Artificial nano-vesicles derived from human umbilical cord mesenchymal stem cells induced by natural products and application of artificial nano-vesicles in ischemic diseases

Abstract

The invention discloses a natural product-induced artificial nano-vesicle derived from human umbilical cord mesenchymal stem cells and application of the natural product-induced artificial nano-vesicle in ischemic diseases. The preparation method of the nano vesicle derived from the human umbilical cord mesenchymal stem cells comprises the following steps: S1, inoculating the human umbilical cord mesenchymal stem cells to a 3D microcarrier; then adding traditional Chinese medicine active ingredients to activate the human umbilical cord mesenchymal stem cells, continuing to culture, cracking the microcarrier, and harvesting cells; s2, dispersing the human umbilical cord mesenchymal stem cells obtained in S1 to obtain a suspension, extruding the suspension through a polycarbonate film, and then discarding large particles by adopting a gradient centrifugation method to obtain the nano-vesicles. Compared with naturally secreted exosomes, the exosome has the advantages that the yield is increased, the treatment efficiency is improved, the production cost is reduced, and compared with stem cells, the problems of low transplantation rate and the like are avoided. The stem cells are pretreated based on the traditional Chinese medicine, the vesicles are extracted by utilizing an extrusion method, and the yield and the treatment efficiency of the nano vesicles are remarkably improved.

Description

Technical Field

[0001] The present invention relates to an artificial nanovesicle derived from human umbilical cord mesenchymal stem cells induced by natural products and its application in ischemic diseases, belonging to the fields of medical biotechnology, biopharmaceutical technology, and cell engineering. Background Art

[0002] Ischemic heart diseases, including acute myocardial infarction (MI) and myocardial ischemic / reperfusion infarction (MI / RI), seriously threaten human life and health. Among them, oxidative stress is one of the main pathological mechanisms in the processes of MI and MI / RI. Therefore, preventing and treating oxidative stress is the key to the treatment of ischemic diseases. Currently, no effective treatment method for inhibiting oxidative stress to treat ischemic heart diseases has been found.

[0003] Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles have the potential to treat ischemic heart diseases. However, their low survival rate in vivo after transplantation, difficulty in accurately delivering to the designated site, and ethical considerations severely limit the application of MSCs; the low yield and high extraction cost of MSCs-derived extracellular vesicles affect their clinical translation.

[0004] Therefore, there is an urgent need to provide a preparation method for improving the yield and treatment efficiency of nanovesicles. Summary of the Invention

[0005] The purpose of the present invention is to provide an artificial nanovesicle derived from human umbilical cord mesenchymal stem cells induced by natural products, a method for combining natural products with nanoengineering technology to improve the preparation efficiency and treatment efficiency of artificial nanovesicles derived from human umbilical cord mesenchymal stem cells, and to solve the problem of treating ischemic diseases by regulating oxidative stress based on mesenchymal stem cells.

[0006] The preparation method of the nanovesicle derived from human umbilical cord mesenchymal stem cells provided by the present invention includes the following steps:

[0007] S1. Inoculate human umbilical cord mesenchymal stem cells on a 3D microcarrier; then add the active ingredient of traditional Chinese medicine to activate the human umbilical cord mesenchymal stem cells, continue culturing, lyse the microcarrier, and harvest the cells;

[0008] S2. Disperse the human umbilical cord mesenchymal stem cells obtained in step S1 to obtain a suspension, extrude the suspension through a polycarbonate membrane, and then use gradient centrifugation to discard large particles to obtain nanovesicles.

[0009] In the preparation method of the present invention, in step S1, the inoculation concentration of the human umbilical cord mesenchymal stem cells is 6000 - 10000 cells / cm 2 ;

[0010] The 3D microcarrier is a 3D porous spherical microcarrier based on recombinant collagen micro raw materials.

[0011] In the preparation method of the present invention, in step S1, the traditional Chinese medicine active ingredient is added 24 hours after inoculating the human umbilical cord mesenchymal stem cells.

[0012] In the preparation method of the present invention, in step S1, the concentration of the traditional Chinese medicine active ingredient is 100 - 1000 μM, preferably 125 - 1000 μM, more preferably 125 μM.

[0013] In the preparation method of the present invention, in step S1, after adding the traditional Chinese medicine active ingredient, the cells are cultured for 3 - 5 days.

[0014] In the preparation method of the present invention, in step S1, the traditional Chinese medicine active ingredient is the active ingredient of ginseng, the active ingredient of salvia miltiorrhiza or the active ingredient of astragalus membranaceus;

[0015] The active ingredient of ginseng can be ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, etc.;

[0016] The active ingredient of salvia miltiorrhiza can be tanshinone IIA, neocrytanshinone, salvianolic acid A, salvianolic acid B, etc.

[0017] The active ingredient of astragalus membranaceus can be calycosin.

[0018] In the preparation method of the present invention, in step S2, the human umbilical cord mesenchymal stem cells are dispersed in PBS phosphate buffer to obtain the suspension;

[0019] The cell density in the suspension is 1×10 6 -8×10 6 cells / mL.

[0020] In the preparation method of the present invention, in step S2, the suspension is extruded through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm in sequence;

[0021] The steps of the gradient centrifugation method are as follows: centrifuge at 300 g for 10 minutes; take the supernatant, centrifuge at 2000 g for 10 minutes; take the supernatant, centrifuge at 10000 g for 10 minutes; take the supernatant, centrifuge at 120000 g for 70 minutes; discard the supernatant, take the precipitate, resuspend with PBS, centrifuge at 120000 g for 70 minutes, discard the supernatant, and take the precipitate.

[0022] The nanovesicles prepared by the method of the present invention also fall within the protection scope of the present invention.

[0023] The preparation method of the mesenchymal stem cell-derived nanovesicles provided by the present invention not only improves the yield of the nanovesicles derived from mesenchymal stem cells, but also improves the therapeutic efficiency of the nanovesicles by regulating oxidative stress.

[0024] The nanovesicles derived from stem cells provided by the present invention significantly increase the yield of nanovesicles, and the nanovesicles can treat ischemic diseases (such as ischemic heart disease) by alleviating oxidative stress. The present invention is different from mesenchymal stem cells and exosomes derived from mesenchymal stem cells. The artificially synthesized nanovesicles are nanostructures, avoiding the influence of low transplantation rate on the therapeutic effect, etc.; compared with the exosomes naturally secreted by mesenchymal stem cells, the artificially synthesized nanovesicles significantly increase the yield and have better antioxidant stress efficacy.

[0025] Compared with the prior art, the method for pretreating mesenchymal stem cells with active ingredients of traditional Chinese medicine and preparing artificially synthesized nanovesicles by extrusion provided by the present invention improves the yield and therapeutic efficiency of nanovesicles. Compared with the exosomes secreted naturally, the present invention improves the therapeutic efficiency while increasing the yield and reducing the production cost. Compared with stem cells, problems such as low transplantation rate are avoided. The present invention is based on pretreating stem cells with traditional Chinese medicine and extracting vesicles by extrusion, significantly increasing the yield and therapeutic efficiency of nanovesicles. Description of the Drawings

[0026] Figure 1 It is a fluorescence image of three-dimensional cultured cells in Example 1 of the present invention (green is calcein live cell dye, and red is propidium iodide dead cell dye);

[0027] Figure 2 It is a statistical chart of the number of three-dimensional cultured cells in Example 1 of the present invention;

[0028] Figure 3 It is the effect of ginsenoside Rg1 at different concentrations on promoting the proliferation of mesenchymal stem cells;

[0029] Figure 4 It is a transmission electron microscopy image of the nanovesicles in Example 1 of the present invention;

[0030] Figure 5 It is the NTA result of the artificially synthesized nanocapsules derived from mesenchymal stem cells in Example 1 of the present invention;

[0031] Figure 6 It is the HPLC result of the artificially synthesized nanocapsules derived from mesenchymal stem cells in Example 1 of the present invention;

[0032] Figure 7 It is the protein marker of the artificially synthesized nanovesicles derived from mesenchymal stem cells detected by Western blotting in Example 1 of the present invention;

[0033] Figure 8Statistical results of the therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on the reactive oxygen species damage of human cardiomyocyte line AC16 and rat cardiomyocyte line H9c2. The absorbance value of CCK8 was detected using a microplate reader.

[0034] Figure 9 Therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on the reactive oxygen species damage of human cardiomyocyte line AC16 and rat cardiomyocyte line H9c2. ROS fluorescence signal was detected using flow cytometry analysis.

[0035] Figure 10 Cardiac function therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on rat myocardial ischemia-reperfusion - echocardiogram.

[0036] Figure 11 Therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on the myocardial tissue structure of rat myocardial ischemia-reperfusion - HE staining.

[0037] Figure 12 Therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on the myocardial tissue fibrosis level of rat myocardial ischemia-reperfusion - Masson staining.

[0038] Figure 13 Therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on the myocardial tissue oxidative stress of rat myocardial ischemia-reperfusion - ROS staining.

[0039] Figure 14 Therapeutic effect of mesenchymal stem cell-derived synthetic nanovesicles in Example 1 of the present invention on the oxidative stress of rat myocardial ischemia-reperfusion - detection of CAT, SOD, GSH and MDA in plasma. Specific embodiments

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0041] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0042] The reagents in the following examples were purchased from Thermo Fisher Scientific, the cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd., the data were expressed as mean ± standard deviation, and one-way ANOVA was used for multiple group comparisons. Statistical analysis was performed using GraphPad Prism 8.0.

[0043] The 3D microcarrier in the following examples is a 3D porous spherical microcarrier 3D with recombinant collagen as the raw material. The microcarrier W01 was purchased from Beijing Huakan Biotech Co., Ltd.

[0044] Example 1: Preparation of nanovesicles

[0045] (1) Human umbilical cord mesenchymal stem cells were seeded at a density of 8000 cells / cm 2 onto 3D microcarriers and cultured using a bioreactor. After 24 hours of seeding, the MSCs were activated with 125 μM ginsenoside Rg1 (the seeding day was D0), and continuous culture was continued for 5 days. Samples of 2 mL were taken on the 1st, 3rd, and 5th days respectively. 200 μL of the carrier suspension was added with 0.1 μL of calcein-AM and 0.1 μL of propidium iodide (PI) to stain the 3D microcarriers, and observed with a fluorescence microscope (as Figure 1 ); further, 1 mL of the carrier suspension was added with lysis buffer (3D Digest Lysis Buffer R001, Beijing Huakan Biotech Co., Ltd.) to a final concentration of 5 mg / ml to lyse the carriers. After complete lysis, the cells were harvested by centrifugation at 1500 rpm for 5 minutes, resuspended in PBS, and the cell number was counted using trypan blue (as Figure 2 ).

[0046] Figure 1 Figure of the fluorescence of three-dimensional cultured cells in Example 1 (green is calcein live cell dye, red is propidium iodide dead cell dye). It can be seen that the mesenchymal stem cells grow well on the 3D microcarriers.

[0047] Figure 2 Statistical chart of the number of three-dimensional cultured cells in Example 1. It can be seen that based on the cell number statistics, ginsenoside Rg1 stimulation can increase the yield of mesenchymal stem cells.

[0048] Figure 3 Shows the effects of different concentrations of ginsenoside Rg1 on promoting the proliferation of mesenchymal stem cells. It can be seen that different concentrations all have the effect of promoting MSC proliferation, but 125 μM is the most economically valuable.

[0049] (2) The MSCs cells activated by traditional Chinese medicine in step (1) were dispersed in phosphate-buffered saline (PBS), and the cell density was 1×10 6cells / mL; Use a syringe to aspirate the MSCs suspension and extrude the MSCs through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm respectively; Use gradient centrifugation to discard larger particles. The specific operation is as follows: Centrifuge at 300 g for 10 minutes; Take the supernatant and centrifuge at 2000 g for 10 minutes; Take the supernatant and centrifuge at 10000 g for 10 minutes; Take the supernatant and centrifuge at 120000 g for 70 minutes; Discard the supernatant, take the precipitate, resuspend it with PBS, and centrifuge at 120000 g for 70 minutes to obtain nanovesicles. Detect the morphology and surface markers of the nanovesicles and further calculate the yield.

[0050] Figure 4 Figure 1 is the transmission electron microscopy imaging of the nanovesicles in Example 1. It can be seen that the artificially synthesized nanovesicles have a structure similar to that of naturally secreted vesicles, a cup-shaped structure wrapped by a bilayer phospholipid membrane.

[0051] Figure 5 Figure 2 is the NTA result of the nanovesicles in Example 1. It can be seen that, similar to naturally secreted vesicles, the diameter of the artificially synthesized vesicles is between 100 - 200 nanometers.

[0052] Figure 6 Figure 3 is the HPLC result of the nanovesicles in Example 1. It can be seen that the artificially synthesized vesicles do not encapsulate ginsenoside Rg1. Among them, a C18 chromatographic column was used, with 20% acetonitrile and 80% water as the mobile phase, a flow rate of 1 mL / min, an injection volume of 10 μL, and detection was carried out using an ultraviolet detector at a wavelength of 210 nm.

[0053] Figure 7 Figure 4 is the protein markers of the mesenchymal stem cell-derived artificially synthesized nanovesicles detected by Western blotting. It can be seen that the artificially synthesized nanovesicles have surface markers similar to those of naturally secreted vesicles, including TSG101, CD9, CD63, CD81, and GAPDH.

[0054] Table 1 is the statistics of the number of nanovesicles prepared using mesenchymal stem cells in Example 1. Table 1 Statistics of the number of nanovesicles prepared using mesenchymal stem cells

[0055]

[0056] Example 2: Pharmacodynamic verification cell experiment of nanovesicles

[0057] Verify the pharmacodynamics of the nanovesicles by measuring the level of apoptosis and changes in oxidative stress.

[0058] 1. Seed human cardiomyocyte AC16 cell line and rat cardiomyocyte H9c2 cells at a density of 1×10 4Inoculate at a concentration of 2×10 cells / ml into a 96-well plate. After 24 hours of inoculation, observe the cell status and distribution. If the cells are in good condition and evenly distributed, proceed with the subsequent drug efficacy experiments.

[0059] First, verify the apoptosis situation: Divide into a blank group, a model group, and a treatment group. The blank group is given a complete medium with 10% serum; the model group is added with 1600 μM of H2O2; the treatment group is added with 20 μg / mL of nanovesicles (Rg1-ACDVs); the control group is added with 20 μg / mL of extracellular vesicles (EVs). After 24 hours of treatment, use CCK8 to detect the relative viability of AC16 and H9c2 cells (as Figure 5 ).

[0060] 2. Inoculate the human cardiomyocyte AC16 cell line and the rat cardiomyocyte H9c2 cell line at a concentration of 2×10 5 cells / ml into a 6-well plate. The grouping and treatment are the same as above; verify the drug efficacy by comparing the content of reactive oxygen species (ROS) in the blank group, the model group, and the drug administration group (as Figure 6 ).

[0061] Figure 8 This is the statistical result of the therapeutic effect of the mesenchymal stem cell-derived artificial synthetic nanovesicles prepared in Example 1 of the present invention on the reactive oxygen species damage of the human cardiomyocyte cell line AC16 and the rat cardiomyocyte cell line H9c2. It can be seen that compared with the naturally secreted vesicles, the Rg1-activated artificial synthetic nanovesicles can better alleviate the cardiomyocyte damage induced by H2O2.

[0062] Figure 9 This is the therapeutic effect of the mesenchymal stem cell-derived artificial synthetic nanovesicles prepared in Example 1 of the present invention on the reactive oxygen species damage of the human cardiomyocyte cell line AC16 and the rat cardiomyocyte cell line H9c2. It can be seen that compared with the naturally secreted vesicles, the Rg1-activated artificial synthetic nanovesicles can better alleviate the effect of oxidative stress of cardiomyocytes induced by H2O2.

[0063] Example 3. Pharmacodynamic verification of nanovesicles - animal experiment

[0064] The wild-type SD rats used were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0065] The rats were maintained in a specific pathogen - free environment at a temperature of 25°C with a 12 - hour light / dark cycle. They were adaptively fed for 3 days before the experiment. Next, a rat myocardial ischemia - reperfusion model was constructed. The rats were intraperitoneally injected with 1% sodium pentobarbital, fixed on a rat fixing plate, the hair on the left anterior chest was shaved, disinfected with 75% alcohol, and then connected to a ventilator. Blunt instruments were used to dissect the muscles between the third and fourth intercostal spaces to gain access to the thoracic cavity. The left atrial appendage was ligated with 5 - 0 surgical sutures at a position approximately 1 to 2 mm below the left atrial appendage. After 30 minutes of ligation, synthetic nanovesicles activated by Rg1 were injected via intramyocardial injection. Subsequently, the sutures were removed, and the thoracic cavity was sutured with 2 - 0 surgical sutures. The control group only underwent puncture but not ligation and was injected with an equal volume of normal saline. Samples were obtained 24 hours after reperfusion for efficacy testing.

[0066] Figure 10 For the echocardiogram results, it can be seen that the synthetic vesicles can alleviate ischemia - reperfusion - induced cardiac function injury.

[0067] Figure 11 For the HE staining results, it can be seen that the synthetic vesicles can alleviate ischemia - reperfusion - induced disordered myocardial tissue arrangement.

[0068] Figure 12 For the Masson staining results, it can be seen that the synthetic vesicles can alleviate ischemia - reperfusion - induced myocardial tissue fibrosis.

[0069] Figure 13 For the ROS tissue section staining results, it can be seen that the synthetic vesicles can alleviate ischemia - reperfusion - induced myocardial tissue oxidative stress.

[0070] Figure 14 For the results of serum oxidative stress indicators (CAT, SOD, GSH, and MDA), it can be seen that the synthetic vesicles can reduce the oxidative stress level induced by ischemia - reperfusion.

Claims

1. A method for preparing nanovesicles derived from human umbilical cord mesenchymal stem cells, comprising the following steps: S1. Inoculate human umbilical cord mesenchymal stem cells on a 3D microcarrier; then add a traditional Chinese medicine active ingredient to activate the human umbilical cord mesenchymal stem cells, continue culturing, lyse the microcarrier, and harvest the cells; S2. Disperse the human umbilical cord mesenchymal stem cells obtained in step S1 to obtain a suspension, extrude the suspension through a polycarbonate membrane, and then use gradient centrifugation to discard large particles to obtain nanovesicles.

2. The preparation method according to claim 1, characterized in that: In step S1, the seeding concentration of the human umbilical cord mesenchymal stem cells is 6,000 - 10,000 cells / cm 2 ; The 3D microcarrier is a 3D porous spherical microcarrier with recombinant collagen as the raw material.

3. The preparation method according to claim 1 or 2, characterized in that: In step S1, the traditional Chinese medicine active ingredient is added 24 hours after inoculating the human umbilical cord mesenchymal stem cells.

4. The preparation method according to any one of claims 1-3, characterized in that: In step S1, the concentration of the traditional Chinese medicine active ingredient is 100 - 1000 μM.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step S1, continue culturing for 3 - 5 days after adding the traditional Chinese medicine active ingredient.

6. The preparation method according to any one of claims 1-5, characterized in that: In step S1, the traditional Chinese medicine active ingredient is a ginseng active ingredient, a salvia miltiorrhiza active ingredient, or an astragalus membranaceus active ingredient.

7. The preparation method according to any one of claims 1-6, characterized in that: In step S2, disperse the human umbilical cord mesenchymal stem cells in PBS to obtain the suspension; The cell density in the suspension is 1×10 6 -1×10 7 cells / mL.

8. The preparation method according to any one of claims 1-7, characterized in that: In step S2, extrude the suspension through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm in sequence; The steps of the gradient centrifugation method are as follows: centrifuge at 300 g for 10 minutes; take the supernatant, centrifuge at 2000 g for 10 minutes; take the supernatant, centrifuge at 10000 g for 10 minutes; take the supernatant, centrifuge at 120000 g for 70 minutes; discard the supernatant, take the precipitate, resuspend with PBS, and centrifuge at 120000 g for 70 minutes.

9. Nanovesicles prepared by the method according to any one of claims 1 - 8.

10. Use of the nanovesicles according to claim 9 in the manufacture of a medicament for treating ischemic diseases or as a medicament for treating ischemic diseases.

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