CN112899224A - Extracellular nano vesicle for loading coronary stent, preparation method and application - Google Patents

Abstract

The invention relates to the field of cell culture, in particular to an extracellular nano vesicle for loading a coronary stent, a preparation method and application. The extracellular nano vesicle for loading the coronary artery stent is secreted by umbilical cord Wharton's jelly source mesenchymal stem cells. The extracellular nano vesicle from the umbilical cord Wharton jelly source mesenchymal stem cell prepared by the invention has the functions of anti-inflammation, regeneration and immunoregulation, promotes the regeneration of endothelial cells and the repair of microenvironment, inhibits the proliferation of smooth muscle cells, resists the proliferation of fibrous tissues, promotes the conversion of inflammatory macrophages to anti-inflammatory M2, protects the integrity of target lesion intima, and realizes the synchronization of revascularization and the prevention of restenosis in a stent and the formation of new atherosclerosis and thrombus.

Description

Extracellular nano vesicle for loading coronary stent, preparation method and application

Technical Field

The invention relates to the field of cell culture, in particular to an extracellular nano vesicle for loading a coronary stent, a preparation method and application.

Background

Coronary heart disease is the first killer to threaten human life. Percutaneous Coronary Intervention (PCI) is the most important means for treating coronary heart disease. PCI surgical devices have evolved over 40 years and are continually being updated as a component of PCI core technology, the scaffold. Nevertheless, the development of new coronary atherosclerosis (NA) and late thrombosis following stenting has always affected the mid-to-long term efficacy after PCI surgery.

Evidence suggests that even with the latest drug eluting stents, Optical Coherence Tomography (OCT) finds 27.4% to 50% of new atherogenesis in the stent. Even 1/4 patients clinically require repeated treatment for restenosis due to the formation of new plaques in the stent. Recently, it has been discovered that the pharmacological effect of the second generation drug eluting stent, such as the rapamycin derivative drug eluting stent like zotarolimus eluting stent and everolimus eluting stent, or the novel polymer coating-free drug eluting (sirolimus and paclitaxel) stent, which inhibits the proliferation of endothelial cells and smooth muscle cells, triggers the central mechanism of atherosclerosis, i.e. the increase of oxidation stress product ROS, the activation of macrophage inflammation of M1 type, the accumulation of a large amount of oxidized low-density lipoprotein subintium and the phagocytosis of macrophages. Significantly reduced cytoskeletal adaptor protein Nck, mTOR, rictor expression and mTORC2 formation ultimately led to the formation of characteristic intra-stent neoatherosclerotic plaques (NA) characterized by relatively thin fibrous caps and extensive yellow lipid-accumulated atherosclerotic plaques. Thus, late NA with intimal rupture and thrombosis are the most common mechanisms of late stent thrombosis, also associated with the high frequency of ST-elevation myocardial infarction. Thus, there is an urgent clinical need to innovate the loading of existing drug eluting stents.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an extracellular nano vesicle for loading coronary stents.

The second purpose of the invention is to provide the application of the extracellular nanovesicle in the preparation of a coronary artery drug-loaded stent.

The third invention of the present invention is directed to a coronary drug-loaded stent.

The fourth invention of the present invention is to provide a method for preparing the extracellular nanovesicle.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the invention provides an extracellular nano vesicle for loading a coronary stent, which is secreted by umbilical cord Wharton's jelly source mesenchymal stem cells.

Optionally, the preparation of the extracellular nanovesicle is lyophilized powder.

Optionally, the lyophilized powder further contains an excipient, the excipient is preferably at least one of mannitol, sorbitol, glucose and lactose, and the weight ratio of the extracellular nano vesicles to the excipient is 1: 50-5000, preferably 1: 100 to 1000.

Optionally, the extracellular nanovesicle is obtained by processing and then separating the umbilical cord Wharton jelly source mesenchymal stem cell by using a culture medium containing cytochalasin B.

Optionally, the concentration of cytochalasin B is 5-20 mug/mL, preferably 10 mug/mL; preferably, the medium is selected from DMEM medium.

Optionally, the separation is selected from membrane affinity separation; preferably, the separation is performed using an exoEasy spin column.

The invention also relates to application of the extracellular nanovesicle in preparation of a coronary artery drug loaded stent, and preferably, the extracellular nanovesicle completely replaces chemical drug loading and the inside of the stent.

The invention also relates to a coronary artery drug-loaded stent, which adopts the extracellular nano vesicles loaded in the nano micro channels of the stent, and the stent is selected from cobalt-chromium alloy or platinum-chromium alloy.

The invention also relates to a preparation method of the extracellular nanovesicle, which at least comprises the following steps:

s1, obtaining umbilical cord Wharton jelly source mesenchymal stem cells;

s2, processing the umbilical cord Wharton jelly source mesenchymal stem cells by adopting a culture medium containing cytochalasin B; the treatment temperature is preferably 37 ℃, the time is preferably 20-40 minutes,

s3, separating the extracellular nanovesicles.

Optionally, the umbilical cord Wharton jelly-derived mesenchymal stem cells are obtained by culturing according to the following method:

s11, taking the Huatong glue tissue, shearing the Huatong glue tissue into pieces, and performing tissue adherent culture in a plate;

s12, after the bottom of the bottle is filled with 80% of cells, cleaning the bottle to prepare a cell suspension, inoculating the cell suspension into 3D hydrogel, and performing amplification culture at 37 ℃ under the conditions of carbon dioxide with volume fraction of 5% and saturated humidity;

and S13, digesting and washing the cells by using a hydrogel enzyme when 80% of the cells are fused, carrying out passage, and culturing the second generation cells and the third generation cells in a 5% hypoxic cell culture box for 10-18 hours.

The invention has at least the following beneficial effects:

the extracellular nano vesicles (WJMSCs-EV) from the umbilical cord Wharton jelly source mesenchymal stem cells prepared by the invention can be used in a coronary artery drug-loaded stent, and the WJMSCs-EV is rich in various biological factors, mRNA and microRNA, has anti-inflammatory, regeneration and immunoregulation functions, promotes the regeneration of endothelial cells and the repair of microenvironment, inhibits the proliferation of smooth muscle cells, resists the proliferation of fibrous tissues, promotes the conversion of inflammatory macrophages to anti-inflammatory M2, protects the integrity of target lesion intima, has a multi-target point synergistic effect, and realizes the synchronization of blood transportation reconstruction, restenosis in a pre-released stent, new atherosclerosis and thrombosis.

The preparation method of the extracellular nano vesicle from the umbilical cord Wharton jelly source mesenchymal stem cells adopts a cell relaxant B combined membrane affinity separation method to extract WJMSCs-EV. The preparation method overcomes the technical defects of damage, small quantity and difficult purification of the shear force extracted by an ultradifferential centrifugation method at present, and can obtain more extracellular nano vesicles and maintain the purity.

The preparation of the invention can be stored for a long time at the temperature of 20 ℃ below zero and applied at normal temperature.

The coronary artery drug-loaded stent of the invention puts the prepared extracellular nano vesicles into a stent nano micro-channel storage tank, uniformly controls and releases the extracellular nano vesicles, and exerts multi-target biological effects such as substance and information transfer by fusing and internalizing with target vascular endothelial cells and entering into a subendothelial intercellular microenvironment.

Drawings

FIG. 1 is a morphological image of extracellular nanovesicles under a transmission electron microscope;

FIG. 2 is a standard curve of detection by a BCA protein detection kit after extraction of extracellular nanovesicles;

FIG. 3 shows the results of western blot detection of extracellular nanovesicles;

FIG. 4 shows HUVECs under a light mirror;

FIG. 5 shows immunofluorescent-stained vWF staining of HUVECs;

FIG. 6 is an immunofluorescent staining for HUVECs CD 31;

FIG. 7 is a graph showing the results of co-staining of WJMSC-EV with umbilical vein-derived intimal epithelial cells (HUVECs) with immunofluorescent staining of CD31+ CD9 and CD31+ CD 63;

FIG. 8 shows the proliferation rate of HUVECs measured by CCK8 method after the co-culture of HUVECs and WJMSC-EV.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In recent years, research shows that in vitro Vesicles (EV) derived from Mesenchymal Stem Cells (MSCs) have biological effects similar to those of the MSCs, such as reduction of apoptosis, reduction of inflammatory reaction, promotion of angiogenesis, inhibition of fibrosis, improvement of tissue repair potential and the like. According to different generation modes, sizes or functions of EV, the EV can be divided into 3 subgroups of extracellular nano vesicles originated from endocytic pathway, micro vesicles released from plasma membrane and apoptotic bodies generated by cell apoptosis. Currently, MSCs-EV is of high international interest as a new, multi-targeted biological agent. MSCs-EV contain a variety of biologically active molecules, such as: protein, lipid, mRNA, microRNA, siRNA, miRNA, ssDNA and dsDNA, and transmits the protein, lipid, mRNA, microRNA, siRNA, miRNA, ssDNA and dsDNA to target cells, mediates intercellular communication and maintains the stability of extracellular microenvironment. Has the important functions of anti-inflammation, regeneration, immunoregulation, tissue repair and the like. The diameter of the extracellular nano vesicles is 20-100 nm, and the diameter of the microbubbles is 200-1000 nm. It was found that EV functional properties depend on the mother cell from which it was derived. The EV characteristics and functions of the MSCs from different tissue sources are greatly different. The mesenchymal stem cells (WJMSCs) of the Huatong glue source are mesenchymal stem cells of a special source. WJMSCs are primitive mesenchymal stem cells (WJMSCs) that persist in the connective tissue at the interface between the perivascular mother and fetus in the umbilical cord, early in fetal embryonic development. It highly expresses a variety of sternness genes: OCT4, SSEA3, SSEA4, SOX2, NANOG, but at lower levels than ESC, did not produce teratomas. WJMSCs secrete a large number of growth factors and immunoregulatory factors, such as Hepatocyte Growth Factor (HGF), tumor necrosis factor activator-6 protein (TSG-6), prostaglandin E-2(PGE-2), monocyte chemotactic protein 1(MCP1), Vascular Endothelial Growth Factor A (VEGFA), interleukin-10 (IL-10), indoleamine 2, 3-dioxygenase (IDO), transforming growth factor-beta (TGF-beta, soluble vascular cell adhesion molecule-1 (sVCAM1) and the like, EV (WJMSCs-EV) which is the best original characteristic MSCs currently called by the international cell biology community contains a large number of bioactive factors and mRNA, microPNA, can promote endothelial cell repair and regeneration, inhibit smooth muscle cell proliferation and migration, repair subendothelial extracellular matrix, inhibit fibroblast proliferation, obviously has anti-inflammatory effect, the polarized macrophage differentiates to anti-inflammatory M2, and has inhibitory effect on autoimmune inflammatory monocyte and acquired immune T, B cell. Thereby protecting the integrity of the intima of the target lesion, and antagonizing the initiation and progression of atherosclerotic inflammatory lesions and late thrombosis at multiple target sites. Therefore, the present invention selects WJMSCs-derived EV as a biological agent to replace the original chemical as the coronary stent cargo.

The invention applies the biomedical technology for the first time, the WJMSCs-EV is loaded into the stent with the nanometer micro-channels, and the WJMSCs-EV is released into the target lesion part of the stent along with the stent placement, and the purposes of promoting endothelial regeneration, repairing the microenvironment inside and outside the blood vessel wall cells, resisting inflammation, inhibiting fibrous tissue proliferation and preventing NA and thrombosis from being integrated synchronously are achieved through the synergistic effect of multiple action mechanisms of the EV.

Specifically, the embodiment of the invention provides an extracellular nanovesicle loaded with a coronary stent, which can be prepared by the following steps: the method comprises the following steps: separating gelatin-like mucus (Wharton jelly) tissues from three parts including intervascular, perivascular and inferior amniotic fluid in umbilical cord of newborn, and performing tissue, 3D culture and hypoxia culture to obtain Wharton jelly source MSCs (WJMSCs); step two: extracting WJMSCs-EV by a cell relaxant B + membrane affinity separation method, adding a mannitol low-temperature protective agent to prepare WJMSCs-EV freeze-dried powder; and adopts standardization to identify the effect and safety of WJMSCs-EV.

Specifically, the concentration of cytochalasin B is 5-20 mug/mL, preferably 10 mug/mL. The extracellular nano vesicles extracted by the method have less cytochalasin B concentration, and the apoptosis is increased due to the excessive cytochalasin B concentration.

Specifically, the culture medium is selected from DMEM medium. DMEM is a basic culture medium in the cell culture process, maintains the basic cell growth environment, enables cell relaxin B to have enough time to act on cell membranes, and promotes the release of extracellular nano vesicles.

Specifically, the treatment temperature is 37 ℃, and the treatment time is 20-40 minutes, preferably 30 minutes.

Specifically, the extracellular nanovesicles are separated by a membrane affinity separation method; preferably an exoEasy spin column.

The preparation of the extracellular nano vesicle of the embodiment of the invention can be freeze-dried powder, and other preparation modes for ensuring the activity of the extracellular nano vesicle can also be adopted, and the WJMSCs-EV freeze-dried powder is prepared and loaded into a nano micropore channel groove of the stent, and the WJMSCs-EV is controlled to enter a target lesion blood vessel along with the placement of the stent, so as to be fused and internalized with endothelial cells to generate a multi-mechanism synergistic effect, thereby realizing the synchronization of revascularization and the prevention of new atherosclerosis and thrombosis in the stent.

Preferably, the excipient of the freeze-dried powder is selected from at least one of mannitol, sorbitol, glucose and lactose, and mannitol is preferred. The weight ratio of the extracellular nano vesicles to the excipient is 1: 50-5000, preferably 1: 100 to 1000. The preparation method of the freeze-dried powder comprises the following steps: adding mannitol with the final concentration of 0.1-1 g/mL into the extracellular nano vesicle with the protein concentration of 0.5-2 mg/mL, and carrying out freeze drying treatment. The concentration of the extracellular nanovesicles is preferably 1mg/mL, and the final concentration of mannitol is 0.5 g/mL.

The conditions for freeze-drying are preferably: freezing the extracellular nano vesicle at-80-60 deg.C for more than 12 hr, and placing in a freeze dryer at-50 deg.C and 8 × 10^-1Freeze-drying under MPa for 72 hr, and storing at-20 deg.C.

The embodiment of the invention also relates to application of the extracellular nano-vesicle or the preparation in preparation of a coronary artery drug-loaded stent.

The embodiment of the invention also relates to a coronary artery drug-loaded stent, the preparation is loaded in the nanometer micro-channels of the stent, and the stent is selected from cobalt-chromium alloy or platinum-chromium alloy.

Reagents, kits, and equipment sources used in the examples section.

Serum-free medium (Youkang Hexagon, China), cell culture incubator (Thermo Fisher, USA), hypoxic culture incubator (ThermoFisher, USA), flow cytometer (Beckman, USA), trypsin (Gibco, USA), phosphate buffer (Gibco, USA), sterile plates, cell culture flasks, pipettes, centrifuge tubes (Corning-Costar, USA), CD73, CD90, CD44, CD105, HLA-ABC, CD34, CD45, CD80, CD86, HLA-DR, CD24, CD108, CD40(BD, USA), hydrogels (sigma-Aldrich USA), cross-linkers, cytochalasin B (Soroban, China), XBP buffer (qiagen, Germany), exoEasy spin columns (qiagen, Germany), BCA protein detection kit (Solebao, China).

Example 1: the extraction, culture and amplification method of WJMSCs comprises the following steps:

1. separating glue-like mucus (huatong glue) tissue from three parts of blood vessels in umbilical cord, blood vessel periphery and amniotic membrane of newborn, and placing the tissue into a sterile plate;

2. tissue adherent culture: placing the mucilage into a sterile plate, and shearing into 1mm with sterile ophthalmic scissors³Placing the tissue blocks with bottom area of 75cm²Culturing in an aseptic cell culture bottle in an adherent manner, adding 15mL of serum-free culture medium, and culturing in a cell culture box with the temperature of 37 ℃, the volume fraction of carbon dioxide of 5% and the saturation humidity.

3. 3D culture; after the cells are paved to 80% of the bottom of the bottle, removing the culture medium, adding 5mL of phosphate buffer solution to clean the bottom of the cell culture bottle, removing the cleaning solution, adding 3mL of 0.125% trypsin to the culture bottle for digestion for 1 minute, adding 3mL of serum-free culture medium to stop digestion after the cells shrink, forming cell suspension, uniformly pumping and mixing by using a pipette, transferring the cell suspension into a 50mL centrifuge tube, centrifuging for 10 minutes by 100g of centrifugal force, removing the supernatant, adding 20mL of phosphate buffer solution, uniformly mixing the cells, repeating the above washing steps, washing once, removing the supernatant, adding hydrogel culture medium to uniformly mix, and enabling the cell concentration to be 0.8-1X 10/mL⁵Cells, this is a hydrogel cell suspension, with a new basal area of 75cm²10mL of hydrogel cell suspension is added into each culture bottle, and the number of the inoculated cells in each culture bottle is (0.8-1) × 10⁶Adding 0.1mL of cross-linking agent into a culture bottle to promote hydrogel to form a jelly-like shape, adding 5mL of serum-free cell culture medium on the surface of the hydrogel in the culture bottle, placing the culture bottle containing the aqueous gel cell suspension into a culture box with the temperature of 36-37 ℃ and the volume fraction of 5% of carbon dioxide and the saturated humidity for amplification culture, and observing the growth condition of the cells every day. After 3D amplification culture for 6-7 days, adding 10mL of 36-37 ℃ phosphate buffer solution when cell fusion is 70-80%, and placing at 36-37 DEG CAnd (3) in a water bath, decomposing the hydrogel after 40-60 min to form a cell suspension, sucking by using a pipettor, uniformly mixing the cell suspension, transferring the suspension into a centrifugal tube, centrifuging for 10-20min by using a centrifugal force of (100-200) g, discarding the supernatant, and entering the next procedure.

4. WJMSCs hypoxic culture: adding serum-free cell culture medium into the cells after 3D culture, and adjusting cell concentration to 0.8-1 × 10⁵Adding 10mL of the suspension into a cell culture flask, wherein the total number of the cells is (0.8-1) × 10⁶Firstly, placing a cell culture bottle into a carbon dioxide saturated humidity incubator with the volume fraction of 5% at 37 ℃ for culture, placing the cell culture bottle into a hypoxia incubator with the oxygen volume concentration of 5% and the saturated humidity at 37 ℃ for culture for 12 hours after 60% fusion of adherent culture of cells, and washing and entering the next procedure. By the low-oxygen culture, the cell tolerance, activity and viability are improved.

And (3) identifying by adopting a flow cytometer:

a. detecting that the common marker of the WJMSCs expresses CD73, CD90, CD44, CD105 and HLA-ABC by a flow cytometer, and does not express CD34, CD45, CD80, CD86 and HLA-DR as the WJMSCs.

b. The purity of WJMSCs detected by a flow cytometer is higher than 70% of CD24+ and CD108+ and lower than 20% of CD 40%.

Example 2: pre-treatment of WJMSCs by cytochalasin B

1. And (3) culturing the WJMSCs by using a serum-free culture medium, and pretreating the cells when the cell confluency reaches more than 80%.

2. The above cells were washed twice with PBS, incubated with DMEM medium containing 10. mu.g/mL cytochalasin B, and incubated at 37 ℃ for 30 minutes.

3. After the incubation is finished, the cell supernatant is aspirated, 100g is centrifuged for 20min, and the supernatant is taken for standby.

Example 3: extracting source EV of WJMSCs, and performing membrane affinity separation:

1. and (3) filtering: filtering the collected supernatant with 0.2 μm filter membrane to remove macromolecular particles;

2. binding to a membrane affinity spin column: adding XBP buffer solution 1:1 into the culture supernatant, and slightly inverting the test tube to fully stir the test tube; 16mL of the sample and XBP mixture were applied to an exoEasy spin column and centrifuged at 500g for 1 min. Discarding the flow-through, and then placing the exoEasy spin column into the collection tube;

3. cleaning: add 10mL XWP buffer and centrifuge at 5000g for 5min to remove residual buffer, discard flow-through and collection tubes;

4. transfer exoEasy spin columns to new collection tubes;

6. and (3) elution: to exoEasy spin column, 400. mu.L of Buffer XE was added and incubated for 1 min. Centrifuging at 500g for 5min, and collecting eluate;

7. the eluate was re-applied to exoEasy spin column and incubated for 1 min, centrifuged at 5000g for 5min to collect the eluate and transferred to appropriate tubes;

and 8, measuring the concentration of the extracellular nanovesicles by using a BCA method, and storing at-80 ℃.

Example 4: WJMSCs-EV identification:

WJMSCs-EV characteristics

And (3) morphological observation: and observing the morphology of the extracellular nanovesicles by using a transmission electron microscope. As shown in fig. 1.

2. Phenotypic identification

The WJMSCs-EV phenotype is observed by adopting flow cytometry, and the extracellular nanovesicles are small and cannot be directly captured by a flow cytometer, so that aldehyde/sulfate latex beads and the extracellular nanovesicles need to be combined firstly, then anti-CD 9 and anti-CD 63 antibodies are incubated together, and the phenotype is identified by the flow cytometer.

CD9 and CD63 were identified to be co-expressed at 76%.

3. Protein level identification

After 12mL of cell culture medium is taken to extract extracellular nano vesicles, protein detection is carried out according to the operation flow of the BCA protein detection kit specification, and in the detection result, FIG. 2 is a standard curve, and the standard curve reaches R2 to 0.9991 as shown in FIG. 2, so that the detection is effective.

The OD450 of the sample was 0.351, 0.349 and 0.352, respectively, and the calculated protein concentration of the sample was 0.399mg/mL, i.e., the number of cells was 7.5X 10⁶。

Western Blot: the WJMSCs-EV is identified by detecting CD9 and CD63 by Western Blot, and the experimental result is shown in FIG. 3.

As can be seen from fig. 3, both positive bands were present after incubation with CD9 and CD63 antibodies, thus demonstrating successful extraction of extracellular nanovesicles.

Example 5: preparing WJMSCs-EV freeze-dried powder:

1. adding mannitol with final concentration of 0.5g/mL into extracellular nano vesicle with protein concentration of 1mg/mL, and freeze drying.

2. Freezing the extracellular vesicle at-80 deg.C, and placing in a freeze dryer at-50 deg.C and 8 × 10^-1Freeze-drying under MPa for 72 hr, and storing at-20 deg.C.

Example 6: and (3) identifying the biological effect of the WJMSCs-EV freeze-dried powder on endothelial cells:

1. extracting and culturing umbilical vein derived endothelial cells:

1.1 aseptic extraction of umbilical cord of a parturient born by caesarean section, and storing the umbilical cord with the length of 15-20 cm in aseptic PBS solution.

1.2 puncture into the umbilical vein with a blunt needle and wash 3 times with PBS solution.

1.3 one end of the cord was clamped with surgical clamps, like collagenase I (15mL,1mg/mL) added to the umbilical vein tube, digested at room temperature for 15-20 minutes.

1.4 after digestion, the forceps are loosened, the digestive juice is collected in a 50mL centrifuge tube, and the umbilical cord is washed 2-3 times by PBS solution.

1.5 the collection was centrifuged for 3 minutes (2000 rpm).

1.6 discard the supernatant, add 10mL of ECM medium containing 5% fetal bovine serum, blow off the cells, transfer all the liquid to a culture flask coated with gelatin, and culture at 37 ℃.

1.7 after 24 hours of incubation, the medium was discarded and washed 2-3 times with sterile PBS, red blood cells and dead cells were washed off, and 10mL of ECM medium containing 5% fetal bovine serum was added.

1.8 when the cell confluency reaches 90%, the pancreatin subculture is continued, and the experiment is started when the cell confluency reaches the third generation.

HUVECs identification:

2.1 HUVECs under light mirror: HUVECs were photographed at different magnification using a light mirror. The results of the experiment are shown in FIG. 4.

2.2HUVECs immunofluorescent staining for vWF, CD31 staining:

2.21 inoculating cells on the NC treated slide glass, and rinsing the cells for 3 times with PBS (3 min each time) when the confluency of the cells reaches 85 percent;

fixing and climbing with 2.224% paraformaldehyde for 15min, and washing the slide with PBS for 3 times, each time for 3 min;

2.230.5% Triton X-100 is transparent for 20min at room temperature;

2.24 soaking and washing the slide with PBS for 3 times, each time for 3min, sucking dry PBS with absorbent paper, dripping goat serum for sealing on the slide, and sealing at room temperature for 30 min;

2.25 dropping a sufficient amount of diluted antibody of vWF and CD31 and putting the mixture into a wet box, and incubating overnight at 4 ℃;

2.26 soaking and washing the slide with PBS for 3 times, each time for 3min, dripping diluted fluorescent secondary antibody after absorbing the redundant liquid on the slide with absorbent paper, incubating for 1h at room temperature, rinsing with PBS for 3 times, each time for 3 min;

2.27 dripping DAPI and incubating for 5min in dark, staining the core of the specimen, and rinsing with PBS for three times;

2.28 blotting the liquid on the slide with absorbent paper, sealing the slide with a sealing liquid containing an anti-fluorescence quencher, and observing and collecting the image under a fluorescence microscope. The results of the experiment are shown in fig. 5 and 6.

3. And (3) detecting results of internalization functions:

3.1 incubation of 10. mu.g/ml WJMSC-EV with umbilical vein derived endothelial cells (HUVEC) overnight.

3.2 cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 20min at room temperature.

3.3 soaking and washing the slide with PBS for 3 times, each time for 3min, sucking dry PBS with absorbent paper, dripping goat serum for sealing on the slide, and sealing at room temperature for 30 min;

3.4 dropping enough diluted CD9 and CD31 antibodies into a wet box, and incubating overnight at 4 ℃;

3.5 soaking and washing the slide with PBS for 3 times, 3min each time, dripping diluted fluorescent secondary antibody after absorbing the redundant liquid on the slide with absorbent paper, incubating for 1h at room temperature, rinsing with PBS for 3 times, 3min each time.

3.6 dropping DAPI, incubating for 5min in dark, staining the specimen with nucleus, and rinsing with PBS for three times.

3.7 blotting the liquid on the slide with absorbent paper, sealing the slide with a sealing liquid containing an anti-fluorescence quencher, and observing and collecting the image under a fluorescence microscope. The results of the experiment are shown in FIG. 7.

As seen from FIG. 7, the internalization effect of WJMSC-EV was excellent.

After HUVECs and WJMSC-EV are co-cultured, the proliferation rate of HUVECs is determined by a CCK8 method:

4.1 cells in logarithmic growth phase were taken at 2X 10 per well⁴Cells were seeded in 96-well plates and cultured to normal growth stage.

4.2 Add WJMSC-EV to incubate overnight.

4.3 Add 10. mu.L of CCK8 solution to each well and incubate for 2 hours in an incubator.

4.4 Absorbance at 450nm was measured using a microplate reader.

4.5 calculate the cell proliferation rate. The results of the experiment are shown in FIG. 8.

As can be seen from FIG. 8, the proliferation rate of HUVECs was significantly increased after co-culture of HUVECs and WJMSC-EV.

Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.

Claims (10)

1. An extracellular nanovesicle for supporting a coronary stent, wherein the extracellular nanovesicle is secreted by umbilical cord Wharton's jelly-derived mesenchymal stem cells.

2. The extracellular nanovesicle according to claim 1, wherein the preparation of the extracellular nanovesicle is a lyophilized powder.

3. The extracellular nanovesicle according to claim 2, wherein the lyophilized powder further comprises an excipient, preferably at least one of mannitol, sorbitol, glucose and lactose, and the weight ratio of the extracellular nanovesicle to the excipient is 1: 50-5000, preferably 1: 100 to 1000.

4. The extracellular nanovesicle according to claim 1, wherein the extracellular nanovesicle is obtained by treating the umbilical cord Wharton's jelly-derived mesenchymal stem cells with a medium containing cytochalasin B and then isolating the cells.

5. The extracellular nanovesicle according to claim 4, wherein the concentration of cytochalasin B is 5-20 μ g/mL, preferably 10 μ g/mL; preferably, the medium is selected from DMEM medium.

6. The extracellular nanovesicle according to claim 4, wherein the separation is selected from the group consisting of membrane affinity separation; preferably, the separation is performed using an exoEasy spin column.

7. Use of the extracellular nanovesicles according to any one of claims 1 to 6 for the preparation of a coronary drug loaded stent, preferably, the extracellular nanovesicles completely replace chemical drug loading and the interior of the stent.

8. A coronary artery drug-loaded stent is loaded in the nano-micro channels of the stent by using the extracellular nano-vesicles as claimed in any one of claims 1 to 6, wherein the stent is selected from cobalt-chromium alloy or platinum-chromium alloy.

9. The method for preparing extracellular nanovesicles according to any one of claims 1 to 6, comprising at least the following steps:

s1, obtaining umbilical cord Wharton jelly source mesenchymal stem cells;

s2, processing the umbilical cord Wharton jelly source mesenchymal stem cells by adopting a culture medium containing cytochalasin B; the treatment temperature is preferably 37 ℃, the time is preferably 20-40 minutes,

s3, separating the extracellular nanovesicles.

10. The method for preparing according to claim 9, wherein the umbilical cord Wharton jelly-derived mesenchymal stem cells are cultured by the following method:

s11, taking the Huatong glue tissue, shearing the Huatong glue tissue into pieces, and performing tissue adherent culture in a plate;

s12, after the bottom of the bottle is filled with 80% of cells, cleaning the bottle to prepare a cell suspension, inoculating the cell suspension into 3D hydrogel, and performing amplification culture at 37 ℃ under the conditions of carbon dioxide with volume fraction of 5% and saturated humidity;

and S13, digesting and washing the cells by using a hydrogel enzyme when 80% of the cells are fused, carrying out passage, and culturing the second generation cells and the third generation cells in a 5% hypoxic cell culture box for 10-18 hours.

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