Novel in-stent restenosis resistant protein coating stent

Abstract

The invention discloses a novel in-stent restenosis resistant protein coating stent, and particularly discloses a medical stent, the stent comprises: (a) a support structure, the surface of the support structure contains a coating, and the coating contains polydopamine; (b) a CCN5 recombinant protein, and the recombinant protein is fixed on the supporting structure by being combined with polydopamine. The medical stent is used for preventing in-stent restenosis after PCI (percutaneous coronary intervention), and is also used for promoting proliferation and migration of human aortic endothelial cell lines (HAECs), and / or promoting endothelialization to inhibit in-stent restenosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to a novel protein-coated stent for preventing in-stent restenosis. Background Art

[0002] Atherosclerotic coronary heart disease myocardial infarction is a major disease threatening human health globally and ranks first among the causes of death. Percutaneous coronary intervention (PCI) coronary revascularization has become the primary choice for the treatment of coronary heart disease myocardial infarction. After PCI, the absence and damage of vascular endothelium induce abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) to form a neointima, leading to vascular restenosis, which is the main reason for the recurrence of myocardial ischemia in patients after PCI stenting in the long term.

[0003] Currently, for the solutions to in-stent restenosis after PCI, there are the following several solutions: using a drug-eluting stent (DES), with a broad-spectrum cell proliferation inhibitory drug coated on the stent surface, which can release the drug to inhibit the occurrence of vascular restenosis. Commonly used drugs include sirolimus, rapamycin, etc.; stent dilation, by performing balloon angioplasty again to expand the stenotic part within the stent to restore vascular patency; placing a new stent again at the in-stent restenosis site to reopen coronary blood flow.

[0004] Currently, the drugs on the surface of drug-eluting stents have a broad-spectrum inhibitory effect. While inhibiting the proliferation of smooth muscle cells, they also inhibit the repair of vascular endothelium, resulting in poor endothelial repair within the stent in the long term after PCI and increasing the risk of in-stent restenosis in the long term after PCI.

[0005] For patients, balloon dilation or reimplantation of a stent requires them to undergo a second invasive treatment, resulting in increased costs, and it cannot solve the risk of in-stent restenosis occurring again in the long term after the second treatment. The present invention only requires one treatment, reducing the possibility of repeated treatment for patients.

[0006] Therefore, there is an urgent need in this field to develop a new method that can promote endothelial repair while inhibiting smooth muscle proliferation and reduce in-stent restenosis in the long term after PCI. Summary of the Invention

[0007] The object of the present invention is to provide a new method that can promote endothelial repair while inhibiting smooth muscle proliferation and reduce in-stent restenosis in the long term after PCI.

[0008] In the first aspect of the present invention, a medical stent is provided, and the stent includes:

[0009] (a) A support structure, the surface of the support structure contains a coating, and the coating contains polydopamine;

[0010] (b) CCN5 recombinant protein, which is fixed to the support structure by binding to polydopamine.

[0011] In another preferred example, the recombinant protein covalently binds to polydopamine and is fixed to the support structure.

[0012] In another preferred example, the support structure is selected from the group consisting of: coronary artery stents, carotid artery stents, abdominal aortic stents, femoral artery stents, renal artery stents, pulmonary artery stents, cerebral artery stents, or combinations thereof.

[0013] In another preferred example, the quinone group of polydopamine in the coating undergoes a Michael addition reaction with the amino group of the CCN5 recombinant protein under alkaline conditions, thereby coating the CCN5 recombinant protein on the support structure.

[0014] In another preferred example, the sequence of the CCN5 recombinant protein is as shown in SEQ ID NO.1.

[0015] In another preferred example, the loading amount of the CCN5 recombinant protein is 150 - 1000 ng / cm 2 , preferably, 250 - 800 ng / cm 2 , more preferably, 300 - 500 ng / cm 2 .

[0016] In another preferred example, the number of layers of polydopamine coated on the support structure is 1 - 6 layers, preferably, 2 - 5 layers; more preferably, 3 - 4 layers.

[0017] In another preferred example, the weight ratio of polydopamine to CCN5 recombinant protein in the medical stent is 0.1% - 2%, more preferably, 0.2% - 1.5%, most preferably, 0.5% - 1%.

[0018] In another preferred example, the material of the medical stent is selected from the group consisting of: metals, non - metals.

[0019] In another preferred example, the metals include: stainless steel stents, titanium alloy stents, cobalt - chromium alloy stents.

[0020] In another preferred example, the non - metals include: polylactic acid stents, polyhydroxybutyrate stents.

[0021] In another preferred example, the shape of the medical stent is selected from the group consisting of: circular, elliptical, or combinations thereof.

[0022] The second aspect of the present invention provides a method for manufacturing a medical stent, comprising the steps of:

[0023] (a) providing a support structure and a CCN5 recombinant protein, wherein the surface of the support structure contains a coating, and the coating contains polydopamine;

[0024] (b) incubating the support structure and the CCN5 recombinant protein under alkaline conditions, such that the quinone groups of the polydopamine in the coating undergo a Michael addition reaction with the amino groups of the CCN5 recombinant protein under alkaline conditions, thereby coating the CCN5 recombinant protein on the support structure to obtain the medical stent.

[0025] In another preferred embodiment, the alkaline conditions refer to a pH of 7.5 - 9.5, preferably 7.5 - 8.5.

[0026] In another preferred embodiment, the incubation time is 2 - 8 hours, preferably 3 - 6 hours.

[0027] The third aspect of the present invention provides a use of the medical stent according to the first aspect of the present invention for preparing a medical device for preventing in-stent restenosis after endovascular interventional therapy.

[0028] In another preferred embodiment, the medical device is further used for one or more uses selected from the following group:

[0029] (a) promoting the proliferation and migration of human aortic endothelial cell line (HAECs);

[0030] (b) promoting endothelialization to inhibit in-stent restenosis.

[0031] In another preferred embodiment, the endovascular interventional therapy includes PCI, neurovascular stent implantation, and peripheral vascular stent implantation.

[0032] In another preferred embodiment, the peripheral vascular stent implantation is selected from the following group: renal artery stent implantation, lower extremity artery stent implantation, carotid artery stent implantation, or a combination thereof.

[0033] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: The CCN5 recombinant protein was successfully immobilized on the surface of a metal stent or a circular metal sheet. A: It was demonstrated by X-ray Photoelectron Spectroscopy (XPS) that the CCN5 recombinant protein was successfully immobilized on the surface of a metal stent or a circular metal sheet; B: Quartz crystal microbalance showed that the loading amount of the CCN5 recombinant protein on the surface of the stent metal material was approximately 330 ng / cm 2 .

[0035] Figure 2 : Using a simple circular metal sheet, a circular metal sheet with polydopamine (PDA), or a circular metal sheet coated with bovine serum albumin (BSA) as controls, in vitro experiments showed that the circular metal sheet with a CCN5 recombinant protein coating could promote the adhesion, proliferation, and migration of human aortic endothelial cell line (HAECs). A: HAECs were seeded on the surface of each group of circular metal sheets, and the adhered HAECs were observed after 2 hours. The results showed that the CCN5 recombinant protein coating promoted the adhesion of HAECs; B-C: HAECs were seeded on the surface of each group of circular metal sheets, and Ki67 staining (B) and MTT assay (C) confirmed that the CCN5 recombinant protein coating significantly promoted the proliferation of HAECs; D: HAECs were confluently seeded on the surface of each group of circular metal sheets, and the scratch assay showed that the CCN5 recombinant protein coating significantly promoted the migration of HAECs.

[0036] Figure 3 : The CCN5 recombinant protein-coated stent had good safety. A: Scanning electron microscopy showed that the surface of the CCN5 recombinant protein-coated stent (CCN5rp) had good flatness; B: The CCN5 recombinant protein-coated stent and the simple PDA stent (control group) were placed in a purified and uniform red blood cell suspension and gently shaken at room temperature for 1 hour. Then, the absorbance in the supernatant was observed by spectrophotometry. A 0.1% triton solution (which would cause lysis of all red blood cells) was used as the control group to observe the hemolysis rate of red blood cells. The results showed that neither the CCN5 recombinant protein-coated stent nor the simple PDA stent caused hemolysis. C: The CCN5 recombinant protein-coated stent was implanted into the right coronary artery of pigs through percutaneous coronary intervention (PCI). Coronary angiography before surgery, immediately after surgery, and on the seventh day after surgery showed that the CCN5 recombinant protein-coated stent did not cause obvious in-stent thrombosis.

[0037] Figure 4: The CCN5 recombinant protein-coated stent promotes endothelialization and inhibits in-stent restenosis. A bare metal stent (BMS) was used as the control group, and a sirolimus eluting stent (SES) was used as the positive control group. Each group of stents was implanted into the porcine coronary artery. Optical Coherence Tomography (OCT) was performed 7 days after the operation to observe the degree of re-endothelialization in the stent; OCT and coronary angiography were performed 90 days after the operation to observe the degree of in-stent restenosis in vivo, and the porcine coronary artery was collected for sectioning and HE staining to observe and statistically analyze the degree of in-stent restenosis in vitro. A: The OCT results 7 days after PCI showed that the CCN5 recombinant protein-coated stent increased the intimal coverage rate of the stent struts and promoted re-endothelialization in the stent. B: Coronary angiography 90 days after PCI showed that, similar to the sirolimus eluting stent, the CCN5 recombinant protein-coated stent inhibited in-stent vascular stenosis. C: OCT 90 days after PCI showed that, similar to the sirolimus eluting stent, the CCN5 recombinant protein-coated stent inhibited neointima formation in the stent. D: HE staining 90 days after PCI showed that, similar to the sirolimus eluting stent, the CCN5 recombinant protein-coated stent inhibited neointima formation in the stent. The statistical graphs were, from left to right, the neointimal area, the in-stent restenosis rate, the ratio of neointima to media in the blood vessel, and the media area of the blood vessel. Detailed implementation manner

[0038] Through extensive and in-depth research, the present inventor unexpectedly developed for the first time a medical stent comprising a support structure and a CCN5 recombinant protein. The surface of the support structure is provided with a coating containing polydopamine, and the recombinant protein is covalently bonded to the polydopamine and fixed to the support structure. The medical stent of the present invention is used to prevent in-stent restenosis after endovascular interventional therapy, and is also used to promote the proliferation and migration of human aortic endothelial cell line (HAECs), and / or promote endothelialization and inhibit in-stent restenosis. On this basis, the present inventor completed the present invention.

[0039] Terms

[0040] Polydopamine

[0041] Polydopamine is a bioamine compound with a polyphenol structure, having adhesiveness and biocompatibility, and thus is widely used in the biomedical field.

[0042] CCN5 recombinant protein

[0043] CCN5 is an extracellular matrix protein and is one of the members of the extracellular matrix protein family. It is also known as WISP-2 (Wnt1 inducible signaling pathway protein-2) and is involved in various biological processes such as cell proliferation, cell migration, apoptosis, and cell differentiation. The CCN5 recombinant protein is a CCN5 protein that is highly expressed and purified by genetic engineering technology and can be used to study the role of CCN5 protein in cell biology, physiology, and pathophysiology.

[0044] In a preferred embodiment, the sequence of the CCN5 recombinant protein of the present invention is as shown in SEQ ID NO.1.

[0045] MQLCPTPCTCPWPPPRCPLGVPLVLDGCGCCRVCARRLGEPCDQLHVCDASQGLVCQPGAGPGGRGALCLLAEDDSSCEVNGRLYREGETFQPHCSIRCRCEDGGFTCVPLCSEDVRLPSWDCPHPRRVEVLGKCCPEWVCGQGGGLGTQPLPAQGPQFSGLVSSLPPGVPCPEWSTAWGPCSTTCGLGMATRVSNQNRFCRLETQRRLCLSRPCPPSRGRSPQNSAF (SEQ ID NO.1)

[0046] Endovascular interventional therapy

[0047] It is a method of treating from inside the blood vessel through a catheter, including interventional surgery, stent implantation, balloon dilation, etc. This treatment method is usually used for the treatment of various vascular diseases such as coronary artery disease, cerebrovascular disease, and peripheral vascular disease.

[0048] Medical stent and its preparation

[0049] The "medical stent" described in the present invention is a stent that can promote the migration of human aortic endothelial cell line (HAECs) and / or promote endothelialization to inhibit in-stent restenosis.

[0050] In the present invention, the medical stent includes:

[0051] (a) A support structure, the surface of the support structure contains a coating, and the coating contains polydopamine;

[0052] (b) CCN5 recombinant protein, and the recombinant protein is fixed to the support structure by binding to polydopamine.

[0053] In the present invention, the loading amount per unit area of the CCN5 recombinant protein on the support structure determines the therapeutic effect of the medical stent. The loading amount per unit area of the CCN5 recombinant protein on the support structure is not particularly limited, and the typical loading amount is 150 - 1000 ng / cm 2 , preferably 250 - 800 ng / cm 2 , more preferably 300 - 500 ng / cm 2 . A preferred loading amount per unit area is that when the loading amount per unit area of the CCN5 recombinant protein on the support structure is 300 - 500 ng / cm 2 , the stent has the best effect in promoting endothelialization and inhibiting in-stent restenosis.

[0054] In the present invention, the medical stent is prepared by the following method:

[0055] (a) Provide a support structure and a CCN5 recombinant protein. The surface of the support structure contains a coating, and the coating contains polydopamine;

[0056] (b) Incubate the support structure and the CCN5 recombinant protein under alkaline conditions, so that the quinone group of the polydopamine in the coating undergoes a Michael addition reaction with the amino group of the CCN5 recombinant protein under alkaline conditions, thereby coating the CCN5 recombinant protein on the support structure to obtain the medical stent.

[0057] The medical stent prepared by the present invention can promote the proliferation and migration of human aortic endothelial cell line (HAECs), and / or promote endothelialization and inhibit in-stent restenosis of the stent, and can be used to prevent in-stent restenosis after endovascular interventional therapy.

[0058] The main advantages of the present invention include:

[0059] (1) The present invention develops for the first time a medical stent including a support structure and a CCN5 recombinant protein. The surface of the support structure contains a coating, and the coating contains polydopamine. The recombinant protein is covalently bonded to the polydopamine and fixed on the support structure. The medical stent of the present invention is used to prevent in-stent restenosis after PCI, and is also used to promote the proliferation and migration of human aortic endothelial cell line (HAECs), and / or promote endothelialization and inhibit in-stent restenosis.

[0060] (2) The present invention inhibits smooth muscle proliferation while promoting endothelial repair, and reduces long-term in-stent restenosis after PCI.

[0061] (3) The present invention provides a CCN5 recombinant protein-coated stent constructed by covalently bonding and fixing human CCN5 recombinant protein to a coronary artery metal stent using a polydopamine coating as a mediator, which is used to prevent in-stent restenosis after PCI.

[0062] (4) Indirectly immobilize human CCN5 recombinant protein onto the surface of a metallic coronary stent through a polydopamine coating to construct a CCN5 recombinant protein-coated stent.

[0063] (5) The protein-coated stent is used to reduce the occurrence of in-stent restenosis after percutaneous coronary intervention (PCI).

[0064] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0065] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention are all commercially available products.

[0066] Example 1 Fabrication method and process of the CCN5rp-coated stent

[0067] Immerse CoCr discs and stents in a solution containing 2 mg / mL dopamine (10 mM Tris buffer, pH 8.5) at 25 °C for 12 hours, take out, ultrasonically treat in PBS for 15 minutes, and then wash with water to remove weakly bound dopamine. Perform 4 consecutive immersion coating steps to form 4 layers of PDA film, and dry the 4 layers of PDA film at 60 °C for 1 hour.

[0068] Sterilize the PDA film-coated CoCr discs and stents by ultraviolet irradiation for 1 hour, and immerse them in a solution containing human CCN5rp (SEQ ID NO.1, purchased from PeproTech, catalog number 120-16) (1 μg / ml, 5% trehalose PBS buffer, pH = 8.0) at 37 °C for 4 hours under sterile conditions to chemically immobilize CCN5rp on the PDA film.

[0069] Example 2 Detection of the loading amount of the CCN5rp-coated stent

[0070] Use an X-ray photoelectron spectrometer (XPS, Thermo Scientific K-Alpha, USA) to determine the chemical composition of the CCN5 coating ( Figure 1 A), and use a quartz crystal microbalance (QCM-D, Q-sense AB, Sweden) to verify the loading density of CCN5rp on the CCN5rp-coated stent material in real time ( Figure 1 B).

[0071] Figure 1From the high-resolution C1s image of A, it can be seen that after CCN5 is immobilized on PDA, compared with the PDA sample, the peak intensities at 287.5 eV and 284.1 eV decrease, indicating that the quinone groups in PDA are consumed during the reaction; the peak intensity at 285.6 eV decreases and shifts to a higher binding energy position, indicating that the aliphatic (aromatic) C-N in PDA is consumed during the reaction; the peak area increases at 284.4 eV, indicating that CCN5 containing a high content of C-H and C-C is successfully attached. Figure 1 From the high-resolution N1s spectrum of A, it can be seen that for the PDA-CCN5 relative to the PDA sample, the peak area of aliphatic C-N / C=N decreases, the peak area of aromatic (aliphatic) C-N increases and the binding energy shifts towards the binding energy, which may be caused by the abundant peptide bonds present in CCN-5; the C-NH3 at 400.10 eV + After CCN-5, it shifts to 401.68 eV, which can be attributed to the transfer of protons from acids (catechol, C-OH, COOH) to amines (C-NH2). Based on the above, it is proved that the CCN5 recombinant protein is successfully loaded on the surface of the metal stent or circular metal sheet (A), and the loading amount corresponding to the plateau period when the mass of the metal crystal no longer changes after washing with PBS is about 330 ng / cm 2 around (as indicated by the red arrow), indicating that the loading amount of the CCN5 recombinant protein is about 330 ng / cm 2 (B).

[0072] Example 3 In vitro evaluation of CCN5 recombinant protein-coated CoCr discs and stents

[0073] Human aortic endothelial cells (HAECs) ( PCS-100-011 TM ) were seeded onto CCN5rp-coated CoCr discs at a density of 1×10 5 cells / well and cultured in endothelial cell medium (Sciencell, Cat No. 1001, USA) for 4 hours, and then washed with PBS to remove non-adherent cells. Phalloidin (1:200, Cat No. 40735ES75, Yeasen, China) and Hoechst (10 μg / ml) were used to stain the adherent cells, and the number of cells was counted in every 5 random fields under a fluorescence microscope to observe the cell adhesion on the membrane ([[]] Figure 2 A).

[0074] Human aortic endothelial cells (HAECs) were seeded onto CCN5rp-coated CoCr discs at a density of 1×10 5 cells / well, and the corresponding spectrophotometric values were measured at 0 h, 24 h, and 48 h respectively by the MTT method to calculate the cell proliferation ability (Figure 2 B).

[0075] Human aortic endothelial cells (HAECs) were seeded onto CCN5rp-coated CoCr discs at a density of 1×10 5 cells / well, cultured in serum-free medium for 24 hours, then stimulated with 10% fetal bovine serum. After 6 hours, cells were fixed with 4% paraformaldehyde and subjected to immunofluorescence ki67 staining. The proportion of endothelial cells entering the proliferative phase was calculated ( Figure 2 C).

[0076] Human aortic endothelial cells (HAECs) stably overexpressing green fluorescent protein were seeded onto CCN5rp-coated CoCr discs at a density of 1×10 6 cells / well. After 24 hours of starvation, the scratch area was observed under a fluorescence microscope at the 24th hour through a scratch assay, and the cell migration ability was calculated ( Figure 2 D).

[0077] Figure 2 The results showed that circular metal sheets with CCN5 recombinant protein coating could promote the adhesion (A), proliferation (B, C), and migration (D) of human aortic endothelial cell line (HAECs).

[0078] Example 4 Surface Smoothness and Biocompatibility Analysis of CCN5rp-Coated CoCr Discs and Stents

[0079] The surface morphologies of the above-mentioned PDA-coated stents and CCN5-coated stents were observed using a scanning electron microscope (SEM, ZEISS GeminiSEM 300, Germany) ( Figure 3 A).

[0080] Furthermore, a quantitative blood hemolysis test was performed to analyze the biocompatibility of the coated stents. Briefly, whole blood was gently mixed with PBS solution at pH 7.4 at a ratio of 1:2, centrifuged at 3000 rpm for 6 minutes at room temperature to obtain red blood cells. The above steps were repeated 5 times to completely remove the serum to obtain a red blood cell suspension. The 10-fold diluted red blood cells were used as a 5% hematocrit solution and placed in a 24-well plate. PDA-coated CoCr discs and CCN5rp-coated CoCr discs were placed in the corresponding small holes of the 24-well plate and incubated on an oscillator (medium speed) at 37°C for 1 hour. Then, they were centrifuged at 6000 rpm for 5 minutes, and the supernatant was collected. The hemoglobin absorbance shown when the excitation light was 540 nm was recorded on an ELISA microplate reader (Tecan Infinite M200). The hemolysis percentage (HP) of each sample was calculated using the following formula: HP (%) = (At - Anc) / (Apc - Anc), where At is the absorbance of the test sample, Apc is the absorbance of the positive control (0.1% Triton), and Anc is the absorbance of the negative control (PBS).Figure 3 B)

[0081] Finally, in a porcine coronary artery model, we performed percutaneous coronary stenting through the porcine femoral artery inlet, and observed the blood flow and thrombosis in the vascular segment corresponding to the implanted stent immediately after stent implantation and on the 7th day after surgery ( Figure 3 C).

[0082] Figure 3 The results showed that the CCN5 recombinant protein-coated stent had good safety, specifically manifested as good surface flatness (A), no hemolysis (B), and no in-stent thrombosis before PCI, immediately after PCI, and on the 7th day after surgery in pigs (C).

[0083] Example 5 verified the effectiveness of the CCN5rp-coated stent through in vivo experiments on porcine coronary arteries

[0084] Five Chinese domestic pigs with a body weight between 30 and 35 kg were selected and provided by Shanghai Jiagan Biotechnology Co., Ltd. (Shanghai, China). They were fed a standard laboratory diet (purchased from Shanghai Jiagan Biotechnology Co., Ltd.) for 7 days before the experiment. The dual antiplatelet therapy regimen for 3 days before surgery and throughout the study period was specifically 100 mg of aspirin per day and 75 mg of clopidogrel per day. Animal feeding and all in vivo experiments were carried out at Shanghai Anzhu Medical Technology Co., Ltd. (Shanghai, China).

[0085] On the day of the experiment, all animals were pre-anesthetized by intramuscular injection of tiletamine, zolazepam (25 mg / kg), and xylazine (2 mg / kg). Mechanical ventilation was established, and anesthesia was maintained by inhaling isoflurane (1 - 2%) during the operation. After local disinfection, femoral artery puncture was performed, and a 6F arterial sheath (Terumo Corporation, Japan) was inserted. 5000 UI of unfractionated heparin was injected intravenously. Three main epicardial coronary arteries were located under digital subtraction angiography (Optima CL323i, GE, USA).

[0086] For each animal, the CCN5rp-coated stent, sirolimus-coated stent, and bare metal CoCr stent were randomly implanted into the left anterior descending artery (LAD), left circumflex artery (LCX), and right coronary artery (RCA). After angiography, quantitative coronary angiography analysis (QCA) was used to measure the coronary artery diameter. A stent was placed in the main trunk of the coronary artery supply area, and the stent-to-vessel ratio was 1.1 - 1.2:1. After catheterization, hemostasis was achieved by manual compression, and the sheath was removed. The animals were allowed to recover from anesthesia.

[0087] Optical coherence tomography (OCT) examination was performed 7 days after surgery to observe the intimal coverage rate on the support structure ( Figure 4 A), and coronary angiography and OCT examination were performed 90 days after surgery to observe and calculate the in-stent neointimal thickening (Figure 4 B-C). At the end of the last step, the animals under deep anesthesia were euthanized with a lethal dose of potassium chloride. The hearts were obtained, and the coronary arteries at least 10 mm distal and proximal to the stent were carefully dissected, fixed, and used for tissue sectioning and HE staining to observe and statistically analyze the severity of neointimal hyperplasia within the stent ( Figure 4 D).

[0088] Figure 4 The results showed that the CCN5 recombinant protein-coated stent promoted endothelialization 7 days after PCI in pigs (A) and inhibited in-stent restenosis 90 days after PCI in pigs (B-D).

[0089] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A medical stent, characterized in that, The stent comprises: (a) A support structure, the surface of which contains a coating, and the coating contains polydopamine; (b) CCN5 recombinant protein, which is fixed to the support structure by binding to polydopamine.

2. The medical stent according to claim 1, wherein The recombinant protein covalently binds to polydopamine and is fixed to the support structure.

3. The medical stent according to claim 2, wherein, The support structure is selected from the group consisting of: coronary artery stent, carotid artery stent, abdominal aortic stent, femoral artery stent, renal artery stent, pulmonary artery stent, cerebral artery stent, or a combination thereof.

4. The medical stent according to claim 1, wherein The quinone group of polydopamine in the coating undergoes a Michael addition reaction with the amino group of CCN5 recombinant protein under alkaline conditions, thereby coating the CCN5 recombinant protein on the support structure.

5. The medical stent according to claim 1, characterized in that, The sequence of the CCN5 recombinant protein is as shown in SEQ ID NO.

1.

6. The medical stent according to claim 1, wherein The loading amount of the CCN5 recombinant protein is 150 - 1000 ng / cm 2 , preferably, 250 - 800 ng / cm 2 , more preferably, 300 - 500 ng / cm 2 .

7. The medical stent according to claim 1, characterized in that, The number of layers of polydopamine coated on the support structure is 1 - 6 layers, preferably 2 - 5 layers; more preferably 3 - 4 layers.

8. The medical stent according to claim 1, characterized in that, The weight ratio of polydopamine to CCN5 recombinant protein in the medical stent is 0.1% - 2%, more preferably 0.2% - 1.5%, and most preferably 0.5% - 1%.

9. A manufacturing method of a medical stent, characterized in that, Comprising the steps of: (a) Providing a support structure and CCN5 recombinant protein, the surface of which contains a coating, and the coating contains polydopamine; (b) Incubating the support structure and CCN5 recombinant protein under alkaline conditions, such that the quinone group of polydopamine in the coating undergoes a Michael addition reaction with the amino group of CCN5 recombinant protein under alkaline conditions, thereby coating the CCN5 recombinant protein on the support structure to obtain the medical stent.

10. Use of the medical stent according to claim 1, characterized in that, For preparing a medical device for preventing in-stent restenosis after endovascular interventional therapy.

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