CN1491728A - Genetically modified tissue engineered blood vessels - Google Patents

Abstract

The characteristics of the genetically modified tissue engineered blood vessels involved in the present invention are as follows: (1) acquisition of vascular stent materials; (2) surface modification of blood vessels: the surface of the blood vessel cavity is modified by biodegradable growth factors Surface modification mixture composed of released anticoagulant polymer material, adhesion short peptide, growth factor protein and A20 gene plasmid DNA; (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

Genetically modified tissue engineered blood vessels

technical field

The invention relates to a method for constructing genetically modified tissue engineering blood vessels.

technical background

At present, various vascular grafts are preferably the human body's own blood vessels. However, the length and diameter of the body's own non-essential blood vessels are extremely limited, and some patients have systemic vascular disease and even have no suitable autologous vascular grafts. For large blood vessels, although artificial blood vessels such as PTFE (polytetrafluoroethylene) can meet clinical needs to a certain extent, they cannot be degraded and exist as foreign bodies in the human body all the time, and inhibit the regeneration function of vascular cells. The anticoagulant mechanism of PTFE and other artificial blood vessels is to let the blood form a layer of thrombus membrane on the inner wall of the artificial blood vessel. Therefore, the inner diameter of this artificial blood vessel should usually not be less than 6mm, otherwise it will cause blood vessel blockage. Satisfactory substitute for artificial blood vessels. At present, clinical vascular grafts still use their own arteries and veins as much as possible. Although autologous arterial grafts have incomparably excellent results, autologous vein grafts are still the most widely used vascular grafts for coronary artery bypass grafting. Intimal hyperplasia occurs in most bypass vessels, which eventually leads to vascular stenosis and occlusion. The long-term patency rate of vein grafts is low, which is another problem that plagues cardiac surgeons.

In addition, artificial blood vessels are used for the treatment of acute vascular injury or for some patients who cannot provide suitable seed cells. The constructed artificial blood vessels cannot be planted with autologous cells and must be resistant to allografting. Coronary artery bypass grafting requires small-diameter blood vessels, but the thrombosis rate of artificial blood vessels with a diameter of less than 6mm is as high as 40% after 6 months of transplantation, and there is still no satisfactory artificial blood vessel substitute. It is not easy to form small-diameter blood vessels with artificial materials, and it is difficult to match the compliance. Therefore, how to improve the mechanical properties and biocompatibility of grafted blood vessels and prevent vascular occlusion is the main problem in the construction of small-diameter blood vessels.

The key issues in the study of tissue engineered blood vessels are seed cells and scaffold materials. Seed cells include adult vascular cells and stem cells. In terms of scaffold materials, the final product developed by vascular tissue engineering is a blood vessel with three-dimensional shape and spatial structure. Localized growth and proliferation, at the same time, the material can make the cells in the space of the scaffold be arranged in an orderly manner, differentiate with specific functions and synthesize an appropriate extracellular matrix, the tissue or organ formed by the cells and the extracellular matrix is consistent with the shape of the three-dimensional scaffold, tissue engineering blood vessel transplantation In vivo, the stent material also has the function of mechanical support, anti-blood flow pressure and so on. At present, the scaffold materials used in tissue engineering blood vessels include natural materials and artificial synthetic materials. There are two main types of artificial synthetic materials, one is non-degradable materials such as polymethyl methacrylate, polytetrafluoroethylene, etc. Materials such as polyethylene glycol acid, polyglycolic acid, etc. and copolymers of the above materials. Such substances are widely used because of their low toxicity, no immune response, good safety and good biocompatibility. Although artificial materials have many advantages, there are still some problems. There are still some shortcomings in the biocompatibility, bioactivity, biodegradability and mechanical matching with host blood vessels of artificial synthetic materials. The bionic production of bionics is still difficult. At the same time, when some artificial materials (such as polylactic acid, etc.) are used in large quantities, the acidic substances produced during the degradation process in the body accumulate, which is not conducive to cell attachment, division, and proliferation; The force is weak and the mechanical strength is insufficient. In addition, as a synthetic polymer material, it lacks cell recognition signals, and its high price also limits its wide application. Therefore, scaffold materials have become a major problem hindering the progress of tissue engineering blood vessel research.

Because vascular replacement therapy cannot change the internal pathogenic environment of the body, Ratliff et al. observed 115 cases of the great saphenous vein used as coronary artery bypass grafts and found that 99% of the grafts had atherosclerotic (GAS) lesions. It shows that despite the replacement therapy, due to the damage during the transplantation process and the internal pathogenic environment of the body still exists, it is very likely to cause restenosis of the graft vessel again, resulting in graft failure. Although there are currently reports on the construction of small-diameter blood vessels, they are all limited to modifying and strengthening cell planting on the surface of blood vessels, and there is no good solution to the restenosis and occlusion of intimal hyperplasia after blood vessel transplantation.

The A20 gene belongs to the family of zinc finger proteins, and there are related reports on anti-inflammation, but there are no reports on the application of the A20 gene to vascular tissue engineering at home and abroad.

Contents of the invention

The purpose of the present invention is to improve the deficiencies in the prior art and provide a genetically modified tissue engineered blood vessel with good mechanical properties, anti-thrombosis and vascular restenosis, which is used for clinically repairing vascular defect or vascular bypass.

The technical scheme adopted for realizing the above-mentioned purpose of the present invention is such that a kind of genetically modified tissue engineering blood vessel is 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 surface modification mixture composed of biodegradable anticoagulant polymer materials that control the release of growth factors, adhesion short peptides, growth factor proteins and A20 gene plasmid DNA on the surface of the blood vessel lumen; its ratio The relationship by weight percentage is: 70-90% of biodegradable anticoagulant macromolecule materials that control the release of growth factors, 0.3-1% of adhesion short peptides, 0.003-0.008% of growth factor proteins, and 0.001% of A20 gene plasmid DNA -0.003%, the balance is deionized water. Fully mixed into an aqueous solution, poured into the treated heterogeneous or homogeneous vascular cavity or the vascular cavity made of artificial synthetic materials, cross-linked on the surface of the vascular cavity by ultraviolet light reaction, and vacuum-dried for later use;

3. Smooth muscle cell planting: after the surface modification of the vascular cavity is completed, smooth muscle cells are seeded on the vascular media;

4. Planting of endothelial cells: After smooth muscle cells grow well, A20 gene-modified vascular endothelial cells are planted on the surface of the vascular lumen.

In the above preparation method, the artificial synthetic material described in step 1 can use non-degradable materials such as polymethyl methacrylate, polytetrafluoroethylene, or degradable materials such as polyethylene glycol acid, polyglycolic acid, etc. and the above-mentioned The copolymer of material; The anticoagulant macromolecular material of the biodegradable control growth factor release in step 2 is N-sulfonic acid-photocrosslinking-chitosan sulfate; Adhesive short peptide is Gly-Arg- Gly-Asp, or Arg-Gly-Asp, growth factor protein is endothelial growth factor or endothelial growth factor plus a small amount of transforming growth factor;

The main problem to be solved by the present invention is the mechanical properties and biocompatibility of blood vessels, and compared with the prior art, it has the following advantages:

1. In terms of genetic modification of tissue engineering blood vessels, the A20 gene has anti-thrombosis and vascular restenosis functions. The present invention combines genetic engineering methods and tissue engineering to transfer or overexpress the A20 gene in seed cells, which can not only resist vascular bypass Injury factors in the patient's blood can damage the endothelium of grafted vessels, inhibit the pathological proliferation of smooth muscle cells, and at the same time effectively inhibit the allograft injury faced by acute vascular replacement therapy.

2. The prepared stent has weak antigenicity, good tissue affinity and binding force, and its natural pore structure, size and shape are correct, providing natural three-dimensional growth for the adhesion, proliferation and differentiation of seed cells Spatial structure. This material is rich in sources, easy to manufacture, easy to shape, and is superior to artificial synthetic materials in terms of functional adaptability, tissue compatibility, physical and chemical properties, biodegradability, and cost.

3. N-sulfonic acid-photocrosslinking-chitosan sulfate surface modifier for anticoagulant and growth factor release control is synthesized by sulfonation and sulfation of photocrosslinked chitosan (Az-CH-LA) , so it has strong hydrophilicity and anticoagulant properties, and can quickly become a flexible hydrogel that is not easy to dissolve after being irradiated by ultraviolet light, and can control the release of drugs. However, the currently commonly synthesized heparanoid N-sulfonic acid-chitosan sulfate has anticoagulant properties but does not have the function of controlling drug release. At present, there is no report on the synthesis of chitosan with both controlled drug release and anticoagulation at home and abroad. Therefore, the present invention chemically modifies the light-crosslinked chitosan (Az-CH-LA) material into a heparanoid structure to form a new biomaterial heparanoid N-sulfonic acid-photocrosslinked that can be used in contact with blood. Linked-chitosan sulfate can control the release of growth factors or drugs on the one hand, and has anticoagulant properties and degradability on the other hand. It can be used for surface modification of blood vessels constructed in the present invention, artificial blood vessels, other tissue engineering blood vessels and intravascular stents.

4. The vascular stent itself has good cell-inducing properties. After further compounding with surface modification substances, it can provide a good microenvironment for seed cell adhesion, proliferation and anticoagulant function. The surface modification in the present invention has the following advantages. The synthetic anticoagulant polymer material N-sulfonic acid-photocross-linking-chitosan sulfate can control the release of growth factors on the one hand, and cross-link on the endothelium on the surface of blood vessels on the other hand. Insufficient seed cells or cell detachment will not cause thrombus formation, cross-linked adhesion short peptide can promote cell adhesion in vitro and in vivo, endothelial growth factor protein can induce seed cell growth in vitro and induce blood endothelial cells in vivo Progenitor cells adhere and differentiate (or plasmid DNA) A20 gene on the surface of blood vessels can effectively resist various factors to damage blood vessels. The present invention can directly transplant blood vessels into the patient's body through surface modification when acute blood vessel replacement or when the number of endothelial cells taken from the patient is insufficient. Growth factors and adhesion peptides can induce endothelial progenitor cells in the blood and adjacent endothelial cells to replenish the endothelial loss or non-implanted sites of transplanted vessels. The cross-linked A20 gene DNA facilitates the absorption of adherent and differentiated endothelial progenitor cells, and rapidly forms small-diameter blood vessels with anti-atherosclerosis function. This method can also induce the adhesion and differentiation of endothelial progenitor cells in blood to grow into endothelial cells in vivo, without the need for isolation and culture in vitro, which can greatly improve the efficiency of tissue engineering. None of these assumptions or experimental results have been reported yet.

5. Use the above-mentioned good scaffold materials to plant smooth muscle cells, and then plant endothelial seed cells in vitro or induce endothelial progenitor cells in vivo to become living gene-modified tissue engineered blood vessels, which can be widely used in clinical repair of blood vessel damage.

Detailed ways:

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

1. Acquisition of vascular stent materials: take fresh heterogeneous (such as pig blood vessels) or homogeneous blood vessels under sterile conditions, remove the soft tissue attached to them, wash the blood vessels with sterile PBS, remove thrombus clots, put them in sterile packaging at -80°C save. Rewarm at 37°C and treat with 0.1mol/L PMSF solution for 1-3 hours, treat with antibiotic hypotonic and hypertonic buffers for 3-5 hours respectively, change the solution every 1 hour; soak in sterile distilled water, put Maintain 37°C, 5% _CO2 , treat with 0.03%-0.05% trypsin for 16-24 hours, change the medium every 8 hours, soak in sterile distilled water; digest with 0.005%-0.01% RNase 1 --3 hours, soak in sterile distilled water, digest with 0.006%-0.01% DNase for 1-3 hours, soak in sterile distilled water, digest with 0.005%-0.01% lipase for 2-4 hours, Rinse with sterile distilled water. 15-20GY γ-ray radiation treatment for 10-15 minutes to disinfect and remove the antigenicity of remaining vascular matrix collagen and elastin, and finally obtain the vascular stent.

The vascular stent obtained by the above method was detected by morphological methods. It can be seen that the treated vascular cells have been removed, the vascular stromal fibers are looser than before, but the distribution is complete, no fiber breakage is seen, and the content of elastic fibers and collagen fibers has no obvious changes. There was no significant difference in the length and inner diameter of posterior vessels. The pressure-volume experiment was used to detect its compliance and control before treatment. The pressure-volume curve of blood vessels before and after treatment was very close to the ascending branch of the parabola. The arterial PV data was fitted with the quadratic parabolic relationship V=aP ² +bP+c, The correlation coefficients are all greater than 0.95, indicating that the fitting effect is good, and the corresponding calculation method of arterial compliance is C=dv/dp=2ap+b. After the treatment, the compliance of blood vessels decreased, but the difference was not significant (P>0.05). When the pressure was 100mmHg, the compliance before and after treatment was 6.25±0.42, 5.80±0.67 (×10 ^-4 ml/mmHg), the difference was Not significant (P>0.05). Western blot detection showed that pig vascular MHC antigen and xenograft hyperacute rejection α-Gal antigen were strongly expressed before treatment, but no expression was seen after treatment.

2. Vascular surface modification:

2.1. The biodegradable anticoagulant polymer material that controls the release of growth factors, one of the surface modifiers, is essentially the N-sulfonic acid-photocrosslinked chitosan obtained after sulfonation and sulfation of photocrosslinked chitosan. Bi-Chitosan Sulfate. Embodiment is: 1., with the synthesis of 6-O-sulfonated photocrosslinked chitosan (6-O-SM-Az-CH-LA): the photocrosslinked chitosan (Az-CH-LA) of purification ) was dissolved in 2% formic acid solution, then an appropriate amount of 1mol/L CuSO4.5H2O solution was added dropwise at room temperature, stirred for 15--20 hours, filtered and washed with water, acetone and ether in sequence. The resulting product is completely dispersed in dry DMF solution, and then the SO3.Pyridine solution dissolved in DMF is in the ratio of 1:4 (1:4=the molar ratio between chitosan molecular repeating unit and SO3.Pyridine) Add dropwise to the solution at low temperature, stir for 1--3 hours, then heat and stir for 15--hours under nitrogen protection at 50°C. The cooled solution was adjusted to pH 8.2 with NaHCO3, and dialyzed with an appropriate amount of water for 3--5 days. After removing the copper ions by the method of chromatography, the obtained solution was freeze-dried (the yield rate of this step was 80%) and waited until the product. 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 reaction is completed, 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, and freeze-dry to obtain the final product. 15--20Gy gamma-ray radiation treatment for 15--20 minutes for disinfection and standby.

2.2. Preparation of A20 gene plasmid DNA: The cultured endothelial cells were stimulated with tumor necrosis factor TNF for 3-5 hours to stimulate the expression of A20 gene. Extract RNA from cells, design primers upstream: 5'-AGTTGTCCCATTCGTCATTCC-3', downstream: 5'-TTTGAGCAATATGCGGAAAGC-3', use RT-PCR method to clone A20 gene cDNA, with A-terminal DNA cloning vector pUCm-T Vector (No. BS434 (purchased from Shanghai Bioengineering Co., Ltd.), A20 gene cDNA was connected to pUCm-T Vector, stained with x-gal, and blue and white spots screened successfully connected colonies, extracted plasmid DNA after amplification, and used EcoR I and BamH I was digested to obtain a large amount of A20 gene cDNA fragments comprising EcoR I and BamH I restriction sites. The green fluorescent protein eukaryotic expression vector pEGFP-N1 (catalog#6085-1 purchased from American Gene Co., Ltd.) includes 4.7kb. This vector includes multiple restriction sites and can be inserted into foreign fragments. This experiment mainly uses vector polycloning The EcoR I site at 631 and the BamH I site at 661bp in the site, use EcoR I and BamH I to double digest the linearized pEGFP-N1 vector, and then use ligase to cut the site containing EcoR I and BamH I The cDNA fragment of the A20 gene was cloned 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.

2.3. Synthesize the surface modification mixture under sterile conditions. Its ratio relationship is N-sulfonic acid-photocrosslinking-chitosan sulfate 70-90% (w/v), Gly-Arg-Gly-Asp (GRGD) 0.3-1%, endothelial growth factor 0.003- 0.008%, A20 gene plasmid DNA is 0.001-0.003%, and the rest is made up with water. Mixed into an aqueous solution, fully mixed and treated, poured into the treated blood vessel cavity, cross-linked on the surface of the blood vessel cavity by ultraviolet light reaction, and vacuum-dried for later use. This modification method can also be used to modify the inside of the blood vessel stent synthesized by biodegradable materials, the surface of the artificial blood vessel lumen and the surface of the blood vessel stent.

After the surface modification of the blood vessel through the above steps, it can be seen through scanning electron microscopy that the inner cavity of the blood vessel is completely covered by the surface modification and adheres evenly.

3. Smooth muscle cell planting. In the self-constructed vascular culture system, the primary cultured smooth muscle cells are expanded and passaged for 3-6 passages, and injected into the vascular lumen at a concentration of 3×10 ⁶ cell/ml to keep the vascular lumen. Cultivate under certain pressure. Another smooth muscle cell seeding method is to microinject the same number of cells into the vascular media at the same interval and culture them in a vascular cell bioreactor.

Morphological detection of smooth muscle cells planted in the above steps: perfuse the vascular media planted with smooth muscle cells with 0.1% silver nitrate solution for 30 seconds, develop color under sunlight for 30 minutes, separate and spread the slices with microscopic instruments, and routinely dehydrate and transparently seal the slides , digital microscope camera photography. At the same time, blood vessels implanted with smooth muscle were stained by HE method. The result of silver staining showed that the smooth muscle cells in the vascular lumen were normal in shape, densely arranged, and distributed along the long axis of the blood vessel, indicating that under the above conditions, the blood vessels were successfully transformed into smooth muscle cells. HE staining showed that the smooth muscle cells in the vascular media were normal in shape, spindle-shaped, densely arranged, and distributed along the long axis of the blood vessels. Demonstrating successful smooth muscle cellization on vascular matrix material

4. Planting of endothelial cells. 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. During the cultivation process, the intraluminal 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 , Construction of genetically modified tissue engineered blood vessels.

The endothelial cells planted in the above steps were detected by light microscope and electron microscope. It can be seen that the endothelial cells in the vascular lumen were normal in shape, densely arranged, and distributed along the long axis of the blood vessel, indicating that under the above conditions, the blood vessels were successfully endothelialized. Planted on vascular stroma. Fluorescence microscopy revealed that endothelial cells seeded on vascular matrix material secrete extracellular matrix. Indicating that endothelial cells can grow normally on the vascular stroma.

The circumferential tension of the blood vessels obtained after the above steps was tested. In the experiment, the constructed transgenic tissue engineering blood vessels were gradually stimulated with increasing force. When the hydrostatic pressure reached 2000mmHg, no blood vessels ruptured, indicating the anti-burst strength of the constructed blood vessels. / Hydrostatic pressure greater than 2000mmHg.

Alternatively, when a sufficient number of human endothelial cells cannot be obtained, the vascular scaffold can be prepared according to the above-mentioned method and the surface is modified, and only smooth muscle cells can be planted in the media. The lumen of the blood vessel mainly induces endothelial progenitor cells and Adjacent to endothelial cell seeding.

The following examples are used to illustrate, but the application of the present invention is not limited to this.

Example 1. A kind of preparation of genetically modified tissue engineering blood vessels, according to the above (1) method to prepare porcine blood vessels or allogeneic blood vessel matrix materials, after performing surface modification according to step (2), planting monkey smooth muscle cells according to (3), and according to step (4) Seed endothelial cells.

The obtained blood vessels showed that the smooth muscle cells were in good shape, and the distribution of vascular endothelial cells was complete along the long axis of the blood vessels. Transplanted into the common carotid artery of monkeys and tested after 12 months. It can be seen that the distribution of vascular endothelial cells is intact, the shape is normal, the shape of vascular smooth muscle cells is normal, distributed along the long axis of the blood vessels, the blood vessels remain unobstructed, and there is no vascular intimal hyperplasia and thrombosis.

Comparisons were made with xenograft vessels (directly from pig to monkey) with allograft vessels. It can be seen that the lumen of the xenograft blood vessels was completely occluded, there was thrombus formation in the blood vessels, the intima of the blood vessel walls was destroyed, no hyperplasia was seen, and a large number of inflammatory cells infiltrated the blood vessel walls. However, after 6 months of allografting, the blood vessels were occluded, and the intima of the vessels showed typical graft atherosclerotic pathological changes, with uniform and diffuse intima thickening, regular lesions, centripetal ring shape, and rich foam in the lesions cells, the inflammatory reaction is very obvious, the fiber formation is insufficient, and the calcification is not obvious. There is a small circular space in the middle of the thickened intima, which is filled with thrombus. . However, the HE staining of blood vessels 1 month after allografting showed that the blood vessels were not occluded, the intimal layer was thickened, light-stained vacuolar cells appeared under the endothelium, the media was significantly hyperplastic, and the smooth muscle cells were arranged in disorder and protruded toward the intima, forming plaques There are obvious calcifications in the media in some parts, and foam-like cells appear around the calcifications. There was inflammatory cell infiltration, and immunohistochemical detection showed that there were CD4-positive cells in varying degrees in the adventitia layer of the blood vessel. Tunnel detection showed that there were basically no apoptotic cells in the intima layer of allograft vessels, but a large number of apoptotic cells could be seen in the media layer and adventitia layer. The thickened part of the intimal layer of blood vessel HE staining was Brdu-positive cells detected by Brdu monoclonal antibody, and the proliferated intimal layer cells were confirmed as α-actin positive cells by α-actin monoclonal antibody detection by immunofluorescence. It shows that the hyperplastic intimal layer is mainly proliferating smooth muscle cells.

However, 12 months after the transplantation of the genetically modified tissue engineered blood vessel constructed by this method, there was basically no thickening of the intimal layer of the blood vessel and obvious smooth muscle cell hyperplasia by HE staining. And inflammatory cell infiltration, no CD4 expression in blood vessels was detected by immunohistochemistry. There were basically no apoptotic cells in blood vessels. After vascular transplantation, HE staining showed no thickening of the intimal layer of the blood vessel, and no red fluorescence was detected in the intimal layer by immunofluorescence using Brdu and α-actin as monoclonal antibodies. It shows that there is no proliferating smooth muscle cells in the intimal layer, which is consistent with the results of HE staining. The above results show that the present invention can transfer or overexpress the A20 gene in the seed cells through the combination of genetic engineering method and tissue engineering, which can resist the damage of the injury factors in the blood of patients with vascular bypass to the endothelium of grafted vessels, resist thrombosis, and inhibit smooth muscle Pathological proliferation of cells, anti-vascular restenosis. At the same time, it can effectively inhibit the allograft injury faced by acute vascular replacement therapy.

Example 2. Preparation of genetically modified tissue engineered blood vessels that can induce endothelialization in vivo. Prepare pig blood vessels or allogeneic blood vessel matrix materials with corresponding diameters according to the above-mentioned step (1). After surface modification according to step (2), press ( 3) Plant rat smooth muscle cells instead of endothelial cells. Transplanted to the common carotid artery of rats, and tested after 6 months, it was found that the blood vessels remained unobstructed, and the scanning electron microscope showed that the blood vessels had been completely endothelialized, and there was no vascular intimal hyperplasia and thrombus formation. In contrast, in the control group without surface modification and gene modification, no endothelial layer was formed, the vascular lumen was narrowed, the intimal hyperplasia, and part of the blood vessels were completely occluded.

Example 3. A kind of preparation of genetically modified tissue engineering blood vessels, according to the implementation of (1) method to prepare porcine blood vessels or the same kind of blood vessel matrix material, after carrying out surface modification according to (2), planting human smooth muscle cells according to (3) and (4), the same kind of Endothelial cells. For dialysis patients, no thrombus formation was seen, while the control group had thrombus formation.

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 surface modification mixture composed of biodegradable anticoagulant polymer materials that control the release of growth factors, adhesion short peptides, growth factor proteins and A20 gene plasmid DNA on the surface of the blood vessel cavity; The proportioning relationship by weight percentage is: 70-90% of biodegradable anticoagulant macromolecule materials that control the release of growth factors, 0.3-1% of short adhesion peptides, 0.003-0.008% of growth factor proteins, and A20 gene plasmid DNA It is 0.001-0.003%, and the balance is deionized water. Fully mixed into an aqueous solution, poured into the treated heterogeneous or homogeneous vascular cavity or the vascular cavity made of artificial synthetic materials, cross-linked on the surface of the vascular cavity by ultraviolet light reaction, and vacuum-dried for later use; (3) Smooth muscle cell planting: After the surface modification of the vascular cavity is completed, smooth muscle cells are inoculated on the vascular media; (4) Planting of endothelial cells: After smooth muscle cells grow well, 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 surface modification mixture composed of biodegradable anticoagulant polymer materials that control the release of growth factors, adhesion short peptides, growth factor proteins and A20 gene plasmid DNA on the surface of the blood vessel cavity; The proportioning relationship by weight percentage is: 70-90% of biodegradable anticoagulant macromolecule materials that control the release of growth factors, 0.3-1% of short adhesion peptides, 0.003-0.008% of growth factor proteins, and A20 gene plasmid DNA It is 0.001-0.003%, and the balance is deionized water. Fully mixed into an aqueous solution, poured into the treated heterogeneous or homogeneous vascular cavity or the vascular cavity made of artificial synthetic materials, cross-linked on the surface of the vascular cavity by ultraviolet light reaction, and vacuum-dried for later use; (3) Implantation of smooth muscle cells: After the surface modification of the vascular lumen is completed, smooth muscle cells are seeded on 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 step 1 of its preparation method can be non-degradable materials such as polymethyl methacrylate, poly Tetrafluoroethylene, or degradable materials such as polyethylene glycol acid, polyglycolic acid, etc., and copolymers of the above materials; the anticoagulant polymer material that can be biodegradable in step 2 to control the release of growth factors is N-sulfonic acid Acid-photocrosslinking-chitosan sulfate; the short adhesion 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: in the preparation method step (1), the blood vessel stent material is obtained in the following manner: Under aseptic conditions, take fresh heterogeneous (such as pig blood vessels) or homologous blood vessels, remove the attached soft tissue, rinse the blood vessels with sterile PBS, remove thrombus clots, and store them in aseptic packaging at -80°C. Rewarm at 37°C and treat with 0.1mol/L PMSF solution for 1-3 hours, treat with antibiotic hypotonic and hypertonic buffers for 3-5 hours respectively, change the solution every 1 hour; soak in sterile distilled water, put Maintain 37°C, 5% _CO2 , treat with 0.03%-0.05% trypsin for 16-24 hours, change the medium every 8 hours, soak in sterile distilled water; digest with 0.005%-0.01% RNase 1 --3 hours, soak in sterile distilled water, digest with 0.006%-0.01% DNase for 1-3 hours, soak in sterile distilled water, digest with 0.005%-0.01% lipase for 2-4 hours, Rinse with sterile distilled water. 15-20GY γ-ray radiation treatment for 10-15 minutes to disinfect and remove the antigenicity of remaining vascular matrix collagen and elastin, and finally obtain the 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 The N-sulfonic acid-photocrosslinking-chitosan sulfate obtained after the chitosan is carried out sulfonated and sulfated, the specific method is: ①, 6-O-sulfonated photocrosslinked chitosan (6-O-SM-Az-CH-LA) synthesis: the purified photocrosslinked chitosan (Az-CH-LA) was dissolved in 2% Formic acid solution, then add an appropriate amount of 1mol/L CuSO4.5H2O solution dropwise at room temperature, stir for 15--20 hours, filter and wash with water, acetone and ether in sequence. The resulting product is completely dispersed in dry DMF solution, and then the SO3.Pyridine solution dissolved in DMF is in the ratio of 1:4 (1:4=the molar ratio between chitosan molecular repeating unit and SO3.Pyridine) Add dropwise to the solution at low temperature, stir for 1--3 hours, then heat and stir for 15--hours under nitrogen protection at 50°C. The cooled solution was adjusted to pH 8.2 with NaHCO3, and dialyzed with an appropriate amount of water for 3--5 days. After removing the copper ions by the method of chromatography, the resulting solution is freeze-dried (the yield of this step is 80%) and waits until the product; 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 reaction is completed, 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, and 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 A20 gene plasmid DNA in step (2) of the preparation method is as follows: Stimulation of cultured endothelial cells with tumor necrosis factor TNF for 3-5 hours can stimulate the expression of A20 gene. Extract RNA from cells, design primers upstream: 5'-AGTTGTCCCATTCGTCATTCC-3', downstream: 5'-TTTGAGCAATATGCGGAAAGC-3', use RT-PCR method to clone A20 gene cDNA, with A-terminal DNA cloning vector pUCm-T Vector (No. BS434), A20 gene cDNA was connected with pUCm-T Vector, successfully connected colonies were screened out, plasmid DNA was extracted after amplification, digested with EcoR I and BamH I, and a large number of enzymes containing EcoR I and BamH I were obtained. The cDNA fragment of the A20 gene at the locus; the green fluorescent protein eukaryotic expression vector pEGFP-N1 (catalog #6085-1) includes 4.7kb. This vector includes multiple restriction sites and can be inserted into foreign fragments. Among them, this experiment mainly uses the vector The EcoR I site at 631 and the BamH I site at 661bp in the multiple cloning site were digested with EcoRI and BamH I to linearize the pEGFP-N1 vector, and then digested with EcoR I and BamH I with ligase The cDNA fragment of the A20 gene at the site was cloned into the green fluorescent protein eukaryotic expression vector pEGFP-N1, positive colonies were screened out, amplified and extracted to obtain the eukaryotic expression vector plasmid 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-built vascular culture system, the primary cultured smooth muscle cells were expanded and passaged for 3-6 passages, injected into the vascular lumen at a concentration of 3×10 ⁶ cell/ml, and cultured while maintaining a certain pulsating pressure in the lumen; Another smooth muscle cell seeding method is to microinject the same number of cells into the vascular media at the same interval and culture them 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 is gradually increased from 0.033 to 0.1ml/s, and the corresponding shear stress on the vessel wall is between 1×10 ^-2 N/m ² and 4×10 ^-2 N/m ² , constructing a genetically modified tissue Engineering blood vessels.