Application of VEGF-C156S in preparation of medicine for relieving vein graft stenosis

Abstract

The invention discloses application of VEGF-C156S in preparation of a medicine for relieving vein graft stenosis, and relates to the technical field of biology. The invention discloses an application of VEGF-C156S (vascular endothelial growth factor-C 156S) in preparation of a medicine for preventing or treating vein graft stenosis. The gel containing VEGF-C156S is administered, local incubation can be achieved, drug delivery positioning is accurate, better targeting performance is achieved, the treatment effect is good, single-time use is achieved, reuse is not needed, and the gel is easy to prepare and has good application prospects in preparation of drugs for preventing or treating vein graft stenosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to VEGF-C 156S Application in the preparation of drugs for alleviating venous graft stenosis. Background Art

[0002] Coronary artery bypass grafting is an effective terminal treatment for occlusive coronary artery disease. Compared with percutaneous coronary intervention, it has many advantages such as more stability, longer duration and fewer complications. The main sources of grafts for coronary artery bypass grafting are the internal mammary artery, radial artery and saphenous vein. The maintenance of patency after arterial transplantation is significantly better than that of saphenous vein. However, due to the limited number of sources of autologous arteries, saphenous vein is the most mainstream source of grafts today. However, venous grafts are prone to lesions or occlusion after surgery.

[0003] In the current clinical surgical treatment process, the traditional protective measure is to take antiplatelet and lipid-lowering drugs and other systemic drugs after surgery. In order to improve coronary artery bypass surgery, endoscopic venous blood vessel collection, extracorporeal / non-extracorporeal circulation catheters, minimally invasive and robotic surgery have been implemented, and research on extravascular stents has been carried out. However, due to high operating costs and demanding technical requirements, it has not yet been effectively and widely implemented in clinical practice.

[0004] In current research, no clear drug has been found to significantly reduce the occlusion rate of vein grafts. Most studies focus on lipids, inflammation, etc., but the efficacy of related drugs is poor. Since traditional systemic medications often fail to achieve the desired effect, there is an urgent need for an effective, convenient, and safe drug to reduce intimal hyperplasia of the graft vessels and prolong the service life of the vein graft, thereby reducing the probability of secondary surgery and the occurrence of adverse events in patients.

[0005] Chinese patent CN118178381A discloses the use of kaempferol in the preparation of drugs for preventing or treating restenosis of transplanted veins, and specifically relates to the use of kaempferol or its pharmaceutically acceptable salts for preventing and / or treating restenosis of transplanted veins. The invention is verified by in vivo and in vitro experimental methods. Kaempferol has a good preventive / therapeutic effect on restenosis of transplanted veins, and is of great significance for improving the therapeutic effect of coronary artery bypass surgery, prolonging the life of patients after coronary artery bypass surgery, and reducing the risk of secondary revascularization of patients, providing more clinical options for the clinical treatment of restenosis of transplanted veins.

[0006] VEGF-C is not disclosed in the prior art 156S Application in the preparation of drugs for alleviating venous graft stenosis. Summary of the invention

[0007] The purpose of the present invention is to provide VEGF-C156S Application in the preparation of drugs for alleviating venous graft stenosis.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides VEGF-C 156S Application in the preparation of medicines for preventing or treating venous graft stenosis.

[0009] Specifically, the medicine also includes a thickener and an alkaline compound.

[0010] Furthermore, the thickener is selected from one or more of carbomer 940, carbomer 934, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and sodium carboxymethyl cellulose.

[0011] Furthermore, the thickener is Carbomer 934.

[0012] Furthermore, the alkaline compound is selected from one or more of diethylamine, triethylamine, triethanolamine, sodium hydroxide and potassium hydroxide.

[0013] Furthermore, the alkaline compound is triethanolamine.

[0014] According to some embodiments of the present invention, the drug comprises VEGF-C 156S , carbomer 934 and triethanolamine.

[0015] Furthermore, the VEGF-C in the drug 156S The concentration of is 5-20 μg / ml, the concentration of carbomer 934 is 2-15 μg / ml, and the concentration of triethanolamine is 2-15 μg / ml.

[0016] According to some embodiments of the present invention, VEGF-C in the drug 156S The concentrations are 5μg / ml, 10μg / ml, and 20μg / ml.

[0017] According to some embodiments of the present invention, the concentration of Carbomer 934 in the drug is 2 μg / ml, 5 μg / ml, 7 μg / ml, 10 μg / ml, and 15 μg / ml.

[0018] According to some embodiments of the present invention, the concentration of triethanolamine in the drug is 2 μg / ml, 5 μg / ml, 7 μg / ml, 10 μg / ml, or 15 μg / ml.

[0019] Furthermore, the VEGF-C in the drug 156S The concentration of is 10 μg / ml, the concentration of carbomer 934 is 7 μg / ml, and the concentration of triethanolamine is 7 μg / ml.

[0020] According to some embodiments of the present invention, the mass ratio of carbomer 934 to triethanolamine in the drug is 1:1.

[0021] Specifically, the dosage form of the drug is drops, tincture, injection, ointment, powder, emulsion, lyophilized powder injection, gel or aerosol.

[0022] Furthermore, the dosage form of the drug is a gel.

[0023] Specifically, when the drug is a gel, the preparation method of the gel comprises the following steps: (1) Add Carbomer 934 to water and mix well to obtain a fully swollen liquid; (2) adding triethanolamine to the liquid obtained in step (1) to adjust the pH to obtain a swelling liquid 1; (3) VEGF-C 156S Dissolve in water to obtain liquid 2; (4) Mix the swelling liquid 1 and liquid 2 to obtain.

[0024] Furthermore, in step (1), the mixture is mixed by stirring.

[0025] Furthermore, the stirring temperature is 20-30°C, 100-200r / min, and the stirring is 2-3h.

[0026] Furthermore, the stirring temperature is 20°C, 100r / min, and the stirring time is 2-3h.

[0027] Furthermore, in step (2), the pH is 6.5-7.5; furthermore, in step (2), the pH is 7.0.

[0028] Specifically, the drug is administered at the anastomosis site.

[0029] Specifically, the medicine may further include a pharmaceutically acceptable carrier.

[0030] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0031] Specifically, VEGF-C in the drug 156S As active ingredient.

[0032] Furthermore, the drug is used to inhibit vascular stenosis after coronary artery bypass grafting surgery.

[0033] In another aspect, the present invention provides a drug for preventing or treating venous graft stenosis, wherein the drug contains VEGF-C 156S .

[0034] Specifically, the medicine also includes a thickener and an alkaline compound.

[0035] Furthermore, the thickener is selected from one or more of carbomer 940, carbomer 934, hydroxypropyl methylcellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, etc.

[0036] Furthermore, the thickener is Carbomer 934.

[0037] Furthermore, the alkaline compound is selected from one or more of diethylamine, triethylamine, triethanolamine, sodium hydroxide and potassium hydroxide.

[0038] Furthermore, the alkaline compound is triethanolamine.

[0039] Specifically, the dosage form of the drug is drops, tincture, injection, ointment, powder, emulsion, lyophilized powder injection, gel or aerosol.

[0040] Furthermore, the dosage form of the drug is a gel.

[0041] Furthermore, the VEGF-C in the drug 156S The concentration of is 5-20 μg / ml, the concentration of carbomer 934 is 2-15 μg / ml, and the concentration of triethanolamine is 2-15 μg / ml.

[0042] Furthermore, the VEGF-C in the drug 156S The concentration of is 10 μg / ml, the concentration of carbomer 934 is 7 μg / ml, and the concentration of triethanolamine is 7 μg / ml.

[0043] The beneficial effects of the present invention are: 1. VEGF-C 156S It is a protein that only stimulates VEGFR3 but does not bind to VEGFR2. Therefore, it can achieve very good therapeutic effects simply by promoting the growth of local lymphatic vessels.

[0044] 2. Through the method of local incubation on the outer membrane of the vascular anastomosis, better therapeutic effects can be guaranteed with the minimum amount of drugs; and the lymphatic vessels grow from the anastomosis to the vein, so the treatment of the anastomosis is more targeted; the outer membrane is the site where the lymphatic vessels grow, and local incubation and drug administration are precisely positioned, which has better targeting than systemic medication.

[0045] 3. The gel of the present invention is easy to prepare, can be stored in a refrigerator, and has a long shelf life.

[0046] 4. The product prepared by the present invention only needs to be sutured during the operation and applied immediately by the operator after checking the blood flow condition to achieve the therapeutic effect. It is a single use and does not need to be reused. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The growth of lymphatic vessels; in the figure, A is a comparison of the macroscopic fluorescence staining of the control group and the adeno-associated virus VEGFC 156S A shows the lymphatic vessel growth in the two groups 4 weeks after vein transplantation. The yellow signal is the lymphatic vessel network (the green positive signal is CD31, the red positive signal is LYVE-1, and the overlapping part is the yellow signal, i.e., the lymphatic vessel). B is its statistical graph. P value > 0.05 indicates no significant difference, and P value < 0.05 indicates a significant difference.

[0048] Figure 2 is the expression level of VEGF-C protein; in the figure, A is the WB analysis comparing the control group and adeno-associated virus VEGF-C 156S Figure 3 Changes in VEGF-C protein expression levels in the two groups. B is its statistical graph. P value > 0.05 indicates no significant difference, and P value < 0.05 indicates a significant difference.

[0049] Figure 3 This is the pathological staining 4 weeks after vein transplantation. In the figure, A is the control group and adeno-associated virus VEGFC 156S Pathological staining of the group 4 weeks after vein transplantation was performed to analyze the degree of intimal thickening and fibrosis, including HE staining and Masson staining. B is the statistical graph, P value>0.05 means no significant difference, P value<0.05 means significant difference.

[0050] Figure 4 CD11b flow cytometry analysis; A shows CD11b flow cytometry analysis, comparing the control group and adeno-associated virus VEGFC 156S The infiltration of mononuclear macrophages in group A 4 weeks after vein transplantation. B is the statistical graph, P value>0.05 means no significant difference, P value<0.05 means significant difference.

[0051] Figure 5 Figure 2 is a diagram of gel effect and decomposition experiment results; in the figure, A is a diagram of gel preparation effect, and B is the gel state at 0 and 24 hours during the gel decomposition experiment.

[0052] Figure 6 The growth of lymphatic vessels and HE pathological staining 4 weeks after vein transplantation; Figure A is a comparison of the macroscopic fluorescence staining of the control group and carbomer 934 loaded VEGF-C 156S The lymphatic vessel growth of the control group and VEGF-C group 4 weeks after vein transplantation. 156SHE pathological staining of the gel group 4 weeks after vein transplantation was performed to analyze the degree of intimal thickening. C is the AB statistical graph. P value > 0.05 represents no significant difference, and P value < 0.05 represents a significant difference. DETAILED DESCRIPTION

[0053] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0054] The manufacturer and item number of the experimental materials: VEGF-C 156S Purchased from R&D system, catalog number 752-VC-025; Carbomer 934 was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number T47989; Triethanolamine was purchased from Sigma with catalog number V900257.

[0055] Example 1 1 Preparation of gel (7% carbomer gel) (1) Take a beaker, put in 50 ml of deionized water, sprinkle 0.7 g of Carbomer 934 evenly on the water surface, put it on a stirrer at 20°C and 100 r / min, and after 2-3 hours, a fully swollen liquid will be obtained; (2) Gradually add 0.7 g of triethanolamine and measure the pH value. Stop when the pH reaches about 7.0. (3) 1 mg VEGF-C 156S , dissolved in 50 ml of deionized water; (4) The swelling liquid and VEGF-C 156S Mix with deionized water to make the volume 100 ml and store in a refrigerator.

[0056] 2 Experimental methods 1. Establishment of the mouse jugular vein bypass model and intraoperative VEGF-C 156S Administration of drug gel (1) All experiments used 8-week-old wild-type C57BL / 6 mice purchased from Zhejiang Weitong Lihua Company and subsequently raised at Zhejiang University Medical Center. All procedures were performed in accordance with the animal experiment protocol. Mice were fasted from food and water for 8 hours before surgery. After anesthesia, the donor mice were heparinized and the inferior vena cava (about 1 cm) of the mice was placed in normal saline; after anesthesia, the recipient mice were freed from the distal end of the right common carotid artery to the proximal end, cut in the middle, and a cuff tube was placed at the proximal and distal ends and fixed with an artery clamp. After the arteries on both sides were flipped over and ligated above the cuff tube, the two ends of the vein were placed on the artery cuff tube and ligated together with 8-0 sutures. The artery clamp was loosened to confirm that the venous bridge had no bleeding or twisting.

[0057] (2) Take about 50-100 μL of VEGF-C 156S The drug gel was applied to the surface of the adventitial anastomosis of the mouse arteriovenous graft and incubated for about 2-5 minutes. After the gel adhered firmly and the venous graft pulsated normally, the skin was sutured continuously with 6-0 surgical sutures. During the operation, the animal was kept warm under a heating lamp until it fully recovered, and 1.5 ml of saline solution was injected subcutaneously for fluid replacement at the end.

[0058] 2. Determination of gel decomposition Place 1 ml of gel in a glass vial and place it in a 37°C oven. Take it out and observe it after a certain period of time (in hours). The end point is when the gel completely disappears and becomes water-like. Record the time when the gel is completely decomposed. The result is as follows: Figure 5 shown.

[0059] 3. Western Blot Experiment The mice were killed and the chest was immediately opened. The right atrial appendage was cut, and the perfusion needle was inserted from the apex to the root of the aortic arch and perfused with saline until the liver turned white. The perfusion was completed. The transplanted vein tissue was removed and the water was completely absorbed. It was soaked in 100 μL of RIPA lysis buffer (Radio Immunoprecipitation Assay) and carefully cut with microscissors. After standing and lysing at 4°C for 30 minutes, it was centrifuged at 12000g for 10 minutes, and the supernatant of the tissue lysate was aspirated for BCA protein quantitative detection (BCA protein concentration determination kit (enhanced), Biyuntian, P0009). Finally, quantification was performed and 5x loading buffer was added to denature the protein (Fude Bio, FD006). Western Blot detection was then performed. The same mass of total protein lysate was loaded for each sample, and the gel was run on SDS-polyacrylamide gel (Yazyme Bio) and transferred to a 0.2μm PVDF membrane (Millipore) using a semi-dry transfer device. The PVDF membrane was blocked in 5% milk at room temperature for 1 h, incubated with primary antibodies (VEGF-C, Santa Cruz Biotechnology, sc-374628, 1:1000; β-ACTIN-HRP, Huabio, ET1702-67, 1:2000) overnight on a shaker at 4°C, washed with TBST for 5 min 3 times, treated with the corresponding secondary antibodies at room temperature for 60 min, and detected after washing with TBST for 5 min 3 times. Enhanced chemiluminescence (ECL) was used for detection, and images were captured using a biospectral imaging system (Solarbio).

[0060] 4. Tissue digestion and flow cytometry Prepare the following solution: ① Papain mixture (stock solution 40 mg / ml, based on papain): papain 40 mg / ml, dithiothreitol 20 mg / ml, bovine serum albumin 10 mg / ml, taurine 6.25 mg / ml, dissolved in PBS and stored at -80°C. Dilute the working solution to 2 mg / ml (based on papain) with PBS and place on ice.

[0061] ② Collagenase I 2mg / ml and dispase II 1mg / ml, dissolved in HBSS balanced solution or Hanks balanced solution.

[0062] Specific process: Four weeks after the vein transplantation surgery, the mice were quickly killed, the chest cavity was opened, the right atrial appendage was cut, the perfusion needle was inserted from the apex to the root of the aortic arch, and saline was used for perfusion until the liver turned white. The samples were collected and placed in 10% FBS+DMEM and temporarily stored on ice. After all the mice were taken, the tissues were placed in PBS for a little rinse, and after absorbing the water, they were placed at the bottom of a 1.5ml EP tube and quickly cut with microscissors. Add an appropriate amount of 2mg / ml of ① working solution, and shake at 37℃ for 15min. After 15min, it can be observed under the microscope that some cells have been dissociated from the surrounding area. At this time, the enzyme solution was aspirated or the supernatant was aspirated after a slight centrifugation, and the enzyme solution was collected in a 15ml centrifuge tube containing a certain amount of 10% FBS+DMEM culture medium and placed on ice. The remaining tissues were added with new enzyme solution and digested for 15min. After 15min, the microscope was continued to observe, and the enzyme solution was collected after the cells were dissociated, and then new enzyme solution was added, and the above steps were repeated. After repeated digestion with solution ① for several times, the tissue blocks gradually become loose. For the outer membrane or small blood vessels that are difficult to digest with solution ① alone, after collecting the enzyme solution, transfer the remaining tissue blocks to a 15ml centrifuge tube, add solution ① and solution ② at a ratio of 1:1, and digest in a 37℃ water bath with shaking. Every 10-15 minutes, collect the enzyme solution in time according to the tissue digestion situation and replace it with new enzyme until the tissue is completely digested. The liquid in the collection tube is sieved with a 40μm cell sieve, centrifuged after collection (250g, 5min, 4℃), and the supernatant is discarded. Lyse the cells with red blood cell lysis buffer (Solarbio, R1010) for 5 minutes at room temperature, centrifuge (250g, 5min, 4℃), discard the supernatant, resuspend in PBS and perform flow staining, stain with direct labeled flow antibody CD11b-APC (BD Pharmingen™, 553312) at 4℃ for 30 minutes, wash once with PBS and resuspend, stain with live / dead stain (LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit, Invitrogen, L34975, 1:1000) at room temperature for 10 minutes, wash once with PBS and finally resuspend in 0.1% bovine serum albumin solution or Hanks balanced solution. Use Cytoflex LX flow cytometer to remove debris, adhesion and dead cells for subsequent flow detection and analysis.

[0063] 5. Tissue clearing and immunofluorescence staining The collected tissue samples were fixed in 4% paraformaldehyde for 2 hours at room temperature, washed with PBS for 1 hour at room temperature, and then soaked in CUBIC-L reagent (10% N-butyldiethanolamine + 10% Triton X-100, single distilled water as solvent) for 24 hours at room temperature for preliminary transparent operation. After washing with PBS for 1 hour at room temperature, immunofluorescence staining was performed with LYVE-1 (Abcam, ab14917, 1:100) and CD31 (R&D, AF3628, 1:50) antibodies at 4°C for 24 hours. After washing with PBS for 12 hours at room temperature, the corresponding Alexa 555 and 647 channel immunofluorescence secondary antibodies were used for staining for 12 hours at room temperature. After washing with PBS for 12 hours, the sections were sealed with CUBIC-R reagent (45% antipyrine + 30% nicotinamide + 0.5% n-butyldiethanolamine, monodistilled water as the solvent), and gross immunofluorescence scanning was performed using a Leica STELLARIS 8 laser confocal microscope. Finally, image processing, three-dimensional presentation and statistics were performed using Imaris software.

[0064] 6. Frozen Section Preparation The samples were collected and fixed with 4% paraformaldehyde at room temperature for 2 hours, and then dehydrated with sucrose / single distilled water solution gradient (20%, 30%, 40% mass volume ratio) in sequence. After the tissues and organs settled to the bottom of the sucrose solution, they were replaced with a higher concentration of sucrose solution. After dehydration, the samples were taken out, dried and placed in an embedding box. After being immersed in OCT at room temperature for 30 minutes, they were quickly frozen in liquid nitrogen and transferred to a freezing microtome. The 8μm thickness mode was selected to prepare frozen sections.

[0065] 3 Experimental Results 1. VEGFC 156S Gene overexpression promotes the growth of lymphatic vessels on the surface of vein grafts VEGF-C 156S It is a point mutation form of VEGF-C and is a selective agonist for VEGFR-3 only. It is thought to have no effect on the adult vasculature but is able to induce lymphatic regeneration.

[0066] To promote the growth of local lymphatic vessels in the adventitia of transplanted blood vessels, prepare 1x10 12 vg / ml 9 types of adeno-associated virus (AAV) were placed on ice, including non-coding control AAV and encoding VEGFC 156S Adeno-associated virus (AAV) was purchased from Hanheng Biotechnology Co., Ltd., and 20 μl AAV (5 × 10 12vg / ml) and 80μl of 25% Pluronic F-127 (PF-127, purchased from Sigma, 9003-11-6) gel were mixed at 4°C for 30 minutes. During the operation, the gel was applied to the surface of the adventitia anastomosis of the mouse arteriovenous graft, with a dosage of about 100ul, and incubated for about 2-5 minutes. After the gel adhered firmly and the venous graft pulsated normally, the skin was sutured continuously with 6-0 surgical sutures, and the intervention was considered complete.

[0067] Four weeks after the vein transplantation surgery, the samples were collected and fixed with 4% paraformaldehyde for 2 hours at room temperature. After washing with PBS for 1 hour, CUBIC-L was used for preliminary transparentization. After washing with PBS for 1 hour at room temperature, immunofluorescence staining was performed with LYVE-1 (Abcam, ab14917, 1:100) and CD31 (R&D, AF3628, 1:50) antibodies at 4°C for 24 hours. After washing with PBS for 12 hours, the corresponding Alexa 555 and 647 channel immunofluorescence secondary antibodies were used for staining at room temperature for 12 hours. After washing with PBS for 12 hours, the slides were sealed with CUBIC-R, and gross scanning was performed with a Leica STELLARIS 8 laser confocal microscope. Finally, image processing and three-dimensional presentation were performed using Imaris software. The results are shown in the figure. Figure 1 As shown, by overexpression of VEGFC at the genetic level 156S The number and density of lymphatic vessels in the adventitia of the transplanted vein increased significantly.

[0068] Four weeks after the vein transplantation, the blood was removed by cardiac perfusion with normal saline and the VEGFC of the control group and 156S For samples in the overexpression group, total protein was extracted and incubated with primary antibody VEGF-C (Santa Cruz Biotechnology, sc-374628, 1:1000). Image J was used to detect WB and statistically analyze the differences in VEGF-C expression between different groups.

[0069] like Figure 2 As shown, VEGFC was found to be used at the genetic level 156S Adeno-associated virus-transplanted veins are effective, VEGF-C protein levels are significantly increased, and the number and density of lymphatic vessels in the adventitia are significantly promoted.

[0070] 2. Promote the growth of lymphatic vessels on the surface of vein bridges and reduce the degree of vascular intimal stenosis and inflammation Four weeks after the vein transplantation, the blood was removed by cardiac perfusion with normal saline and the VEGFC of the control group and 156SOverexpression group samples. After fixation with 4% paraformaldehyde at room temperature for 2 hours, sucrose / single distilled water solution was used for gradient dehydration (20%, 30%, 40% mass volume ratio) in turn. After waiting for the tissues and organs to settle to the bottom of the sucrose solution, a higher concentration of sucrose solution was replaced. After dehydration, the samples were taken out, dried and placed in an embedding box. After immersion in OCT at room temperature for 30 minutes, they were quickly frozen in liquid nitrogen and transferred to a freezing microtome. The 8μm thickness mode was selected to prepare frozen sections. The sections were stained with HE and Masson, and the section areas were randomly selected to avoid human errors. Image J software was used to perform relevant statistics by counting the area of ​​the proliferative endometrium. The results are shown in Figure 3 As shown, VEGFC was overexpressed using adeno-associated virus 156S It can significantly reduce the degree of vascular intimal stenosis.

[0071] The degree of inflammatory infiltration between different groups was further evaluated. After collecting samples, papain, collagenase I and dispase II were used for tissue digestion. After filtering with a 40μm filter, the red blood cell lysis solution (Solarbio, R1010) was used for lysis at room temperature for 5 minutes once, and then flow staining was performed. The direct-labeled flow antibody CD11b-APC (BD Pharmingen™, 553312) was used for staining at 4°C for 30 minutes, washed once with PBS and resuspended, and the live / dead stain (LIVE / DEAD™ FixableNear-IR Dead Cell Stain Kit, Invitrogen, L34975, 1:1000) was used for staining at room temperature for 10 minutes. After washing once with PBS, it was finally resuspended in 0.1% bovine serum albumin solution and loaded onto the machine. The Cytoflex LX flow cytometer was used to remove debris, adhesions, and dead cells for subsequent flow detection and analysis. The results are shown in the figure. Figure 4 As shown, adeno-associated virus overexpressed VEGFC 156S The inflammatory infiltration of macrophages in the graft vein was significantly reduced after treatment.

[0072] 3. VEGF-C 156S Protein-loaded gel promotes lymphatic vessel growth and reduces stenosis of grafted veins 3.1 Preparation of gel (2% carbomer gel) (1) Take a beaker, put in 50 ml of deionized water, sprinkle 0.2 g of Carbomer 934 evenly on the water surface, put it on a stirrer at 20°C and 100 r / min, and after 2-3 hours, a fully swollen liquid will be obtained; (2) Gradually add 0.2 g of triethanolamine and measure the pH value. Stop when the pH reaches about 7.0. (3) 0.5 mg VEGF-C 156S, dissolved in 50 ml of deionized water; (4) The swelling liquid and VEGF-C 156S Mix with deionized water to make the volume 100 ml and store in a refrigerator.

[0073] 3.2 Preparation of gel (15% carbomer gel) (1) Take a beaker, put in 50 ml of deionized water, sprinkle 1.5 g of Carbomer 934 evenly on the water surface, put it on a stirrer at 20°C and 100 r / min, and after 2-3 hours, a fully swollen liquid will be obtained; (2) Gradually add 1.5 g of triethanolamine and measure the pH value. Stop when the pH reaches about 7.0. (3) 2 mg VEGF-C 156S , dissolved in 50 ml of deionized water; (4) The swelling liquid and VEGF-C 156S Mix with deionized water to make the volume 100 ml and store in a refrigerator.

[0074] 3.3 Samples were collected four weeks after vein transplantation surgery, fixed, and transparentized for immunofluorescence staining at 4°C using LYVE-1 (Abcam, ab14917, 1:100) and CD31 (R&D, AF3628, 1:50) antibodies, and then stained at room temperature using the corresponding Alexa 555 and 647 channel immunofluorescence secondary antibodies. The samples were then scanned using a Leica STELLARIS 8 laser confocal microscope, and finally image processed and presented in three dimensions using Imaris software. The results are shown in Figure 2. Figure 6 As shown in Figure A, the number and density of lymphatic vessels in the adventitia of the grafted vein were significantly increased by surface application of the protein.

[0075] Similarly, through the pathological sections and HE staining of the tissue, Image J software was used to calculate the area of ​​the hyperplastic intima. The results are as follows: Figure 6 B- Figure 6 C, found that lymphatic vessel growth and intima area were significantly different, proving that VEGF-C 156S The degree of intimal stenosis of the grafted vein in the gel group was significantly reduced. 156S The gel treatment is effective.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. VEGF-C 156S Application in the preparation of medicines for preventing or treating venous graft stenosis.

2. The use according to claim 1, characterized in that: The medicine also includes a thickener and an alkaline compound.

3. The use according to claim 2, characterized in that: The thickener is selected from one or more of carbomer 940, carbomer 934, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and sodium carboxymethyl cellulose.

4. The use according to claim 3, characterized in that: The thickener is Carbomer 934.

5. The use according to claim 2, characterized in that: The alkaline compound is selected from one or more of diethylamine, triethylamine, triethanolamine, sodium hydroxide and potassium hydroxide.

6. The use according to claim 5, characterized in that: The alkaline compound is triethanolamine.

7. The use according to claim 1, characterized in that: VEGF-C in the drug 156S The concentration of is 5-20 μg / ml, the concentration of carbomer 934 is 2-15 μg / ml, and the concentration of triethanolamine is 2-15 μg / ml.

8. The use according to claim 7, characterized in that: VEGF-C in the drug 156S The concentration of is 10 μg / ml, the concentration of carbomer 934 is 7 μg / ml, and the concentration of triethanolamine is 7 μg / ml.

9. The use according to any one of claims 1 to 8, characterized in that: The dosage form of the medicine is drops, tincture, injection, ointment, powder, emulsion, freeze-dried powder injection, gel or aerosol.

10. The use according to claim 9, characterized in that: The dosage form of the medicine is a gel.

11. The use according to claim 10, characterized in that: When the drug is a gel, the preparation method of the gel comprises the following steps: (1) Add Carbomer 934 to water and mix well to obtain a fully swollen liquid; (2) adding triethanolamine to the liquid obtained in step (1) to adjust the pH to obtain a swelling liquid 1; (3) VEGF-C 156S Dissolve in water to obtain liquid 2; (4) Mix the swelling liquid 1 and liquid 2 to obtain.

12. The use according to claim 11, characterized in that: The pH in step (2) is 6.5-7.

5.

13. The use according to claim 1, characterized in that: The application site of the medicine is the anastomosis site.

14. The use according to claim 1, characterized in that: The medicine also includes a pharmaceutically acceptable carrier.

15. A drug for preventing or treating venous graft stenosis, characterized in that: The drug contains VEGF-C 156S .

16. The drug according to claim 15, characterized in that The medicine also includes carbomer 934 and triethanolamine.

17. The drug according to claim 15, characterized in that VEGF-C in the drug 156S The concentration of is 5-20 μg / ml, the concentration of carbomer 934 is 2-15 μg / ml, and the concentration of triethanolamine is 2-15 μg / ml.

Images