CN1251769C - Tissue engineering blood vessel of gene modification - Google Patents

Abstract

The characteristics of the genetically modified tissue engineered blood vessels involved in the present invention are: prepared by the following method: (1) acquisition of vascular scaffold materials; (2) surface modification of blood vessels: modifying the surface of blood vessel lumens by biodegradable control growth factors released Surface modification mixture composed of anticoagulant polymer material, adhesion short peptide, growth factor protein and A20 gene plasmid; (3) smooth muscle cell planting; (4) endothelial cell planting. The tissue-engineered blood vessel has good mechanical properties, anti-thrombosis, anti-restenosis and other properties, and is used for clinically repairing vascular defects or bypassing blood vessels.

Description

The engineering blood vessel of genetic modification

Technical field

The present invention relates to a kind of engineering blood vessel that makes up genetic modification.

Technical background

Present various blood vessel graft is good with the human body self blood vessel.But the length of the nonessential blood vessel of human body self and diameter are very limited, some patient the whole body vascular lesion is arranged in addition do not have suitable for the body blood vessel graft.For trunk, though PTFE artificial blood vessels such as (politef) can meet clinical needs to a certain extent, can not degrade, exist in the human body as foreign body always, and suppress the regeneration function of vascular cell.Artificial blood vessels' such as PTFE anticoagulant mechanism is to allow blood form one deck thrombosis film at artificial blood vessel's inwall, therefore this artificial blood vessel's internal diameter usually can not be less than 6mm, otherwise can cause angiemphraxis, diameter becomes less than the small vessel disease of 6mm, does not still have satisfied artificial blood vessel's substitute.Present clinical blood vessel graft is still taken self artery and vein as far as possible.Though the autotransplantation of artery thing has unrivaled excellent results, the autogenous vein graft thing remains bypass operation of coronary artery and selects maximum blood vessel grafies for use, but because the source is few, its compliance does not match and transplants the front and back various factors to endothelial cell damage, most of bridge blood vessel generation neointimal hyperplasia, finally cause the angiostenosis obturation, the long-term patency rate of Vein Bridge is low, is again puzzlement cardiac surgeon's a difficult problem.

The artificial blood vessel is used for the acute vascular injury in treating or is used for the patient that some can not provide suitable seed cell in addition, and the artificial blood vessel of structure can not need tolerance homotransplantation with the autogenous cell plantation.Coronary artery bypass grafting needs small-diameter intravascular, but diameter is transplanted after 6 months the thrombosis rate less than the artificial blood vessel of 6mm and still do not had satisfied artificial blood vessel's substitute up to 40%.With artificial material construction small-diameter intravascular easy-formation not, be difficult to simultaneously accomplish that compliance is complementary.Thereby how to improve the mechanical characteristic and the biocompatibility of grafting vessel, and prevent vascular occlusion, be to make up the subject matter that small-diameter intravascular faces.

The key issue of engineering blood vessel research is seed cell and timbering material.Seed cell comprises adult vascular cell and stem cell etc.Aspect timbering material, the product of the final development of intravascular tissue engineering is the blood vessel with 3D shape and space structure, therefore when cultivating cell, three-dimensional vascular stent material with specific three dimensional structure must be provided, inoculating cell can be located, attach, the localization growing multiplication, material can make cell arranged evenly in order in rack space simultaneously, differentiation has specific function and synthetic suitable extracellular matrix, tissue that cell and extracellular matrix form or organ and three-dimensional rack shape are consistent, timbering material also has mechanics support function, anti-blood stream pressure etc. concurrently in the time of in the engineering blood vessel transplant.The timbering material of engineering blood vessel use at present comprises natural material and synthetic material, synthetic material mainly contains two kinds, a kind of is non-degradable material such as polymethyl methacrylate, politef etc., and another kind is the copolymer of degradation material such as Polyethylene Glycol acid, poly-lactic acid etc. and above-mentioned material.Because this class material has low toxicity, no immunoreation, safety is better and have the characteristic of good biocompatibility to be used widely.Although artificial material has many advantages, but also there are some problems, the biocompatibility of synthetic material, biological activity, biological degradability and also have some shortcomings with aspect such as host blood vessel mechanics coupling, and the bionical making at aspects such as porosity and pore sizes also has certain difficulty, simultaneously the acidic materials that produce in the degradation in vivo process when using in a large number of some artificial material (as polylactic acid etc.) are piled up, and are unfavorable for adhering to, divide, breeding of cell; Simultaneously hydrophilic is poor, the cell absorption affinity a little less than, the mechanical strength deficiency.As synthetic family macromolecule material shortage cell recognition signal, its expensive price has also limited its extensive use in addition.Therefore, timbering material has become the subject matter that hinders the engineering blood vessel flow of research.

Because of the blood vessel replacement therapy, can not change the pathogenic environment in inside of body, Ratliff etc. have observed the great saphenous veins of 115 examples as coronary bypass, find that transplant graft atherosclersis (GAS) pathological changes person accounts for 99%.Although illustrate and carried out replacement therapy, environment still exists owing to cause a disease in the inside of damage in the migration process and body, very likely causes grafting vessel generation restenosis once again, thereby causes graft failure.Although the report that makes up small-diameter intravascular is arranged at present, all be confined to blood vessel surface and modify the reinforcement cell seeding, still there is not good way to solve to the neointimal hyperplasia vascular restenosis obturation that takes place after the blood vessel transplantation.

The A20 gene belongs to zinc finger protein family, aspect anti-inflammatory relevant report is arranged, but does not see both at home and abroad at present and any the A20 gene is applied to intravascular tissue engineering research report.

Summary of the invention

Purpose of the present invention just is to improve deficiency of the prior art, provides a kind of and has simultaneously that excellent mechanical performances and antithrombotic form, the engineering blood vessel of the genetic modification of vascular restenosis, is used for repairing clinically vascular defects or vascular bypass.

For realizing that the technical scheme that above-mentioned purpose of the present invention adopts is such, i.e. a kind of engineering blood vessel of genetic modification is characterized in that: adopt following method preparation:

1, vascular stent material obtains: remove antigenicity and cell component by xenogenesis or blood vessel of the same race, the natural network structure of artery-sparing is made, or directly adopts synthetic material to make;

2, blood vessel surface is modified: the finishing mixture that medical polymer material, adhered short peptides, growth factor protein and the A20 gene plasmid that is discharged by the biodegradable control growing factor in the lumen of vessels finishing formed; Its proportion relation is by weight percentage: its proportion relation is the medical polymer material 70-90% that the biodegradable control growing factor discharges, adhered short peptides is 0.3-1%, growth factor protein is 0.003-0.008%, the A20 gene plasmid is 0.001-0.003%, and all the other are deionized water.Fully be mixed into aqueous solution, be filled in the lumen of vessels that the xenogenesis handled well or lumen of vessels of the same race or synthetic material make, on the lumen of vessels surface, vacuum drying is standby through ultraviolet light reaction cross-linking.

3, smooth muscle cell plantation: after finishing the lumen of vessels finishing, again at tunica media inoculation smooth muscle cell;

4, endothelial cell seeding: after smooth muscle cell is grown well, plant the vascular endothelial cell of A20 genetic modification again on the lumen of vessels surface.

In above-mentioned preparation method, the synthetic material described in the step 1 can adopt non-degradable material such as polymethyl methacrylate, politef, or the copolymer of degradation material such as Polyethylene Glycol acid, poly-lactic acid etc. and above-mentioned material; The medical polymer material of releases such as the biodegradable control growing factor in step 2 is N-sulfonic acid-photo-crosslinking-sulfated chitosan; Adhered short peptides is that Gly-Arg-Gly-Asp or Arg-Gly-Asp, growth factor protein are that endothelial cell growth factor (ECGF) or endothelial cell growth factor (ECGF) add a spot of transforming growth factor;

The subject matter that the present invention solves is the mechanical characteristic and the biocompatibility of blood vessel, compared with prior art has following advantage:

1. aspect the genetic modification engineering blood vessel, the A20 gene has antithrombotic formation, vascular restenosis function, the present invention combines by gene engineering method and organizational project and changes over to or overexpression A20 gene at seed cell, then can resist damage factor in the vascular bypass patient blood to the damage of grafting vessel endothelium, the pathologic propagation that suppresses smooth muscle cell can effectively suppress the homotransplantation damage that the acute vascular replacement therapy is faced simultaneously.

2. Zhi Bei intravascular stent, its antigenicity a little less than, favorable tissue affinity and adhesion are arranged, its natural pore structure, size and form are rectified, for adhesion, propagation, the differentiation of seed cell provides natural three dimensional growth space structure.This material source is abundant, and is simple for production, is easy to mouldingly, and is better than synthetic material at aspects such as function adaptability, histocompatibility, physicochemical property, biological degradability, costs.

3. N-sulfonic acid-photo-crosslinking-sulfated chitosan the coating material of the anticoagulation and control growing factor release is synthetic by chitosan (Az-CH-LA) sulfonation and the Sulfation of photo-crosslinking, thereby have very strong hydrophilic and an anticoagulant, the flexible hydrogel of difficult dissolving can be become rapidly through irradiation under ultraviolet ray, the release of medicine can be controlled.And present common synthetic heparinoid body N-sulfonic acid---sulfated chitosan has anticoagulant but does not have the function of control drug release.Do not see as yet both at home and abroad at present and have control drug release and the synthetic report of anticoagulant chitosan simultaneously.Therefore the present invention becomes the heparinoid body structure to form brand-new being used for and the contacted biomaterial heparinoid of blood body N-sulfonic acid-photo-crosslinking-sulfated chitosan the modification of photo-crosslinking chitosan (Az-CH-LA) materials chemistry, the then release of may command somatomedin or medicine on the one hand, have anticoagulation function on the other hand, have degradability simultaneously.Can be used for the finishing of blood vessel, artificial blood vessel, other engineering blood vessels and the endovascular stent of the present invention's structure.

4. intravascular stent itself has good cell myrmecophily, after further composite surface is modified material, is the microenvironment that seed cell adheres to, breeds and performance anticoagulation function provides.The surface is modified with following advantage among the present invention, synthetic medical polymer material N-sulfonic acid-photo-crosslinking-sulfated chitosan is the release of may command somatomedin on the one hand, crosslinkedly on the other hand can not cause thrombosis during at endothelium seed cell deficiency or cell detachment in blood vessel surface, crosslinked adhered short peptides can promote to exsomatize and at the cell adhesion of body, but endothelial cell growth factor (ECGF) albumen in-vitro inducing seed cell growth and induce in the blood endothelial progenitor cells to adhere to differentiation (or plasmid) A20 gene at body and can effectively resist various factors to blood vessel injury in blood vessel surface.The present invention by finishing when acute vascular substitutes or gets patient's endotheliocyte lazy weight, can be directly with blood vessel transplantation in the patient body, N-CM-photo-crosslinking chitosan anticoagulant surface can not cause blood coagulation, and crosslinked endothelial cell growth factor (ECGF) and adhesin polypeptide can be induced the endothelial progenitor cells in the blood and close on endotheliocyte and be replenished the grafting vessel endothelium and lose or do not plant the position as yet.Crosslinked A20 gene plasmid is convenient to adhere to the endothelial progenitor cells absorption of differentiation, forms the small-diameter intravascular with atherosclerosis function rapidly.This method also can induce the endothelial progenitor cells adhesion differential growth in the blood to become endotheliocyte at body, and needn't isolated ex vivo cultivate, and can improve organizational project efficient greatly like this.Do not see that as yet any report has these imaginations or experimental result.

5, utilize above-mentioned good support material, the plantation smooth muscle cell exsomatizes and plants the endothelium seed cell or induce the endothelial progenitor cells plantation at body, becomes lived genetic modification engineering blood vessel, can be widely used in and repair blood vessel injury clinically.

The specific embodiment:

Below in conjunction with embodiment content of the present invention is described further.

1. vascular stent material obtains: get fresh xenogenesis (as the Sanguis sus domestica pipe) or blood vessel of the same race under the aseptic condition, remove appended soft tissue on it, aseptic PBS flushing blood vessel is removed the thrombosis grumeleuse, and-80 ℃ of preservations are put in aseptic packaging.Rewarming in 37 ℃ with 0.1mol/L PMSF solution-treated 1-3 hour, the hypotonic and high osmotic buffer of antibiotic was handled respectively 3--5 hour, changed liquid once in per 1 hour; Sterile distilled water embathes totally, is keeping 37 ℃, 5%CO ₂With 0.03%--0.05% trypsin treatment 16--24 hour, changed liquid once in per 8 hours, sterile distilled water embathes totally; With 0.005%---0.01%RNA enzymic digestion 1--3 hour, sterile distilled water embathes totally, 0.006%--0.01%DNA enzymic digestion 1--3 hour, and sterile distilled water embathes totally, 0.005%--0.01% lipid enzymic digestion 2--4 hour, aseptic antibiotic distilled water embathes totally.The sterilization in 10--15 minute of 15--20GY gamma-rays radiation treatment is also removed the antigenicity that remains vascular stroma collagen protein, elastin laminin, obtains intravascular stent at last.

The visible vascular cell of handling of intravascular stent utilization morphological method detection that said method obtains is removed, the vascular stroma fiber is more preceding loose, but it is complete to distribute, do not see fibrous fracture, elastic fibers and collagen contents are not seen significant change, differ not remarkable with processing back length of vessel and internal diameter before handling.The P-V experiment detects and handles preceding contrast to its compliance, and the pressure-volume curve and the parabolical upstroke of blood vessel are very approaching before and after handling, tremulous pulse P-V data second-degree parabola relational expression V=aP ²+ bP+c carries out match, and its phase relation number average illustrates that greater than 0.95 fitting effect is good, the computational methods C=dv/dp=2ap+b of corresponding arterial compliance.Must handle the back vascular compliance and descend, but differ not remarkable (P＞0.05), wherein when pressure was 100mmHg, the compliance before and after handling was respectively 6.25 ± 0.42,5.80 ± 0.67 (* 10 ^-4Ml/mmHg), differ not significantly (P＞0.05).Show that with Western blot detection the preceding Sanguis sus domestica pipe MHC antigen that is untreated, xenotransplantation hyperacute rejection α-Gal antigen all have than strongly expressed, do not see Table after the processing to reach.

2. blood vessel surface is modified:

2.1. the medical polymer material that the biodegradable control growing factor of one of finishing thing discharges comes down to photo-crosslinking chitosan is carried out N-sulfonic acid-photo-crosslinking-sulfated chitosan of obtaining behind sulfonation and the Sulfation.Embodiment is: 1., 6-O-sulfonation photo-crosslinking chitosan (6-O-SM-Az-CH-LA) synthetic: the photo-crosslinking chitosan (Az-CH-LA) of purification is dissolved in 2% formic acid solution, the CuSO4.5H2O solution that at room temperature dropwise adds an amount of 1mol/L then, stir after 15--20 hour water, acetone and ether washing successively after the filtration.Products therefrom is dispersed in the exsiccant DMF solution fully; to be dissolved in SO3.Pyridine solution among the DMF then in 1: 4 ratio (1: the mol ratio between 4=chitosan molecule repetitive and the SO3.Pyridine) be added drop-wise in the solution at low temperatures; stir after 1--3 hour, nitrogen protection and 50 ℃ following heated and stirred 15--20 hour.It is 8.2 that cooled solution is transferred to pH value with NaHCO3, adds an amount of water dialysis 3--5 days.After gained solution removed copper ion with stratographic method, lyophilization (productive rate in this step is 80%) obtained product.2., 6-o-sulfonic acid photo-crosslinking chitosan that previous step is obtained is dissolved in a certain amount of water, adds an amount of Na2CO3 and SO3.NMe3 complex then, uses nitrogen protection, stirred 15--20 hour down at 70 ℃.Deng the reaction finish after, in deionized water the dialysis 25--35 hour, in rare NaoH solution the dialysis 4--6 hour, at last in deionized water the dialysis 3--5 days, lyophilization gets final products.It is standby that the 15--20Gy gamma radiation is handled sterilization in 15--20 minute.

2.2.A20 the preparation of gene plasmid: stimulated endothelial cells cultured 3--5 hour with TNF, can stimulate A20 gene expression, RNA in the extracting cell, design primer upstream: 5 '-AGTTGTCCCATTCGTCATTCC-3 ', downstream: 5 '-TTTGAGCAATATGCGGAAAGC-3 ', with RT-PCR method clone A20 gene cDNA, give birth to the worker from Shanghai and buy the terminal dna cloning vector pUCm-T of band A Vector (numbering BS434), the A20 gene cDNA is connected with pUCm-T Vector, utilization x-gal dyeing, blue white macula filters out the bacterium colony that successfully connects, amplification back extracting plasmid DNA, carry out enzyme action with EcoR I and BamH I, comprised the A20 gene cDNA fragment of EcoR I and BamH I restriction enzyme site in a large number.Buy green fluorescent protein carrier for expression of eukaryon pEGFP-N1 (catalog#6085-1) from the gene company limited, comprise 4.7kb, this carrier comprises that a plurality of restriction enzyme sites can insert the external source fragment, wherein this tests the EcoR I site at 631 places in the main application vector multiple clone site and the BamH I site at 661bp place, with EcoR I and BamH I double digestion linearisation pEGFP-N1 carrier, the A20 gene cDNA fragment that the reuse ligase will contain EcoR I and BamH I restriction enzyme site is cloned into green fluorescent protein carrier for expression of eukaryon pEGFP-N1, filter out positive bacterium colony, the amplification extracting obtains comprising the carrier for expression of eukaryon pEGFPA20-N1 of A20 gene.

2.3. synthetic surface modified mixture under aseptic condition.Its proportion relation is N-sulfonic acid-photo-crosslinking-sulfated chitosan 70-90% (w/v), Gly-Arg-Gly-Asp (GRGD) 0.3-1%, endothelial cell growth factor (ECGF) is 0.003-0.008%, and A20 gene plasmid pEGFPA20-N1 is 0.001-0.003%, and all the other waters are supplied.Be mixed into aqueous solution, fully be filled into the lumen of vessels of handling well after the mixed processing, on the lumen of vessels surface, vacuum drying is standby through ultraviolet light reaction cross-linking.But this kind method of modifying also in the synthetic intravascular stent of applying biological degradable material and surface, artificial blood vessel chamber and endovascular stent surface is modified.

Blood vessel after above-mentioned steps is carried out finishing detects the visible vessels inner chamber through scanning electron microscope and is all covered by the finishing thing, and adheres to evenly.

3. smooth muscle cell plantation in making up the blood vessel culture systems voluntarily, is got 3-6 generation after former smooth muscle cell amplification of being commissioned to train foster is gone down to posterity, and adds transforming growth factor, with concentration 3 * 10 ⁶Cell/ml injected into blood vessel chamber, holding tube intracavity have certain pressure to cultivate always.Perhaps smooth muscle cell kind method for planting is to adopt identical at interval some microinjection to tunica media with same quantity cell, cultivates in the vascular cell bioreactor.

The smooth muscle cell morphologic detection of above-mentioned steps plantation: the tunica media that will plant smooth muscle cell poured into 30 seconds with 0.1% silver nitrate solution, daylight is colour developing 30min down, the reuse microinstrument separates the shop sheet, the transparent mounting of conventional dehydration, and the digital micrograph camera is taken a picture.Simultaneously the HE method dyes to the blood vessel of plantation smooth muscle, and to dye visible vessels chamber smooth muscle cell form normal for silver as a result, and dense arrangement distributes along the blood vessel major axis, illustrates that under these conditions, blood vessel successfully carry out smooth muscle cellization.HE dyeing shows that the smooth muscle cell form of tunica media is normal, is the shuttle type, and dense arrangement distributes along the blood vessel major axis.Explanation successfully carry out smooth muscle cellization on the vascular stroma material

4. endothelial cell seeding is got 3-6 generation after the transgenic expansion of endothelial cells goes down to posterity, with concentration 3 * 10 ⁶Cell/ml injected into blood vessel chamber, adhere to 90--120min after, in 2--4 days endothelial cell seeding incubation, the intracavity flow velocity increases gradually from 0.033 to 0.1ml/s, is about 1 * 10 at the corresponding shear stress of blood vessel wall ^-2N/m ²To 4 * 10 ^-2N/m ²Between, the engineering blood vessel of structure genetic modification.

Endotheliocyte to the above-mentioned steps plantation detects through light microscopic and Electronic Speculum, and visible vessels intracavity chrotoplast form is normal, dense arrangement, distribute along the blood vessel major axis, illustrate that under these conditions blood vessel has successfully carried out endothelialization, silver dyes and shows that also endotheliocyte successfully is planted on the vascular stroma.Fluorescence microscope shows that the endotheliocyte that is planted on the vascular stroma material can the secretory cell epimatrix.Show endotheliocyte can be on vascular stroma normal growth.

The blood vessel that above-mentioned steps is obtained carries out circumferential tension force detection, in the experiment the progressively increase power of genetically modified organism engineering blood vessel that builds is stimulated, when hydrostatic pressing reaches 2000mmHg, do not see angiorrhexis yet, illustrate that the anti-spalling intensity/hydrostatic pressing of blood vessel that makes up is greater than 2000mmHg.

Select scheme fully, in the time can not obtaining the human endothelial cell of sufficient amount, after can preparing intravascular stent and finishing by preceding method, also can be only at middle film plantation smooth muscle cell, intravascular space is mainly induced in body blood endothelial progenitor cells and is closed on endothelial cell seeding.

Describe for example below, but application of the present invention not only is this.

Embodiment 1.A kind of bio-derivative tissue engineering blood vessel preparation of genetic modification prepares Sanguis sus domestica pipe or allosome vascular stroma material by above-mentioned steps (1) method, after (2) carry out finishing set by step, by (3) plantation monkey smooth muscle cell, (4) plantation endotheliocyte set by step.

The smooth muscle cell form of the visible plantation of the blood vessel that obtains is good, and vascular endothelial cell distributes complete, distributes along the blood vessel major axis.Be transplanted to the monkey common carotid artery, detect after 12 months.It is intact to see that vascular endothelial cell distributes, and form is normal, and the vascular smooth muscle cell form is normal, distributes along the blood vessel major axis, and blood vessel keeps clear, and does not have vascellum endometrial hyperplasia and thrombosis.

Compare with xenotransplantation blood vessel (directly from the pig to the monkey) and homotransplantation blood vessel.As seen the visible tube chamber of xenotransplantation blood vessel is all inaccessible, and thrombosis is arranged in the blood vessel, and film destroy in the blood vessel wall is not seen hypertrophy, and blood vessel wall has a large amount of inflammatory cell infiltrations.And homotransplantation blood vessel vascular occlusion after 6 months, tunica intima is typical transplant graft atherosclersis pathological change, visible evenly diffusivity intimal thickening, the focus rule is centration wheel ring-type, and intralesional is rich in foam cell, inflammatory reaction is fairly obvious, fiber forms insufficient, and calcification is not obvious, and a little circular void is arranged in the middle of the inner membrance that thickens, interior by the thrombosis filling, explanation causes thrombosis, vascular occlusion after angiostenosis arrives to a certain degree.And 1 month blood vessel HE of homotransplantation dyeing visible vessels is not inaccessible, theca interna thickens, the interior light physaliphore that dyes of subcutaneous appearance, the middle obvious hypertrophy of film, smooth muscle cell arrangement disorder and to the inner membrance projection, be speckle shape structure, film has tangible calcification to form in some position, occurs the foam like cell around the calcification district.Inflammatory cell infiltration is arranged, and detecting the visible vessels theca externa through SABC has in various degree CD4 positive cell.Tunnel detects and to show that homotransplantation blood vessel tunica intima layer do not see apoptotic cell substantially, and middle rete, the visible a large amount of apoptotic cell of theca externa.Homotransplantation blood vessel HE dyeing in 1 month blood vessel theca interna thickening is that the monoclonal antibody detection all is the Brdu positive cell through Brdu, and immunofluorescence is that the monoclonal antibody detection determines that outgrowth theca interna cell is α-actin positive cell through α-actin.Illustrate that outgrowth theca interna mainly is the smooth muscle cell of propagation.

And the engineering blood vessel of the genetic modification that this method makes up transplants after 12 months that HE dyeing does not see substantially that the tunica intima layer thickens and tangible smooth muscle cell proliferation, vascular morphology is normal, each layer structural integrity, marshalling, do not see calcification district and inflammatory cell infiltration, SABC detects does not see that blood vessel has CD4 to express.Blood vessel is not seen apoptotic cell substantially.Vascellum tunica interna incrassation is not seen in HE dyeing after the blood vessel transplantation, and immunofluorescence is that monoclonal antibody detects not show color fluorescence of theca interna with Brdu, α-actin.Illustrate that theca interna do not see the smooth muscle cell of propagation, match with the HE coloration result.Above presentation of results the present invention combines by gene engineering method and organizational project and changes over to or overexpression A20 gene at seed cell, then can resist damage factor in the vascular bypass patient blood to the damage of grafting vessel endothelium, the pathologic propagation that suppresses smooth muscle cell can effectively suppress the homotransplantation damage that the acute vascular replacement therapy is faced simultaneously.

Embodiment 2.Can induce the genetic modified organism engineering blood vessel preparation of endothelialization at body, the Sanguis sus domestica pipe or the allosome vascular stroma material that prepare corresponding caliber by above-mentioned steps (1) method, after (2) carry out finishing set by step,, do not plant endotheliocyte by (3) plantation rat smooth muscle cell.Be transplanted to rat carotid artery, detect after 6 months, see that blood vessel keeps clear, scanning electron microscope detects, and blood vessel is endothelialization fully, does not have vascellum endometrial hyperplasia and thrombosis.And matched group not the blood vessel of surface modification and genetic modification do not see that endodermis forms, lumen of vessels is narrow, vascellum endometrial hyperplasia, a part of blood vessel are all inaccessible.

Embodiment 3.The preparation of a kind of engineering blood vessel of genetic modification prepares Sanguis sus domestica pipe or vascular stroma material of the same race by (1) method of enforcement, carry out finishing by (2) after, by (3), (4) plantation human smooth muscular cells, endotheliocyte.Be used for dialysis patient and do not see thrombosis, and matched group thrombosis.

Claims (8)

1. A genetically modified tissue engineered blood vessel, characterized in that it is prepared by the following method: (1) Acquisition of vascular stent materials: It is made from heterogeneous or homogeneous blood vessels to remove antigenic and cellular components, retaining the natural network structure of blood vessels, or directly using artificial synthetic materials; (2) Blood vessel surface modification: modify the anticoagulant polymer material N—sulfonic acid—photocrosslinking—chitosan sulfate, adhesion short peptide Gly- A surface modification mixture composed of Arg-Gly-Asp or Arg-Gly-Asp, growth factor protein and A20 gene plasmid; its proportioning relationship is by weight percentage: its proportioning relationship is biodegradable anticoagulant for controlling the release of growth factors Blood polymer material N—sulfonic acid—photocrosslinking—chitosan sulfate 70-90%, adhesion short peptide Arg-Gly-Asp or Arg-Gly-Asp 0.3-1%, growth factor protein 0.003- 0.008%, A20 gene plasmid is 0.001-0.003%, the rest is deionized water, fully mixed into an aqueous solution, poured into the treated heterogeneous or homogeneous blood vessel cavity or the blood vessel cavity made of artificial synthetic materials, and reacted with ultraviolet light. It is cross-linked on the surface of the vascular lumen and dried in vacuum for later use. The growth factor protein is endothelial growth factor or endothelial growth factor plus a small amount of transforming growth factor, and the A20 gene plasmid is pEGFPA20-N1; (3) Implantation of smooth muscle cells: After the surface modification of the vascular lumen is completed, smooth muscle cells are inoculated in the vascular media; (4) Planting of endothelial cells: After the smooth muscle cells grow well, the A20 gene-modified vascular endothelial cells are planted on the surface of the vascular lumen. 2. A genetically modified tissue engineered blood vessel, characterized in that it is prepared by the following method: (1) Acquisition of vascular stent materials: It is made from heterogeneous or homogeneous blood vessels to remove antigenic and cellular components, retaining the natural network structure of blood vessels, or directly using artificial synthetic materials; (2) Blood vessel surface modification: modify the anticoagulant polymer material N—sulfonic acid—photocrosslinking—chitosan sulfate, adhesion short peptide Gly- A surface modification mixture composed of Arg-Gly-Asp or Arg-Gly-Asp, growth factor protein and A20 gene plasmid; its proportioning relationship is by weight percentage: its proportioning relationship is biodegradable anticoagulant for controlling the release of growth factors Blood polymer material N—sulfonic acid—photocrosslinking—chitosan sulfate 70-90%, adhesion short peptide Arg-Gly-Asp or Arg-Gly-Asp 0.3-1%, growth factor protein 0.003- 0.008%, A20 gene plasmid is 0.001-0.003%, the rest is deionized water, fully mixed into an aqueous solution, poured into the treated heterogeneous or homogeneous blood vessel cavity or the blood vessel cavity made of artificial synthetic materials, and reacted with ultraviolet light. It is cross-linked on the surface of the vascular lumen and dried in vacuum for later use. The growth factor protein is endothelial growth factor or endothelial growth factor plus a small amount of transforming growth factor, and the A20 gene plasmid is pEGFPA20-N1; (3) Implantation of smooth muscle cells: After the surface modification of the vascular lumen is completed, smooth muscle cells are inoculated in the vascular media. 3. The genetically modified tissue engineered blood vessel according to claim 1 or 2, characterized in that: the artificial synthetic material described in the step (1) of its preparation method uses non-degradable materials or degradable materials, wherein the non-degradable materials Be selected from polymethyl methacrylate and polytetrafluoroethylene, degradable material is selected from polyglycolic acid, polyalcolic acid and the copolymer of above-mentioned material; In step (2), release such as biodegradable control growth factor The anticoagulant polymer material is N-sulfonic acid-photocrosslinking-chitosan sulfate; the adhesion short peptide is Gly-Arg-Gly-Asp or Arg-Gly-Asp, and the growth factor protein is endothelial growth factor or Endothelial growth factor plus a small amount of transforming growth factor. 4. The genetically modified tissue engineered blood vessel according to claim 1 or 2, characterized in that: the acquisition of the stent material in the preparation method step (1) adopts the following method: Take fresh porcine blood vessels or allogeneic blood vessels under aseptic conditions, remove the soft tissue attached to them, wash the blood vessels with sterile PBS, remove thrombus and clot, store them in sterile packaging at -80°C, and rewarm them at 37°C with 0.1mol/LPMSF The solution was treated for 1-3 hours, and the antibiotic hypotonic and hypertonic buffer were treated for 3-5 hours respectively, and the solution was changed every 1 hour; soaked in sterile distilled water, and kept at 37°C and 5% CO ₂ with 0.03- -0.05% trypsin treatment for 16-24 hours, change the medium every 8 hours, soak in sterile distilled water; digest with 0.005%---0.01% RNase for 1-3 hours, soak in sterile distilled water, 0.006% --- 0.01% DNase digestion for 1-3 hours, soak in sterile distilled water, 0.005% --- 0.01% lipase digestion for 2-4 hours, soak in sterile antibiotic distilled water, 15- -20Gy γ-ray radiation treatment for 10-15 minutes to sterilize and remove the antigenicity of remaining vascular matrix collagen and elastin, and finally obtain the biologically derived vascular stent. 5. The genetically modified tissue engineered blood vessel according to claim 1 or 2, characterized in that: in the step (2) of the preparation method, the biodegradable anticoagulant polymer material that controls the release of growth factors is prepared by combining light transfusion N-sulfonic acid-photocrosslinking-chitosan sulfate obtained by linking chitosan with sulfonation and sulfate esterification, the specific method is: ①. Synthesis of 6-0-sulfonated photocrosslinked chitosan: Dissolve the purified photocrosslinked chitosan in 2% formic acid solution, and then add an appropriate amount of 1mol/L CuSO4.5H2O dropwise at room temperature solution, stirred for 15--20 hours, filtered and washed with water, acetone and ether in sequence, and the resulting product was completely dispersed in a dry DMF solution, and then the SO3.Pyridine solution dissolved in DMF was in a ratio of 1:4 in Add it dropwise into the solution at low temperature, where 1:4 refers to the molar ratio between chitosan molecular repeating unit and SO3. After hours, the cooled solution was adjusted to pH 8.2 with NaHCO3, and an appropriate amount of water was added for dialysis for 3--5 days. After removing copper ions from the resulting solution by chromatographic methods, freeze-dried to obtain the product, the yield of this step 80%; ②. Dissolve the 6-o-sulfonic acid photocrosslinked chitosan obtained in the previous step in a certain amount of water, then add an appropriate amount of Na2CO3 and SO3.NMe3 complexes, protect with nitrogen, and stir at 70°C for 15-- 20 hours, after the completion of the reaction, dialyze in deionized water for 25--35 hours, dialyze in dilute NaoH solution for 4--6 hours, finally dialyze in deionized water for 3--5 days, freeze-dry to obtain the final product, 15 --20Gy gamma-ray radiation treatment for 15--20 minutes for disinfection and standby. 6. The genetically modified tissue engineered blood vessel according to claim 1 or 2, characterized in that: the preparation of the A20 gene plasmid pEGFPA20-N1 in the step (2) of the preparation method is as follows: Stimulate the cultured endothelial cells with tumor necrosis factor TNF for 3--5 hours, stimulate the expression of A20 gene, extract the RNA in the cells, design primers upstream: 5'-AGTTGTCCCATTCGTCATTCC-3', downstream: 5'-TTTGAGCAATATGCGGAAAGC-3', use The A20 gene cDNA was cloned by RT-PCR, and the A-terminal DNA cloning vector pUCm-T Vector was purchased from Shanghai Sangong, and the A20 gene cDNA was connected to the pUCm-T Vector, stained with x-gal, and the successful connection was selected by blue and white spots After amplification, the plasmid DNA was extracted, digested with EcoR I and BamH I, and a large number of A20 gene cDNA fragments containing EcoR I and BamH I restriction sites were obtained. The green fluorescent protein eukaryotic expression was purchased from Gene Co., Ltd. Vector pEGFP-N1, including 4.7kb, this vector includes multiple restriction sites for inserting foreign fragments, and this experiment mainly uses the EcoR I site at 631 and the BamH I site at 661bp in the multi-cloning site of the vector point, use EcoR I and BamH I to cut the linearized pEGFP-N1 vector, and then use ligase to clone the A20 gene cDNA fragment containing EcoR I and BamH I restriction sites into the green fluorescent protein eukaryotic expression vector pEGFP-N1 , positive colonies were screened out, amplified and extracted to obtain the eukaryotic expression vector pEGFPA20-N1 containing the A20 gene. 7. The genetically modified tissue engineered blood vessel according to claim 1 or 2, characterized in that: the smooth muscle cell planting method in step (3) of the preparation method is as follows: In the self-constructed vascular culture system, the primary cultured smooth muscle cells were expanded and passaged for 3-6 passages, and transformed growth factor was added, and injected into the vascular lumen at a concentration of 3×10 ⁶ cell/ml to keep a certain amount in the vascular lumen. Pressure culture, or smooth muscle cell seeding method is to microinject the same number of cells into the vascular media at the same interval and culture in a vascular cell bioreactor. 8. The genetically modified tissue engineered blood vessel according to claim 1, characterized in that: the method for planting endothelial cells in step (4) of the preparation method is as follows: After the transgenic endothelial cells are expanded and passaged, take 3-6 passages and inject them into the vascular lumen at a concentration of 3×10 ⁶ cell/ml. The internal flow rate gradually increases from 0.033 to 0.1ml/s, and the corresponding shear stress on the vessel wall is about 1×10 ^-2 N/m ² to 4×10 ^-2 N/m ² , constructing genetically modified tissue engineering Blood vessel.