,1%NP-40,0.25%Na-deoxycholate, 150mM NaCl, 1mM EDTA, 1mM Na3VO4,1mM NaF, (protease inhibitor, purchased from Roche company for 1mM PMSF and 1mM proteinase inhibitors cocktails, article No. 04693116001)), cracking on ice 30 minutes, 4 ℃ centrifugal (12,000g) 15 minutes.
3) drawing supernatant is cell pyrolysis liquid, after Bradford method is measured protein concentration, total protein concentration is diluted to 1mg/ml.
4) in lysate, add 20 μ l protein G-Agarose, hatch 1 hour for 4 ℃, remove the protein with 20 μ l protein G-Agarose non-specific bindings.
5) centrifuging and taking supernatant, adds CD146 monoclonal antibody AA1 or the Netrin-1 monoclonal antibody of 2 μ g, hatches 2 hours for 4 ℃.
6) again add 30 μ l protein G-Agarose, hatch 1 hour for 4 ℃.
7) the centrifugal supernatant of abandoning, the agarose beads of precipitation washes 3 times with the PBS containing protease inhibitor, adds sample-loading buffer 100 μ l vortex 2 minutes after each 5 minutes, and 100 ℃ are boiled 10 minutes, centrifuging and taking supernatant.
8) protein sample of handling well and full cell pyrolysis liquid keep sample and one are used from Western blot and detect.
External pull-down experiment:
1) Fc-CD146 protein 20 0ng or Fc protein 20 0ng are melted in the EP pipe of 500 μ l HEPES together with Netrin-1 protein 20 0ng, or 2ug anti-CD146 monoclonal antibody AA1 or AA98 are melted in the EP pipe of 500 μ lPBS together with His-CD146 protein 20 0ng and Netrin-1 protein 20 0ng, hatch 1 hour for 4 ℃.
2) add 20 μ l protein G beads, hatch 1 hour for 4 ℃.
3) 4 ℃ centrifugal 5 minutes, abandon supernatant, the agarose beads of precipitation washes 3 times with the PBS containing protease inhibitor, after each 5 minutes, adds sample-loading buffer 50 μ l vortex 2 minutes, 100 ℃ are boiled 10 minutes.
4) protein sample of handling well detects for Western blot.
Surface plasma resonance experiment:
1) 1ug Fc-CD146 is dissolved in to HEPES solution, is fixed on CM5 sensing chip, be placed in BIACORE3000 instrument.
2) Netrin-1 albumen is diluted to variable concentrations (18.75nM, 37.5nM, 75nM, 150nM and300nM), with the flow velocity of 5 μ l/min, flows through CM5 sensing chip.
3) combination of Netrin-1 and Fc-CD146 is measured by resonance unit (RU), through instrument, calculates, and obtains the interactional affinity constant of Netrin-1 and CD146.
Result shows as Fig. 1, and in HEK293 cell, when catching CD146 with anti-CD146 antibody A A1, Netrin-1 can be detected, vice versa, illustrates that CD146 and Netrin-1 exist interact (A).In pull-down experiment, as shown in (B), between CD146 and Netrin-1, there is direct interaction in vitro.In addition, while utilizing anti-CD146 monoclonal antibody AA98 or AA1 to catch CD146 (C), AA1 group can detect Netrin-1 albumen and AA98 group does not detect, and illustrates that AA98 can block the combination of Netrin-1 and CD146.Utilize the method for surface plasma resonance, the affinity constant of having measured Netrin-1 and CD146 is 1.33nM (D).These results suggest that Netrin-1 and CD146 direct interaction, and anti-CD146 monoclonal antibody AA98 can block interaction between the two.
The endothelial cell proliferation of embodiment 2:Netrin-1 induction, moves and becomes blood vessel to depend on the combination of Netrin-1 and receptor CD146
Angiogenesis is mainly tumor nutrient is provided, and provides approach for what tumor cell shifted, therefore for the growth of tumor, plays vital effect.Suppress angiogenesis and just can suppress significantly the growth of tumor.Netrin-1 is combined by the receptor CD146 with endothelial cell surface, promotes the propagation of endotheliocyte, moves and becomes blood vessel, and the monoclonal antibody of anti-CD146 suppresses the inner skin cell function of Netrin-1 induction by the combination of blocking-up Netrin-1 and CD146.
Main material: Human umbilical vein endothelial cells system (HUVEC, purchased from CellSystems Biotechnolegie Vertrieb), 96 hole transwell plates (Corning HTS Transwell-96Cell Migration Products).
Main agents: recombinant human Netrin-1 albumen, CD146 specific siRNA (synthesizing the company in invitrogen), the antibody A A98 of anti-CD146, Mus IgG (mIgG, purchased from Sigma-Aldrich company, article No. I5381), Matrigel is not (containing somatomedin, BD Biosciences, article No. 354234), CCK8 cell proliferation reagent box.
Main method: cell proliferation, migration experiment, endotheliocyte becomes blood vessel experiment.Concrete grammar is as follows:
Cell proliferation experiment:
1) the HUVEC cell of transient transfection CD146 specific siRNA or contrast siRNA is suspended with complete medium, make single cell suspension (5 * 10
4/ ml).
2) cell is inoculated in to (100 μ l/ holes, three parallel holes are established in every kind of processing) in 96 orifice plates.
3) in cell culture medium, add antibody A A98 or contrast mIgG (50 μ g/ml) and stimulant Netrin-1 albumen (50ng/ml), in 37 ℃ of CO2 gas incubator, cultivate 48 hours.
4) use CCK8 cell proliferation reagent box to measure cell density.
Cell migration experiment:
1) the HUVEC cell of transient transfection CD146 specific siRNA or contrast siRNA is resuspended with complete medium, make single cell suspension (1 * 10
5/ ml).
2) under Transwell, chamber adds the RPMI-1640 culture medium (purchased from Gibco, article No. 31800-022) (200 μ l/ hole) containing 10% hyclone, and upper chamber adds cell suspension (100 μ l/ holes, three parallel holes are established in every kind of processing),
3) upwards in the cell of chamber, add antibody A A98 or AA1 (50 μ g/ml) and stimulant Netrin-1 (50ng/ml).Overnight incubation in 37 ℃ of CO2 gas incubator.
4) cell on film upper strata is wiped with cotton swab, with tweezers, the film of 96 hole transwell plates is taken off, film lower floor is put on microscope slide upward.
5) lower confluent monolayer cells is fixed after 15 minutes with 4% paraformaldehyde room temperature, and by 1% violet staining 15 minutes, microscopy recorded the cell quantity under each visual field.
It is to improve on the basis of the experimental technique set up people such as Nagata that endotheliocyte becomes blood vessel experiment, and concrete operations are as follows:
1) HUVECs cell is resuspended with complete medium, makes single cell suspension (1 * 10
5/ ml).
2) Matrigel (50 μ l/ hole) of coated ice bath in 96 orifice plates, 37 ℃ solidify 30 minutes.
3) in each hole, add 100 μ l cell suspension, correspondingly add stimulant Netrin-1 (200ng/ml) and antibody A A98 or mIgG (50 μ g/ml) simultaneously.
4) overnight incubation in 37 ℃ of incubators is observed afterwards under inverted microscope, takes pictures.
Experimental result is as shown in Fig. 2 (A-C), and Netrin-1 can increase the propagation of endotheliocyte, moves and becomes blood vessel ability, compares with matched group, and transfection CD146 specific siRNA can obviously reduce VEGF and stimulate caused inner skin cell function.Same, anti-CD146 monoclonal antibody AA98 can obviously suppress Netrin-1 stimulates the endothelial cell proliferation causing, moves and becomes blood vessel (D-F).Above result confirms, the angiogenesis mediating for Netrin-1 with the combination of CD146 is necessary.
Embodiment 3:CD146 specific siRNA or anti-CD146 monoclonal antibody AA98 can block the activation that Netrin-1 stimulates the signal path causing
In endotheliocyte, it is upper that Netrin-1 is attached to receptor CD146, can activate the ERM in downstream, p38, ERK, the signaling molecules such as NF-κ B.The expression that the specific siRNA of CD146 can strike CD146 on low endotheliocyte, suppresses the downstream signal path that Netrin-1 induces.Equally, antibody A A98, by the combination of blocking-up Netrin-1 and CD146, can suppress the downstream signal that Netrin-1 activates.
Main material: Human umbilical vein endothelial cells system.
Main agents: recombinant human Netrin-1 albumen, CD146 specific siRNA, the antibody A A98 of anti-CD146, Mus IgG etc.
Main method: the HUVEC that transfection is crossed to CD146 specific siRNA or contrast siRNA cell for Netrin-1 (50ng/ml) stimulate after cell lysis be used for biochemical analysis signal path (A).Or utilize anti-CD146 monoclonal antibody AA98 or mIgG (50 μ g/ml) to hatch respectively HUVEC cell, 37 ℃ after 1 hour, with cell culture medium, clean after three times, then after using Netrin-1 (50ng/ml) to stimulate, cell lysis is used for biochemical analysis signal path.As shown in the figure, Netrin-1 stimulates and can cause downstream signaling molecule ERM, p38, the activation of ERK (10 minutes) and NF-κ B (6 hours).When using transfection to cross CD146 specific siRNA or anti-CD146 monoclonal antibody AA98, can block the downstream signal being caused by Netrin-1 and activate.Illustrate that Netrin-1 activates downstream signal path by being attached on the CD146 of cell surface.
Embodiment 4: in mice arterial ring and Matrigel plug model, the mice or the AA98 that use endothelial cell specific to knock out CD146 can suppress the angiogenesis that Netrin-1 stimulation causes
In assessment body, in the experiment of angiogenesis, arterial ring and Matrigel plug are two models commonly using.Arterial ring experiment, by the total arterial ring of isolated culture mice breast, is observed the quantity that endotheliocyte sprouts.Matrigel plug experiment, by mouse subcutaneous injection Matrigel, is taken out and cuts into slices after certain natural law, observes the situation of angiogenesis.Zoopery confirmation, Netrin-1 can promote angiogenesis in vivo, and this facilitation depends on receptor CD146.
Experimental technique:
Arterial ring experiment: the wild-type mice (C57/BL6J, purchased from dimension tonneau China) and the CD146 endothelium specificity knock-out mice (CD146 that choose 8 weeks sizes
eC-KO, C57/BL6J background), after being put to death, the disconnected neck of mice takes out the total tremulous pulse of breast, be cut into the arterial ring that thickness is 0.5-1 millimeter.Arterial ring is inoculated in 96 orifice plates that are coated with by Matrigel glue to every group of 10 arterial rings.In cell culture medium, correspondence adds antibody A A98 or contrast mIgG (100ug/ml), and stimulant recombinant human Netrin-1 albumen (200ng/ml).In 37 ℃ of incubators, cultivate after 4-6 days, arterial ring is placed in to optical microphotograph Microscopic observation, calculate the endotheliocyte quantity of sprouting.
Matrigel plug experiment: the wild-type mice (C57/BL6J, purchased from dimension tonneau China) and the CD146 endothelium specificity knock-out mice (CD146 that choose 8 weeks sizes
eC-KO, C57/BL6J background), 5 every group.Respectively at back subcutaneous injection 500ul Matrigel glue.In Matrigel glue, be mixed with respectively antibody A A98 or contrast mIgG (100ug/ml), and Netrin-1 (200ng/ml).Inject after 10 days, disconnected neck is put to death mice.The structure that Matrigel is formed is taken out, and after taking pictures, with 4%PFA, fixes 24 hours, paraffin embedding, section, immunohistochemical analysis.Be below the SABC process of paraffin section:
1) take out slice, thin piece, enter 37 ℃ of dewaxings twice of xylene solution, each 30 minutes;
2) enter aquation in dehydrated alcohol * 2-95%-80%-70%-50%-30% and distilled water, each 5 minutes of room temperature;
3) 37 ℃ of lucifuges of 0.3% hydrogen peroxide/methanol solution are processed 30 minutes, eliminate the activity of endogenous peroxydase, and PBS washes three times;
4) 100 ℃ of water-baths of pH6.0 citric acid repair liquid antigen hot repair in 30 minutes is multiple, natural cooling;
5) 37 ℃ of sealings of 5% normal sheep serum (purchased from company of Zhong Shan Golden Bridge, article No. ZLI-9021) are 1 hour;
6) add PBS dilution primary antibodie (the anti-CD31 multi-resistance of rabbit, purchased from Abcam company, article No. ab28364; 1:50 dilution), 4 ℃ of overnight incubation;
7) PBS washes three times; Anti-rabbit-the biotin of goat (purchased from company of Zhong Shan Golden Bridge, article No. ZB-2010; 1:1000 dilution) at 37 ℃, hatch 1 hour, PBS washes three times;
8) Avidin-HRP (purchased from Hyclone-pierce company, article No. N100; 1:1000) at 37 ℃, hatch 45 minutes;
The DAB 9) now joining (purchased from company of Zhong Shan Golden Bridge, article No. ZLI-9032; 1:1000 dilution) lucifuge colour developing 2-7 minute, haematoxylin is redyed.
10) dehydration step by step: 50-70-80-90-100-100% ethanol-dimethylbenzene, dries resinene mounting.
11) in micro imaging system, make film.
Result shows, in wild-type mice, what Netrin-1 can inducing mouse endotheliocyte sprouts and angiogenesis.When using CD146 endothelium specificity knock-out mice, or use while interacting between antibody A A98 blocking-up Netrin-1 and CD146, Netrin-1 promotes the function of angiogenesis to be suppressed.Above result shows, Netrin-1 promotes angiogenesis to depend on the combination with its receptor CD146.
Claims (9)
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710201961.2A CN106913870A (en) | 2014-06-27 | 2014-06-27 | Reagent and its oncotherapy purposes for blocking the interaction between Netrin 1 and CD146 |
| CN201410301523.XA CN104083762A (en) | 2014-06-27 | 2014-06-27 | Reagent for blocking interaction between Netrin-1 and CD146 and tumor treatment application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016086560A1 (en) * | 2014-12-04 | 2016-06-09 | 杭州中翰金诺生物信息技术有限公司 | Marker for diagnosing liver cancer, melanoma cell adhesion molecule, and uses thereof |
| CN110291105A (en) * | 2017-01-05 | 2019-09-27 | 奈特里斯药物公司 | Combination therapy of transin-1 interfering drugs and immune checkpoint inhibitor drugs |
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| CN111850127A (en) * | 2020-08-10 | 2020-10-30 | 叶甲舟 | Application of NTNG1 protein in preparation of liver cancer diagnostic reagent or kit |
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| CN101245101A (en) * | 2008-01-31 | 2008-08-20 | 中国科学院生物物理研究所 | Anti-human CD146 monoclonal antibody, composition comprising same, method for detecting soluble CD146 |
| WO2014000338A1 (en) * | 2012-06-28 | 2014-01-03 | 中国科学院生物物理研究所 | Application of composite of anti-cd146 antibody and anti-vegf antibody in preparing medicine for suppressing tumor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101245101A (en) * | 2008-01-31 | 2008-08-20 | 中国科学院生物物理研究所 | Anti-human CD146 monoclonal antibody, composition comprising same, method for detecting soluble CD146 |
| WO2014000338A1 (en) * | 2012-06-28 | 2014-01-03 | 中国科学院生物物理研究所 | Application of composite of anti-cd146 antibody and anti-vegf antibody in preparing medicine for suppressing tumor |
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| YAN X, LIN Y, YANG D, ET AL: "A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth", 《 BLOOD》, 31 December 2003 (2003-12-31) * |
| ZHENG C, QIU Y, ZENG Q, ET AL: "Endothelial CD146 is required for in vitro tumor-induced angiogenesis: the role of a disulfide bond in signaling and dimerization", 《THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY》, 31 December 2009 (2009-12-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016086560A1 (en) * | 2014-12-04 | 2016-06-09 | 杭州中翰金诺生物信息技术有限公司 | Marker for diagnosing liver cancer, melanoma cell adhesion molecule, and uses thereof |
| CN110291105A (en) * | 2017-01-05 | 2019-09-27 | 奈特里斯药物公司 | Combination therapy of transin-1 interfering drugs and immune checkpoint inhibitor drugs |
| CN110291105B (en) * | 2017-01-05 | 2024-03-01 | 奈特里斯药物公司 | Combination therapy of guide-1 interfering drugs and immune checkpoint inhibitor drugs |
| US12162932B2 (en) | 2017-01-05 | 2024-12-10 | Netris Pharma | Combined treatment with Netrin-1 interfering drug and immune checkpoint inhibitors drugs |
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