Preparation method of artificial blood vessel

Abstract

The invention relates to a preparation method of an artificial blood vessel. The method comprises the following steps: carrying out common electrostatic spinning on a hydrophobic high-molecular polymer and a hydrophilic high-molecular polymer to obtain an artificial blood vessel skeleton, and introducing a smooth muscle cell gene activation system (Myocd-dCas9 / VPR) into the hydrophilic high-molecular fiber; compared with single hydrophobic polymer fiber, the hydrophilic polymer fiber has better biocompatibility and degradability, raw materials are rich and easy to obtain, the immunogenicity is low, and the biocompatibility of the vascular material can be effectively improved; meanwhile, the Myocd-dCas9 / VPR carrier can be used as a Myocd-dCas9 / VPR carrier, so that the activity and the stability of the Myocd-dCas9 / VPR can be maintained. The artificial blood vessel obtained by the invention has better biocompatibility and smoothness.

Description

Technical Field

[0001] The present invention belongs to the technical field related to biomedical materials, and more specifically, relates to a method for preparing an artificial blood vessel. Background Art

[0002] Vascular embolism is one of the important causes of death from cardiovascular diseases, and its main cause is intimal hyperplasia or atherosclerosis. Vascular embolism causes insufficient blood supply to local tissues or organs, leading to tissue necrosis or organ failure. Vascular transplantation is an effective means of treating severe cardiovascular diseases. Artificial blood vessels are widely used in the reconstruction of vascular access due to their easy availability, excellent mechanics, customizability, and low immune rejection. However, existing artificial blood vessels, especially small-caliber artificial blood vessels with a caliber of less than 6 mm, have insufficient biocompatibility, slow regeneration, and hemodynamic problems after in vivo transplantation, resulting in poor clinical effects after transplantation and easy blockage, leading to treatment failure.

[0003] Physical or chemical modification of artificial blood vessels can improve the in vivo regeneration effect of blood vessels, guide them to regenerate structures similar to natural blood vessels, inhibit the formation of thrombi, and improve the patency of artificial blood vessels. Some researchers have improved the anti-thrombotic ability of blood vessels by loading active substances such as heparin anticoagulants inside the tube wall; others have improved the patency by loading active substances such as low molecular weight hyaluronic acid (HA) and miRNA145 in vascular stents to promote smooth muscle phenotype transformation and inhibit vascular intimal hyperplasia; or the secretion of smooth muscle cell contractile proteins can be promoted through oriented structures. However, a single physical or chemical modification method cannot guarantee the long-term patency of artificial blood vessels. At the same time, the preparation process of using multiple modification methods at the same time is complicated, which limits its later clinical application.

[0004] Therefore, it is of great clinical significance to develop a multifunctional artificial blood vessel with controllable process and long-term regulatory effect. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method that can achieve long-term regulation of artificial vascular tissue in view of the deficiencies in the current technology. In this method, the artificial blood vessel selects hydrophobic polymer micron fibers and hydrophilic polymer micron fibers, and introduces the cell gene activation system Myocd-dCas9 / VPR into the hydrophilic polymer fibers; compared with single hydrophobic polymer fibers, the hydrophilic polymer fibers have better biocompatibility and degradation performance, and the raw materials are abundant and easy to obtain, and the immunogenicity is low, which can effectively improve the biocompatibility of vascular materials; at the same time, it also serves as a carrier of Myocd-dCas9 / VPR as a cell gene activation system to maintain the activity and stability of the carrier. The artificial blood vessel obtained by the present invention has good biocompatibility and patency.

[0006] The technical solution of the present invention comprises the following steps:

[0007] A method for preparing an artificial blood vessel, the method comprising the following steps:

[0008] (1) adding a hydrophobic polymer to an organic solvent and dissolving the organic solvent by stirring to obtain a hydrophobic polymer solution; wherein the solid content of the polymer solution is 5% to 30%;

[0009] (2) adding the natural hydrophilic polymer into water and continuing to stir to obtain a solution; wherein the solid content of the natural hydrophilic polymer solution is 0.5%-30%.

[0010] (3) placing the hydrophobic polymer solution and the natural hydrophilic polymer solution on the syringe pump of the electrospinning machine respectively, and performing electrospinning by co-spinning, and obtaining artificial blood vessel skeletons with different inner diameters by selecting receiving rods of different diameters for receiving; immersing the artificial blood vessel skeleton in an ethanol solution containing a cross-linking agent to complete the cross-linking of the natural hydrophilic polymer material, and the cross-linking time is 0.5 to 24 hours, and then placing the cross-linked blood vessel stent in water to swell for 1 to 48 hours, and then freeze-drying;

[0011] Among them, the injection volume ratio of the hydrophobic polymer solution to the natural hydrophilic polymer solution is 1:0.1 to 1:1.5;

[0012] The cross-linking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): N-hydroxysuccinimide (NHS) = 3:1, with a concentration of 3 to 30 mM.

[0013] The receiving rod has a diameter of 1 to 22 mm, a spinning time of 10 to 120 min, and a tube wall thickness of 100 to 1000 microns;

[0014] The operating parameters of electrospinning are: negative voltage: -20KV ~ -5KV, positive voltage: +5 ~ +20KV, hydrogel solution flow rate 0.05-0.5mm / min, translation speed 100-1000mm / min, receiving speed 100-300r / min, temperature setting 20-45℃, spinning time 5-90min;

[0015] (4) Loading the Myocd-dCas9 / VPR system onto the artificial vascular stent obtained in step (3):

[0016] The two plasmids, sgRNA targeting Myocd and dcas9-VPR for completing gene activation, are collectively referred to as the Myocd-dCas9 / VPR system. The two plasmids are dispersed in a cell culture medium or PBS buffer to obtain a Myocd-dCas9 / VPR dispersion. The freeze-dried artificial vascular stent is then immersed in the dispersion and allowed to swell for 0.5-2 hours to obtain an artificial blood vessel carrying the two plasmids Myocd-dCas9 / VPR.

[0017] Among them, in the Myocd-dCas9 / VPR dispersion, the concentration range of the two plasmids is 0.5 μg / mL to 50 μg / mL; the mass ratio of the two plasmids is 1:0.5 to 2;

[0018] The high molecular polymer is one or more of polycaprolactone, polycaprolactone lactide, polyurethane, segmented polyurethane, and polylactic acid;

[0019] The natural hydrophilic polymer compound includes natural polymer materials such as polysaccharide polymers and their derivatives (hyaluronic acid, chitosan, dextran, cellular cellulose), polypeptide polymers and their derivatives (gelatin, collagen, chondroitin sulfate, heparin), one or more of them;

[0020] The organic solvent is one or more of hexafluoroisopropanol, acetone, chloroform, dichloroethanol and methanol.

[0021] The sequence of the guide RNA (sgRNA) for activating the smooth muscle cell Myocardin (Myocd) gene is one of pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9337], pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9344], and pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9345].

[0022] The artificial blood vessel prepared by the method is used as an artificial blood vessel for treating coronary artery stenosis and obstruction, uremia, and peripheral arterial disease (such as lower limb ischemia).

[0023] The essential features of the present invention are:

[0024] The soluble polymer materials in the hydrogel microfilaments in the artificial vascular scaffold are cross-linked through chemical bonds to form a stable hydrogel macromolecular structure. The hydrogel microfilaments lose water during the freeze-drying process. By immersing them in the Myocd-dCas9 / VPR dispersion, they reabsorb the dispersion and swell, thereby fixing the plasmid in the pores between the microfilaments, thereby achieving local delivery of the plasmid complex to the cells on the spinning tube.

[0025] The beneficial effects of the present invention are:

[0026] Different from conventional in situ inducible artificial blood vessels, the present invention activates the gene expressing contractile proteins inside smooth muscle cells through the Myocd-dCas9 / VPR system, making them contractile phenotypes, inhibiting the intimal hyperplasia of blood vessels, and improving the patency of blood vessels. The multi-hole structure is made by freeze-drying of hydrogel electrospinning and promoting its adsorption and activity retention of the gene editing system. This technical solution is not only suitable for artificial blood vessel research, but can also be extended to the regeneration and repair of other tissues, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the preparation process of the artificial blood vessel in this patent.

[0028] Figure 2 This is a photo of an artificial blood vessel with a gene editing system prepared in Example 1 of the present invention;

[0029] Figure 3 The internal microscopic structure of the blood vessel obtained in Example 1;

[0030] Figure 4 The internal microscopic structure of the blood vessel obtained for Example 2;

[0031] Figure 5 The internal microscopic structure of the blood vessel obtained for Example 3;

[0032] Figure 6 The internal microscopic structure of the blood vessel obtained for Example 4;

[0033] Figure 7 The internal microscopic structure of the blood vessel obtained for Example 5;

[0034] Figure 8 The internal microscopic structure of the blood vessel obtained for Example 6;

[0035] Fig. 9 The internal microscopic structure of the blood vessel obtained for Example 7;

[0036] Fig.10 The internal microscopic structure of the blood vessel obtained for Example 8;

[0037] Fig.11 This is a diagram showing the gene editing results of smooth muscle cells during in vitro cell culture of the artificial blood vessels composed of Example 1 and Comparative Example a; the successfully edited cells showed the labeled EGFP green fluorescence, and the expression of the contractile protein MYH was found to be enhanced.

[0038] Fig.12This is a diagram showing the results of transplanting the artificial blood vessel prepared in Example 5 into a rat; the blood vessel has good patency, and the endothelium is smooth and free of thrombus.

[0039] Fig.13 The fluorescence image of MYH (contractile smooth muscle marker) of the artificial blood vessel prepared in Example 6 of the present invention after 12 weeks of in vivo transplantation shows an obvious strong fluorescence signal. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with embodiments.

[0041] Embodiment 1:

[0042] 1) Weigh 1 g of polycaprolactone (PCL) particles and dissolve them in 10 ml of a chloroform-methanol (volume ratio 9:1) mixed solvent. Stir magnetically at room temperature overnight to fully dissolve them, and obtain a PCL solution with a mass concentration of 10%.

[0043] 2) Weigh 1.15 g of gelatin (Gel) particles and 0.1 g of hyaluronic acid (HA) powder and dissolve them in 5 ml of a trifluoroethanol / water mixed solvent (volume ratio 3:2), stir them magnetically at 60° C. for 6 hours to fully dissolve them, and obtain a natural hydrophilic polymer solution with a mass concentration of 25%.

[0044] 3) The above two solutions were sucked into a 5ml syringe respectively, the syringe was fixed on the push pump, a 21G needle was connected, and the electrospinning parameters were adjusted for co-spinning. The spinning rod with an inner diameter of 2mm was used for external reception. The specific parameters were: negative voltage was -5KV, positive voltage was +15KV, hydrogel solution flow rate was 0.1mm / min, PCL solution flow rate was 0.3mm / min, translation speed was 500mm / min, receiving speed was 140r / min, temperature was set to 25℃, and spinning time was 40min.

[0045] 4) After spinning for 40 minutes, the electrospinning rod was separated from the outer spinning tube to obtain a tubular spinning bracket, which was then immersed in a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): N-hydroxysuccinimide (NHS) = 18mM: 6mM solution for cross-linking for 12 hours. After cross-linking, the bracket was sterilized by soaking in 75% alcohol, and then the residual solution was washed away with distilled water. The bracket was then placed in water to swell for 3 hours, and then freeze-dried at -80°C for 2-3 days to obtain a freeze-dried vascular stent.

[0046] 5) Extract two plasmids, sgRNA (pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9337]) and dcas9-VPR (pRP[Exp]-EGFP-CMV>dCas9 / VPR) from glycerol bacteria: First, prepare 1.25g LB medium powder for liquid culture and dissolve it in 50ml distilled water. Glycerol bacteria containing plasmids are cultured in LB medium overnight, and the inoculation concentration is usually 1×10 6 to 1×10 8 CFU / mL, the temperature of the culture bacteria is 37℃, the speed is 250rpm, and the culture time is 14-16h. After the solution is obviously turbid, centrifuge the glycerol bacteria and add 250μl of alkaline lysis buffer solution (containing NaOH and SDS, which can lyse cells and release intracellular DNA, NaOH: 0.2-0.3M, used to break the cell wall and melt DNA to release it from the cell. SDS (sodium dodecyl sulfate): 0.5-1%, used to destroy the cell membrane and release the components in the cell) to lyse the cells. Gently invert to mix and incubate at room temperature for 2-5 minutes. Add 350μl of neutralization buffer (potassium acetate (KAc): 3-5M (usually potassium acetate buffer) to neutralize the alkaline solution and promote the precipitation of proteins and membrane components.) Neutralize the lysate, gently invert to mix, and place on ice for 5 minutes to allow the cell residues to precipitate. Then centrifuge to remove the fragments, precipitate the plasmid DNA with an equal volume of 70% isopropanol, wash the plasmid precipitate with 70% ethanol, mix gently, and centrifuge again. Discard the ethanol and dry the precipitate at room temperature for 1-2 minutes. Dissolve the plasmid DNA with 1 ml of deionized water or TE buffer (pH 8.0). Finally, use a spectrophotometer (NanodropOneC) to measure the DNA concentration of 50-200 ng / μl and the purity (260 / 280 ratio) A260 / A280=1.8-2.0. Use the above method to extract the two plasmids, sgRNA and dcas9-VPR, respectively, and analyze the concentration of the plasmids.

[0047] 6) The concentration of both plasmid sgRNA and dcas9-VPR was dispersed into 20ul / ml DMEM cell culture medium containing Lip2000 at a concentration of 8μg / ml, and a 1cm long freeze-dried vascular stent was immersed in 2ml of the above-obtained plasmid dispersion to allow it to swell fully for 1 hour for use. The obtained artificial blood vessels were evaluated in vitro and in vivo to verify their effectiveness. The electron microscope image of the artificial blood vessel was taken using a cold field scanning electron microscope (model: German ZEISS GeminiSEM 300), and it was found that the material fibers were thick and thin and evenly distributed.

[0048] Figure 2This is a photo of an artificial blood vessel with a gene editing system prepared in Example 1 of the present invention; an electrospun blood vessel is obtained by blending 10% PCL solution with 25% gelatin and hyaluronic acid. Figure 2 It can be seen that an artificial blood vessel with a corresponding inner diameter can be obtained by using a spinning rod with a diameter of 2 mm, which means that the inner diameter of the artificial blood vessel can be adjusted by changing the diameter of the receiving rod. Figure 3 This is a SEM picture of the artificial blood vessel prepared in Example 1. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0049] Embodiment 2:

[0050] 1) Weigh 1.5 g of polycaprolactone lactide (PLCL) particles and dissolve them in 10 ml of hexafluoroisopropanol (HFIP) solvent, and stir them magnetically overnight at room temperature to fully dissolve them, to obtain a 15% PLCL solution.

[0051] 2) Weigh 1.15 g of gelatin (Gel) particles and 0.1 g of hyaluronic acid (HA) powder and dissolve them in 5 ml of a trifluoroethanol / water mixed solvent (volume ratio 3:2), and stir them magnetically at 60° C. for 6 hours to fully dissolve them.

[0052] 3) The above two solutions were sucked into a 5ml syringe respectively, the syringe was fixed on the push pump, a 21G needle was connected, and the electrospinning parameters were adjusted for co-spinning. The spinning rod with an inner diameter of 5mm was used for receiving. The specific parameters were: voltage of +15KV, flow rate of hydrogel solution of 0.1mm / min, flow rate of PCL solution of 0.3mm / min, translation speed of 500mm / min, receiving speed of 140r / min, temperature setting of 25℃, and spinning time of 50min.

[0053] 4)-7) Same as Example 1

[0054] Figure 4 This is a SEM picture of the artificial blood vessel prepared in Example 2. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0055] Embodiment 3:

[0056] 1) Step 1) is the same as in Example 1.

[0057] 2) Weigh 1.15 g of Gel particles and 0.1 g of HA powder and dissolve them in 5 ml of a trifluoroethanol / water mixed solvent (volume ratio 3:2), and stir them magnetically at 60° C. for 6 hours to fully dissolve them.

[0058] 3) The above two solutions were sucked into a 5ml syringe respectively, the syringe was fixed on the push pump, a 21G needle was connected, and the electrospinning parameters were adjusted for co-spinning. The spinning rod with an inner diameter of 1mm was used for receiving. The specific parameters were: negative voltage: -8KV, positive voltage: +10KV, hydrogel solution flow rate: 0.1mm / min, PCL solution flow rate: 0.3mm / min, translation speed: 500mm / min, receiving speed: 140r / min, temperature setting: 25℃, spinning time: 15min

[0059] Steps 4)-7) are the same as in Example 2.

[0060] Figure 5 This is the internal structure of the electrospun blood vessel in Example 3. From this figure, it can be seen that the blood vessel is spun with uniform thickness, and PCL and hydrogel are evenly distributed in the artificial blood vessel wall. The artificial blood vessel can be stably cross-linked for 12 hours.

[0061] Embodiment 4:

[0062] 1)-2) Same as Example 3

[0063] 3) The above two solutions were sucked into a 5ml syringe respectively, the syringe was fixed on the push pump, a 21G needle was connected, and the electrospinning parameters were adjusted for co-spinning. The spinning rod with an inner diameter of 12mm was used for receiving. The specific parameters were: voltage of +15KV, flow rate of hydrogel solution of 0.1mm / min, flow rate of PCL solution of 0.3mm / min, translation speed of 500mm / min, receiving speed of 140r / min, temperature setting of 25℃, and spinning time of 90min.

[0064] 4) The electrospun stent was removed and immersed in a 1% EDC solution for cross-linking. After cross-linking, it was immersed in 75% alcohol for sterilization. The residual solution was then washed away with distilled water and freeze-dried to obtain a freeze-dried vascular stent.

[0065] 5) Extract two plasmids, sgRNA (pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9344]) and dcas9-VPR (pRP[Exp]-EGFP-CMV>dCas9 / VPR) from glycerol bacteria;

[0066] 6) The plasmid solution was dispersed into a cell culture medium containing Lip2000 at a concentration of 50 ul / ml, wherein the concentrations of the plasmid sgRNA and dcas9-VPR were both 10 μg / ml, and a 1 cm long freeze-dried vascular stent was immersed in 2 ml of the culture medium dispersion to allow it to fully swell for 1 hour for use.

[0067] 7) Same as Example 3

[0068] Figure 6 This is a SEM picture of the artificial blood vessel prepared in Example 4. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0069] Embodiment 5:

[0070] The other steps are the same as those in Example 1, except that the amount of polycaprolactone (PCL) polymer is 1.2 g; the masses of gelatin and hyaluronic acid are 1.5 g gelatin (Gel) particles and 0.2 g hyaluronic acid (HA), respectively.

[0071] Figure 7 This is a SEM picture of the artificial blood vessel prepared in Example 5. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0072] Embodiment 6:

[0073] The other steps are the same as those in Example 4, except that the poly(vinylidene fluoride) (PVDF) polymer is 1.2 g and the masses of gelatin and hyaluronic acid are 1.5 g gelatin (Gel) particles and 0.15 g dextran, respectively;

[0074] Figure 8 This is a SEM picture of the artificial blood vessel prepared in Example 6. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0075] Embodiment 7:

[0076] The other steps were the same as those in Example 5, except that 1.5 g of PLCL particles were weighed and dissolved in 10 ml of hexafluoroisopropanol (HFIP) solvent, and magnetic stirring was performed at room temperature overnight to fully dissolve the particles, thereby obtaining a 15% PLCL solution.

[0077] Fig. 9 This is a SEM picture of the artificial blood vessel prepared in Example 7. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0078] Embodiment 8:

[0079] Steps 1)-4) are the same as in Example 1

[0080] 5) Extract two plasmids, sgRNA (pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9345]) and dcas9-VPR (pRP[Exp]-EGFP-CMV>dCas9 / VPR) from glycerol bacteria.

[0081] Steps 6)-7) are the same as in Example 1.

[0082] Fig.10 This is a SEM picture of the artificial blood vessel prepared in Example 8. It can be seen from the SEM picture that the spun fibers of the hydrophilic polymer are thinner, while the spun fibers of the synthetic polymer material are relatively thicker, and the two types of fibers are evenly mixed together.

[0083] Fig.11 The results of Myocd gene activation on smooth muscle cells by the artificial blood vessels of the present invention in the process of in vitro cell culture are shown in Figure 2. Rat smooth muscle cells were cultured at a rate of 1×10 5 / well density was inoculated into a 24-well plate, and 100 μl of transfection mixture was added, which contained 0.8 μg of sgRNA plasmid and dcas9 / VPR plasmid, 5 μl of lip2000 reagent, and immunofluorescence staining was performed after 24 hours of culture. First, the cells were incubated with the primary antibody and then fluorescently labeled with the secondary antibody (the primary antibody used was mouse monoclonal MYH, model Servicebio GB121220, with a dilution ratio of 1:500; the secondary antibody was goat anti-mouse abcamAB150116, diluted at a ratio of 1:200), and photographed by laser scanning confocal microscopy (LSCM) Zeiss LSM710, Germany).

[0084] The results of in vitro cell culture show that the cultured cells produced GFP fluorescence, indicating that Myocd-dCas9 / VPR successfully activated the Mycod gene in smooth muscle cells; at the same time, the staining results of contractile proteins found positive expression of MYH, indicating that the secretion of contractile protein MYH was promoted.

[0085] Example 9

[0086] In this embodiment, artificial blood vessel 1 was transplanted. The artificial blood vessel in Example 1 was transplanted into the abdominal aorta of 12-week-old SD rats weighing about 300g by end-to-end anastomosis. B-ultrasound images of the artificial blood vessel were obtained by Doppler ultrasound at 2 weeks, 4 weeks, and 8 weeks, respectively, to obtain data on the patency of the blood vessel. The test results show that the patency rate of artificial blood vessel 1 was 100% at three time points. The samples were collected at 2 weeks, 4 weeks, and 12 weeks, respectively. After the rats were anesthetized with isoflurane, 0.9ml of sodium heparin solution (50 units / ml of normal saline) was injected through the tail vein for anticoagulation, and after the heart was perfused with 50mL of normal saline, the artificial blood vessel was cut off from the natural blood vessel side connected at both ends. After the artificial blood vessel was collected, it was divided into two sections from the middle, one of which was cut open by longitudinal section, and the artificial blood vessel was photographed by a camera. Fig.12 The artificial blood vessel prepared in the embodiment of the present invention is photographed after in vivo transplantation. The smooth intima without thrombus shows good vascular patency. The artificial blood vessel is placed in a centrifuge tube, OCT embedding agent is added, and the vascular tissue is fixed with liquid nitrogen. The Leica cryostat (Leika 1950) is used to cut the tissue into slices with a thickness of 6 microns, and stained by immunofluorescence. After the cells are incubated with the primary antibody, they are fluorescently labeled with the secondary antibody (the primary antibody used is mouse monoclonal MYH, model ServicebioGB121220, with a dilution ratio of 1:500; the secondary antibody is sheep anti-mouse AbcamAB150116, diluted at a ratio of 1:200), and the tissue slices are photographed by laser scanning confocal microscopy (LSCM) (Zeiss LSM710, Germany) after sealing.

[0087] Fig.13 The fluorescent expression of MYH (contractile smooth muscle marker) after 12 weeks of in vivo transplantation of the artificial blood vessels prepared in the embodiment of the present invention shows an obvious strong fluorescent signal, which shows that the gene expressing the contractile protein is activated and the smooth muscle cells are transformed into a contractile phenotype. The test is tested by immunofluorescence staining, and then the pictures are taken by fluorescence microscopy. The picture shows that the artificial blood vessels play an editing role in vivo and can still play a regulatory role after three months.

[0088] Comparative Example 1

[0089] This comparative example prepares an artificial blood vessel a, which is different from Example 1 in that the Myocd-dCas9 / VPR system is introduced. The specific process is as follows:

[0090] 1) Weigh 1 g of polycaprolactone (PCL) particles and dissolve them in 10 ml of hexafluoroisopropanol (chloroform-methanol (volume ratio 9:1) mixed solvent), and stir them magnetically at room temperature overnight to fully dissolve them, to obtain a 10% PCL solution.

[0091] 2) Weigh 1.15 g of gelatin (Gel) particles and 0.1 g of hyaluronic acid (HA) powder and dissolve them in 5 ml of a trifluoroethanol / water mixed solvent (volume ratio 3:2), and stir them magnetically at 60° C. for 6 hours to fully dissolve them.

[0092] 3) The above two solutions were sucked into a 5ml syringe respectively, the syringe was fixed on the push pump, a 21G needle was connected, and the electrospinning parameters were adjusted for co-spinning. The spinning rod with an inner diameter of 2mm was used for external reception. The specific parameters were: voltage of +15KV, flow rate of hydrogel solution of 0.1mm / min, flow rate of PCL solution of 0.3mm / min, translation speed of 500mm / min, receiving speed of 140r / min, temperature setting of 25℃, and spinning time of 40min.

[0093] 4) After spinning for 40 minutes, the electrospinning rod was separated from the outer spinning tube to obtain a tubular spinning scaffold, which was then immersed in a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): N-hydroxysuccinimide (NHS) = 18mM: 6mM solution for cross-linking for 12 hours. After cross-linking, the scaffold was sterilized by soaking in 75% alcohol, and then the residual solution was washed away with distilled water. The scaffold was freeze-dried at -80°C for 2-3 days to obtain a freeze-dried vascular scaffold. The blood vessel was immersed in saline or PBS to swell before transplantation.

[0094] The technical features of the above-mentioned embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described.

[0095] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0096] Matters not covered by the present invention are known technologies.

Claims

1. A method for preparing an artificial blood vessel, characterized in that: The method comprises the following steps: (1) adding a hydrophobic polymer to an organic solvent and dissolving the organic solvent by stirring to obtain a hydrophobic polymer solution; wherein the solid content of the hydrophobic polymer solution is 5% to 30%; (2) adding the natural hydrophilic polymer into water and continuing to stir to obtain a solution; wherein the solid content of the natural hydrophilic polymer solution is 0.5%-30%. (3) placing the hydrophobic polymer solution and the natural hydrophilic polymer solution on the syringe pump of the electrospinning machine respectively, and performing electrospinning by co-spinning, and obtaining artificial blood vessel skeletons with different inner diameters by selecting receiving rods of different diameters for receiving; immersing the artificial blood vessel skeleton in an ethanol solution containing a cross-linking agent to complete the cross-linking of the natural hydrophilic polymer material, and the cross-linking time is 0.5 to 24 hours, and then placing the cross-linked blood vessel stent in water to swell for 1 to 48 hours, and then freeze-drying; The injection volume ratio of the high molecular polymer solution to the natural hydrophilic polymer solution is 1:0.1 to 1:1.5; The receiving rod has a diameter of 1 to 22 mm, a spinning time of 10 to 120 min, and a tube wall thickness of 100 to 1000 microns; The operating parameters of electrospinning are: negative voltage: -20KV ~ -5KV, positive voltage: +5 ~ +20KV, hydrogel solution flow rate 0.05-0.5mm / min, translation speed 100-1000mm / min, receiving speed 100-300r / min, temperature setting 20-45℃, spinning time 5-90min; (4) Loading the Myocd-dCas9 / VPR system onto the artificial vascular stent obtained in step (3): The two plasmids, sgRNA targeting Myocd and dcas9-VPR for completing gene activation, are collectively referred to as the Myocd-dCas9 / VPR system. The two plasmids are dispersed in a cell culture medium or PBS buffer to obtain a Myocd-dCas9 / VPR dispersion. The freeze-dried artificial vascular stent is then immersed in the dispersion and allowed to swell for 0.5-2 hours to obtain an artificial blood vessel carrying the two plasmids Myocd-dCas9 / VPR. Among them, in the Myocd-dCas9 / VPR dispersion, the concentration of the two plasmids is 0.5μg / mL~50μg / mL; the mass ratio of the two plasmids is 1:0.5~2.

2. The method for preparing an artificial blood vessel according to claim 1, characterized in that: The high molecular polymer is one or more of polycaprolactone, polycaprolactone lactide, polyurethane, segmented polyurethane and polylactic acid.

3. The method for preparing an artificial blood vessel according to claim 1, characterized in that: The natural hydrophilic polymer compound includes one or more of the natural polymer materials, which are polysaccharide polymers and their derivatives (hyaluronic acid, chitosan, dextran, cellular cellulose), polypeptide polymers and their derivatives (gelatin, collagen, chondroitin sulfate, heparin).

4. The method for preparing an artificial blood vessel according to claim 1, characterized in that: The organic solvent is one or more of hexafluoroisopropanol, acetone, chloroform, dichloroethanol and methanol.

5. The method for preparing an artificial blood vessel according to claim 1, characterized in that: The sequence of the guide RNA (sgRNA) for activating the smooth muscle cell Myocardin (Myocd) gene is One of pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9337], pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9344], pRP[gRNA]-EGFP-U6>rMyocd[gRNA#9345].

6. The use of the artificial blood vessel prepared by the method as claimed in claim 1, characterized in that it is used as an artificial blood vessel for treating coronary artery stenosis and obstruction, uremia, and peripheral artery disease.

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