CN117427227A

CN117427227A - Epsin1-Epsin2-shRNA functional gene coating stent and preparation method thereof

Info

Publication number: CN117427227A
Application number: CN202311389181.7A
Authority: CN
Inventors: 张文旭; 潘鹏宇; 梁国标
Original assignee: General Hospital of Shenyang Military Region
Current assignee: General Hospital of Shenyang Military Region
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2024-01-23

Abstract

The invention discloses an Epsin1-Epsin2-shRNA functional gene coating bracket and a preparation method thereof. Wherein, the Epsin1-Epsin2-shRNA functional gene coating bracket is characterized in that: the novel scaffold comprises a scaffold body and an Epsin1-Epsin2-shRNA functional gene coating, wherein the Epsin1-Epsin2-shRNA functional gene coating is coated on the surface of the scaffold body; the preparation method of the bracket comprises the following steps: immersing the stent body in a PrS solution; taking out the bracket main body, and washing with buffer solution; immersing the stent main body taken out in the second step into an Epsin1-Epsin2-shRNA solution; repeating the washing in the second step; repeating the operations from the first step to the fourth step for twelve times to form a single-layer ion layer. The invention explores the application of the Epsin protein in inhibiting vascular endothelial cell proliferation, and discovers that the Epsin knockdown indirectly inhibits proliferation and migration of smooth muscle cells by promoting endothelialization, reducing endothelial inflammation and mitochondrial oxidative stress.

Description

Epsin1-Epsin2-shRNA functional gene coating stent and preparation method thereof

Technical Field

The invention belongs to the technical field of vascular stents, and particularly provides an Epsin1-Epsin2-shRNA functional gene coating stent.

Background

With the aging of the social population, cerebrovascular diseases are becoming a leading cause of death worldwide. Among these cerebrovascular diseases, ischemic stroke is the highest, with about 15-20% of ischemic strokes being due to carotid atherosclerosis and carotid stenosis. Due to the low risk of injury in intravascular interventions, few surgical complications, interventional procedures have become one of the main approaches to treat such diseases. However, neointimal hyperplasia (IH) can lead to restenosis of the lumen, leading to failure of angioplasty. Without preventive intervention, the incidence of restenosis would reach 30-50% within 6 months after surgery. The occurrence of neointimal hyperplasia thus severely affects the long-term efficacy and safety of carotid stenting. The current drugs for preventing stenosis, such as rapamycin (sirolimus) and paclitaxel, applied to drug-coated stents, all prevent neointimal thickening by inhibiting smooth muscle cell proliferation and migration. However, these drugs do not selectively inhibit proliferation, migration of smooth muscle cells and endothelial cells, and cause long-term damage to endothelial cells. Endothelial cells have reduced proliferative migration capacity and the damaged vessel cannot be endothelialized in time, resulting in a greatly increased risk of late thrombosis and late restenosis.

Endothelial dysfunction and endothelial cell loss are also one of the causes of neointimal hyperplasia. If endothelialization is rapid after stent implantation, neointima will be reduced and the risk of post-stent stenosis and late stent thrombosis will be significantly reduced. In addition, rapid regeneration of the endothelium and protection of endothelial cell function may also indirectly inhibit proliferation and migration of SMC to reduce intimal hyperplasia. Thus, protecting endothelial cells functions plays an important role in preventing restenosis.

Drug eluting stents and drug eluting balloons are currently used to reduce neointimal hyperplasia by inhibiting smooth muscle cell proliferation. However, these antiproliferative agents also inhibit endothelial cell growth, resulting in delayed endothelial healing. Long-term clinical studies of stents or balloons employing these drugs often report cases of vascular Smooth Muscle Cell (SMC) fibrosis and advanced stent thrombosis, which raise concerns about the clinical application prospects of these drug eluting stents and drug eluting balloons.

The family of growth regulatory proteins (Epsin) proteins is known for their role as highly conserved, membrane-associated, ubiquitin-binding endocytic adaptors, exhibiting high specificity in binding protein selection. Under different pathophysiological conditions, their expression in different cells is different. In the context of atherosclerosis, epsin1 and Epsin2 expression is increased in endothelial cells and macrophages. Epsin interacts with IP3R1 in endothelial cells and binds to LRP-1 in macrophages. This cell-specific targeting of Epsin has therapeutic potential.

Disclosure of Invention

In order to achieve the aim, the invention provides an Epsin1-Epsin2-shRNA functional gene coating stent, which comprises a stent main body and an Epsin1-Epsin2-shRNA functional gene coating, wherein the Epsin1-Epsin2-shRNA functional gene coating is coated on the surface of the stent main body.

The preparation method of the Epsin1-Epsin2-shRNA functional gene coating stent comprises the following steps:

step one, immersing a stent body in PrS solution;

step two, taking out the bracket main body, and washing with buffer solution;

step three, immersing the stent main body taken out in the step two into an Epsin1-Epsin2-shRNA solution;

step four, repeating the washing in the step two;

repeating the operations from the first step to the fourth step for twelve times, and forming a single-layer ion layer each time to coat twelve layers of Epsin1-Epsin2-shRNA coatings on the surface of the bracket main body;

and step six, cleaning and air-drying the support main body, and placing the support main body into a dryer at room temperature for storage.

Further, the immersion time in the first step is 6 to 10 minutes, most preferably 8 minutes.

Further, the buffer solution in the second step is HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) buffer solution, and the washing process is repeated three times for 1-2 minutes, and most preferably 1 minute.

Further, the immersion time in step three is 6 to 10 minutes, most preferably 8 minutes.

Further, in the step six, cleaning and air-drying are performed by using three distilled water, and air-drying is performed by using nitrogen.

The beneficial effects of using the invention are as follows:

1. exploring the application of inhibiting the expression of the Epsin protein in inhibiting the proliferation of vascular endothelial cells, finding that the Epsin knockdown indirectly inhibits the proliferation and migration of smooth muscle cells by promoting endothelialization, reducing endothelial inflammation and mitochondrial oxidative stress;

2. the Epsin1-Epsin2-shRNA functional gene is used as a coating of the vascular stent, so that the risk of late thrombosis and late restenosis after vascular stent implantation is reduced.

Drawings

FIG. 1 is a schematic image of carotid Epsin expression after 7 days in each group of mice;

FIG. 2 is a graph showing statistics of carotid Epsin expression after 7 days in each group of mice;

FIG. 3 is a schematic image of the effect of knockdown of Epsin on endothelial cell proliferation;

FIG. 4 is a statistical plot of the effect of knockdown of Epsin on endothelial cell proliferation;

FIG. 5 is a schematic image of the effect of knockdown of Epsin on endothelial cell migration;

FIG. 6 is a statistical plot of the number of effects of knockdown of Epsin on endothelial cell migration;

FIG. 7 is a graphical representation of the effect of knockdown of Epsin on endothelial cell mitochondrial morphology;

FIG. 8 is a statistical plot of the quantitative impact of knockdown of Epsin on endothelial cell mitochondrial morphology;

FIG. 9 is a graphical representation of the effect of knockdown of Epsin on endothelial cell mitochondrial potential;

FIG. 10 is a statistical plot of the quantitative impact of knockdown of Epsin on endothelial cell mitochondrial potential;

FIG. 11 is a graphical representation of the effect of Epsin-knockdown endothelial cells on smooth muscle cell proliferation co-cultured therewith;

FIG. 12 is a statistical plot of the number of effects of Epsin knockdown endothelial cells on smooth muscle cell proliferation co-cultured therewith;

FIG. 13 is a graphical representation of the effect of Epsin-knockdown endothelial cells on smooth muscle cell migration in co-culture therewith;

FIG. 14 is a statistical plot of the number of effects of Epsin knockdown endothelial cells on smooth muscle cell migration in co-culture therewith;

FIG. 15 is a graphical representation of transfection efficiency of each group 7 days after stent implantation and 28 days after stent implantation;

FIG. 16 is a schematic image of the surface features of each set 7 days after and 28 days after stent implantation;

FIG. 17 is a schematic image of intimal hyperplasia in each group 7 days and 28 days after stent implantation;

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

Example 1

Inhibiting Epsin protein expression can play a role in inhibiting vascular intimal proliferation;

the Epsin-shRNA is applied to the vascular stent as a functional coating, so that the effects of inhibiting migration and proliferation of vascular endothelial cells and smooth cells are achieved, and the purposes of reducing the risk of late thrombosis and late restenosis after implantation of the vascular stent are achieved.

Epsin proteins specifically refer to Epsin1 and Epsin 2.

Example two

Specifically, the experimental method and experimental procedure are as follows:

1. experimental animals and groups

The study purchased 12 healthy clean male C57BL/6J mice weighing around 24g from the north battlefield general hospital medical laboratory animal center, animals were randomly divided into 2 groups: the animal model was constructed with carotid stenosis model group (injury) (n=6) and sham operation group (sham) (n=6).

2. Method of

2.1 preparation of a mouse carotid stenosis model

C57BL/6J mice were anesthetized with a syringe to extract 1% sodium pentobarbital at a concentration of 50mg/kg by intraperitoneal injection. The fully anesthetized mice were placed on an operating table, the mice were fixed in a supine position, and the razor was used to remove hair around the neck surgical incision and thoroughly disinfect the neck skin. The mice were placed on a 37℃thermostat to maintain their body temperature stable and monitored for stability. Towel around incision, operate under surgical microscope, adjust focal length of microscope, gently separate left cervical tissue with atraumatic microshutter, left common carotid artery (common carotid artery, CCA), external carotid artery (External carotid artery, ECA) and internal carotid artery (internal carotid artery, ICA). In the process, bleeding is avoided as much as possible, the tail end of the left common carotid artery is ligated, and the skin is sewn. The operation of the false operation group is consistent with the operation method of the carotid artery stenosis model except the ligation of the carotid artery on the left side, the mice are sent back to the observation state of the animal room after being awakened, and the materials are obtained after 7 days.

2.2 materials of study object

After the carotid artery ligation model is successfully manufactured for 7 days, 1% concentration pentobarbital sodium is extracted by a syringe according to the proportion of 50mg/kg, the mice are anesthetized by an intraperitoneal injection mode, after the mice are in a complete anesthetic state, the mice are fixed on an operation table in a supine position, hairs at the incision are removed by a razor, neck skin is fully disinfected by soaking with a cotton swab or a medical cotton ball, the skin is cut open, the skin is taken out, the carotid arteries of the mice are rapidly separated under a stereoscopic microscope, and the separated carotid arteries of the mice are respectively put into liquid nitrogen and paraformaldehyde for preservation.

2.3 cell culture

Cell culture was performed using DMEM (Dulbecco's modified eagle medium) medium containing 10% FBS, and the cell culture chamber conditions were constant and set to 5% CO ₂ The growth conditions of the cells are observed by a microscope at regular intervals at 37 ℃, and the cells are replaced, passaged and frozen at intervals according to the growth conditions of the cells so as to be required by experiments.

2.4 passage of cells

When the cells are close to the overgrown cell culture dish, the culture medium in the cell culture dish is sucked out, the PBS (phosphate buffer solution) is used for lightly cleaning the culture medium for three times, the residual culture medium is cleaned, the prepared pancreatin is added for digestion, when the cells are fully digested, the culture medium with serum is added for stopping digestion, 500r and 5min are centrifuged, the supernatant is discarded, the culture medium containing the serum is added for blowing and evenly mixing, and then the cells are transferred into a new culture dish.

2.5 treatment of laboratory cells

The following groupings were formed by transfecting human umbilical vein endothelial cells with siRNA, after VEGF (vascular endothelial growth factor) (50 μm) stimulation and PBS, respectively, for 24 hours: siControl (blank), siEpsin1+ siEpsin2, siepsrol + VEGF, siEpsin1+ siEpsin2+ VEGF. The siRNA transfection is to culture the cells first, and a proper amount of cells are taken one day before transfection and inoculated into a six-hole plate or other containers for cell experiments, and the method takes a 6-hole plate as an example, so that the cell density of a culture dish can reach about 50-70% in the next day, cells are intermittently observed, and when the cells grow to a proper density, the transfection is started. Taking two clean and sterile tubes, respectively adding 150 mu l of culture medium without antibiotics and serum into the two EP tubes, then adding siRNA into one tube, and lightly blowing and uniformly mixing by using a gun; while the other tube was filled with 6. Mu.l Lipo 2000 transfection reagent, gently stirred with a gun and allowed to stand at room temperature for 5min. The siRNA-containing medium was gently added to Lipo 2000 transfection reagent-containing medium with a pipette, followed by gently pipetting and mixing, and allowed to stand at room temperature for 15min, at which time the six well plates with cells cultured were washed three times with PBS per well. The transfection reagent was mixed with 700. Mu.l serum-free antibiotic-free medium and added to a 6-well plate.

2.6 cell cryopreservation

The cell digestion process is consistent with the cell passage method, the upper liquid of the cells is removed after centrifugation, the prepared cell freezing solution (900 mu l of serum and 100 mu l of dimethyl sulfoxide) is added, the cells are resuspended, the cell freezing tube is added, and the cell freezing tube is put into liquid nitrogen for preservation. And taking out the cells when the cells need to be recovered.

2.7 Paraffin section

The carotid artery of the mice fixed with paraformaldehyde is taken out, washed with water, dehydrated with 70% alcohol, 80% alcohol, 90% alcohol and 95% alcohol in sequence for 5min to 10min, and finally placed into xylene for transparency. Immersing the tissue in paraffin, cooling, slicing, taking the cut tissue slice when the tissue slice is cut to a designated position, spreading, and water-bathing. The tissue was fixed to the slide by baking after scooping up the slide.

2.8 HE staining

The tissue sections were dewaxed sequentially with xylene, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, distilled water, stained with hematoxylin for 5min, and rinsed with running water. Adding eosin for dyeing, washing off the dyeing agent with running water, sequentially dehydrating with 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol and xylene, and sealing with neutral resin.

2.9 immunohistochemistry

The dewaxing step is consistent with the HE staining step, according to an immunohistochemical kit, firstly adding a peroxidase blocker for 10min, washing with PBS for 3 times, adding non-immune animal serum for sealing for 10min, incubating with a primary antibody overnight, rewarming, adding a drop of biotin-marked secondary antibody, washing with PBS (phosphate buffer solution) for 3 times, adding streptomycete biotin-peroxidase for 10min, washing for 3 times, developing with DAB solution, and paying attention to the staining time to avoid over-deep or over-shallow color.

2.10 mitochondrial staining and JC-1 staining

Mito-Tracker: after treatment of the cells, when the cells were cultured in a cell culture plate or dish to a certain time point, mito-Tracker working solution was added according to the instructions and incubated at 37℃for 30min. Mito-Tracker working solution was removed, washed 3 times with PBS, and cell culture medium was added. Observations were made with a microscope.

JC-1: when each group of cells reached the experimental target time point, JC-1 staining working solution was added according to the instruction, and incubated in a cell incubator at 37℃for 20min. After the incubation, the staining solution was removed and the JC-1 staining buffer was washed 2 times. Cell culture medium was added and observed under a microscope.

2.11 cell migration

Endothelial cells and smooth cell migration were analyzed by wound healing assay. Cells were spread to an appropriate density, endothelial cells and smooth muscle cells were uniformly injured using a pipette tip having a diameter of 1.15 mm, a standard photograph of the wound area was taken by a microscope, and a picture of the wound site was taken again after 24 hours. The distance traveled by the wound cells was calculated by subtracting the distance of the 24h lesion edge from the distance of the 0h lesion edge.

2.12 Co-culture model of endothelial cells and smooth muscle cells

Endothelial cells were incubated with smooth muscle cells using a Transwell chamber (0.4 μm) for 24h, the smooth muscle cells were placed in the lower chamber and the endothelial cells were placed in the upper chamber. After 3 times of PBS washing, VSMC (smooth muscle cell) proliferation and migration indexes were detected.

2.13 immunofluorescence

Cell seeds were washed 3 times in a cover glass in PBS (phosphate buffer solution), punched with Triton X-100 at a concentration of 0.1%, washed 3 times with PBS after 30min, blocked with goat serum for 2h, incubated overnight on the cells in a 4 degree refrigerator using a prepared primary antibody solution dropwise, washed 3 times with PBS, incubated with a fluorescent secondary antibody for 2h, washed 3 times with PBS, incubated for 5min with a staining solution (preferably a staining solution of DPAI), and blocked. And observing by using a fluorescence microscope.

2.14, preparation of Experimental materials

Two groups of vascular stents are prepared, and shControl and shEpsin1+Epsin2 are respectively coated on the two groups of vascular stents.

2.15 observing males

About 30kg of male Bama pigs are taken to implant the stent into carotid arteries, and the stent is divided into shControl group and shEpsin1+Epsin2 group: detection by pathological tissue morphology analysis.

2.16 statistical analysis

Statistical analysis was performed using GraphPad Prism v9.0 and chart making was performed. All data were analyzed using One-way ANOVA and the differences between groups were compared using multiple comparisons (Tukey's multiple comparisons tests), P <0.05 indicating that the differences were statistically significant.

3. Experimental results

3.1 in order to evaluate the variation of the expression level of Epsin in the carotid stenosis formation process of mice, the experiment shows that the level of immunohistochemistry of the Epsin is remarkably increased in the carotid artery ligation model group mice compared with the sham operation group mice by carrying out 7 days on the two groups of mice, namely the sham operation group mice and the carotid artery ligation model group mice, which shows that the expression of the Epsin in the carotid artery stenosis model of the mice is increased, and the positive correlation exists between the Epsin and the carotid artery stenosis of the mice. Has statistical significance (P < 0.05), and is shown in FIGS. 1 and 2.

3.2 on the basis of clear positive correlation of Epsin and proliferative vascular diseases, in order to further explore the specific mechanism of Epsin's role in proliferative vascular diseases, this study was performed by subjecting endothelial cells to four different treatments, siControl, siEpsin1+siepsin2, sicontrol+vegf, siEpsin1+siepsin2+vegf. Due to the similarity of the functions of Epsin1 and Epsin2, knocking down of Epsin leads to increased expression content of the endothelial cells Ki67, the relative migration distance is prolonged, and knocking down of Epsin promotes proliferation and migration of endothelial cells, which is also the first step of ensuring early endothelialization after vascular injury. See fig. 3, 4, 5 and 6 for details.

3.3 further studies showed that knocking down Epsin stabilizes endothelial cell function by maintaining mitochondrial homeostasis. Mitochondrial performance (especially mitochondrial fission) in endothelial cells is significantly regulated by mitochondrial dynamics. Thus, we studied whether knockdown of Epsin protects endothelial cells for mitochondrial dysfunction by alleviating mitochondrial fission. By observing the mitochondrial morphology through immunofluorescence, the Epsin knockdown obviously increases the area of mitochondria in endothelial cells and increases the activity of the endothelial cells. Meanwhile, the Epsin knockdown protects the mitochondrial potential homeostasis of endothelial cells and maintains the stability of the endothelial cells. See fig. 7, 8, 9 and 10 for details.

3.4, we found that Epsin knockdown endothelial cells indirectly inhibited smooth muscle cell proliferation and migration. See fig. 11, 12, 13 and 14 for details.

3.5 HUVECs were seeded onto scaffolds and transfection of these cells was observed under confocal laser scanning microscopy. It is blue, indicating that the cells on the scaffold are evenly distributed and almost completely covered. Some cells fluoresce green, indicating that they have been transfected.

The scaffold was examined with a scanning electron microscope. No metal support, smooth surface and no fittings. The machined support surface coating is uniform.

Example III

To directly evaluate the Epsin-shRNA functional coated scaffolds, the functional coated scaffolds were compared to shRNA groups and Epsin-shRNA functional coated scaffold groups. At 1 week (W) and 1 month (M), the samples were observed and analyzed. Angiography was performed 15 minutes after successful implantation to confirm that the cavity was clear, free of filling defects, vessel wall dissection, embolism, stent displacement or distal vasospasm. There were no significant differences in heart rate, blood pressure and body weight in the preoperative, intraoperative and follow-up angiography groups. No obvious abnormal behavior, diet and defecation after operation.

Histomorphology analysis

Hematoxylin-eosin (he) staining images showed that the shEpsin scaffold forms a monolayer of endothelial cells on the shEpsin scaffold on days 7 and 7 after 1 month of scaffold implantation, whereas the control group was directly exposed to blood. After one month, the endomembrane proliferation of the shEpsin scaffold was less than that of the control group, figures 15, 16, 17.

In this study, endothelial cell proliferation and migration were increased, which is the first step required for endothelialization, while we found that Epsin1 and 2 endocytic adaptor proteins play a novel role in endothelial cell division into mitochondria, which is common in proliferative vascular diseases such as restenosis following carotid artery injury. Endothelial mitochondrial volume is one of the main mechanisms responsible for abnormal proliferation of blood vessels; however, the underlying molecular mechanisms remain elusive. Plays a key role in the widely accepted model of "endothelial injury". Knocking down Epsin reduces mitochondrial division in endothelial cells. Maintains membrane potential steady state of mitochondria, protects activity of mitochondria, and maintains relative stability of endothelial cells.

Meanwhile, we found that Epsin knockdown indirectly inhibited smooth muscle cell proliferation and migration by promoting endothelialization, reducing endothelial inflammation and mitochondrial oxidative stress. Provides a new target for treating the proliferative vascular diseases.

Targeting Epsin to VEGFR2 in endothelial cells has shown promise in the treatment of various cancers. In the mouse model of atherosclerosis, it reduces activation of endothelial NF- κB and reduces endothelial inflammation. Also, recent studies have found that inhibition of Epsin promotes the formation of a monolayer of endothelium on the surface of vascular grafts.

Example IV

The Epsin1-Epsin2-shRNA functional gene coating stent comprises a stent main body and an Epsin1-Epsin2-shRNA functional gene coating, wherein the Epsin1-Epsin2-shRNA functional gene coating is coated on the surface of the stent main body.

Example five

Specifically, the preparation method of the Epsin1-Epsin2-shRNA functional gene coating stent comprises the following steps:

step one, immersing a stent body in PrS solution;

step two, taking out the bracket main body, and washing with buffer solution;

step three, immersing the stent main body taken out in the step two into an Epsin1-Epsin2-shRNA solution (the solution is a commercially available reagent);

step four, repeating the washing in the step two;

step five, repeating the operations from the step one to the step four for twelve times to form a single-layer ion layer each time, so that twelve layers of Epsin1-Epsin2-shRNA coatings are coated on the surface of the bracket main body;

Specifically, the immersion time in the first step is 6 to 10 minutes, and most preferably 8 minutes;

the buffer solution in the second step is HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) buffer solution, and the washing process is repeated for three times, wherein the time length of each time is 1-2 minutes, and most preferably 1 minute;

the immersion time in the third step is 6-10 minutes, most preferably 8 minutes;

and step six, cleaning and air-drying, namely cleaning by using triple distilled water and air-drying by using nitrogen.

The foregoing is merely exemplary of the present invention, and many variations may be made in the specific embodiments and application scope of the invention by those skilled in the art based on the spirit of the invention, as long as the variations do not depart from the gist of the invention.

Claims

An Epsin1-Epsin2-shRNA functional gene coating stent, characterized in that: the novel scaffold comprises a scaffold body and an Epsin1-Epsin2-shRNA functional gene coating, wherein the Epsin1-Epsin2-shRNA functional gene coating is coated on the surface of the scaffold body.
The preparation method of the Epsin1-Epsin2-shRNA functional gene coating stent comprises the following steps:

step one, immersing a stent body in PrS solution;

step two, taking out the bracket main body, and washing with buffer solution;

step three, immersing the stent main body taken out in the step two into an Epsin1-Epsin2-shRNA solution;

step four, repeating the washing in the step two;

repeating the operations from the first step to the fourth step for twelve times, and forming a single-layer ion layer each time to coat twelve layers of Epsin1-Epsin2-shRNA coatings on the surface of the bracket main body;

and step six, cleaning and air-drying the support main body, and placing the support main body into a dryer at room temperature for storage.
3. The Epsin1-Epsin2-shRNA functional gene coated stent of claim 2, wherein: the immersion time in step one is 6-10 minutes, most preferably 8 minutes.
4. The Epsin1-Epsin2-shRNA functional gene coated stent of claim 2, wherein: the buffer solution in the second step is HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) buffer solution, and the washing process is repeated for three times, wherein each time lasts for 1-2 minutes, and most preferably 1 minute.
5. The Epsin1-Epsin2-shRNA functional gene coated stent of claim 2, wherein: the immersion time in step three is 6-10 minutes, most preferably 8 minutes.
6. The Epsin1-Epsin2-shRNA functional gene coated stent of claim 2, wherein: and step six, cleaning and air-drying, namely cleaning by using triple distilled water and air-drying by using nitrogen.