Activated stem cell microcapsules, methods of making and using same

Abstract

The present application relates to an activated stem cell microcapsule, a preparation method and application thereof. The activated stem cell microcapsule is prepared by the following method: taking mesenchymal stem cells, culturing the mesenchymal stem cells in a culture medium containing resveratrol, collecting the supernatant, and centrifuging the supernatant by differential centrifugation to obtain the activated stem cell microcapsule; the concentration of the resveratrol in the culture medium is less than 1.5 micromoles per liter. Then the activated stem cell microcapsule is mixed with a nitric oxide donor for incubation to obtain an activated stem cell microcapsule loaded with a nitric oxide donor. The activated stem cell microcapsule and the activated stem cell microcapsule loaded with the nitric oxide donor provided by the present application have good blood vessel repair effect and can be used for preparing a drug for treating peripheral vascular lesions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to an activated stem cell microcapsule, a nitric oxide donor-loaded activated stem cell microcapsule and a preparation method and application thereof. BACKGROUND

[0002] Peripheral vascular disease is a vascular disease other than cardiovascular and cerebrovascular disease, which includes diseases of the three systems of arteries, veins and lymphatics. According to the investigation of the World Health Organization, peripheral vascular disease is a highly dangerous high-incidence disease. If it is not cured for a long time, the disease will develop progressively, and severe cases will lead to amputation and even endanger life. According to incomplete statistics, about 500,000 legs are amputated at a high level every year in China due to vascular disease and diabetic foot (gangrene), and 2 million feet (legs) are gangrenous and ulcerated and cannot heal. At present, revascularization surgery, including surgical bypass surgery and interventional stent surgery, is the main clinical treatment scheme. However, due to the lack of autologous vein grafts, extensive disease or high incidence of complications of tibial and peroneal arteries, 25-50% of peripheral vascular disease patients cannot be treated by revascularization surgery. For these patients, the amputation rate can increase to 50%. Therefore, it is of great significance to seek drug treatment for these patients who cannot choose revascularization surgery.

[0003] Stem cell therapy is the most promising method to solve peripheral vascular disease. Stem cells can play a therapeutic role in peripheral vascular disease through differentiation and proliferation, paracrine and mitochondrial transfer. However, although cell therapy based on stem cells has made some progress in a large number of studies, it has not shown significant clinical benefits so far. The main reasons are the lack of oxygen and blood supply in the lesion site, low cell survival rate after stem cell transplantation to the lesion site, insufficient paracrine signal and impaired mitochondrial function of cells. SUMMARY

[0004] Therefore, the present application provides a preparation method of an activated stem cell microcapsule, which activates stem cells by using resveratrol, stimulates them to secrete more cell microcapsules carrying growth factors, and obtains activated stem cell microcapsules by differential centrifugation. Compared with unactivated stem cell microcapsules, the activated stem cell microcapsules prepared by the preparation method of the present application have higher content of vascular growth-promoting factors, better effect of improving the mitochondrial potential of damaged vascular endothelial cells, better effect of improving the function of vascular endothelial cells, and better vascular repair effect.

[0005] Specifically, the technical solutions include the following.

[0006] A preparation method of an activated stem cell microcapsule, comprising the following steps:

[0007] Mesenchymal stem cells are taken, cultured in a medium containing resveratrol, and then the supernatant is collected and centrifuged by differential centrifugation to obtain activated stem cell microvesicles;

[0008] The concentration of resveratrol in the medium is less than 1.5 μmol / L.

[0009] In some embodiments, the concentration of resveratrol in the medium is less than 1 μmol / L.

[0010] In some embodiments, the concentration of resveratrol in the medium is less than 0.5 μmol / L.

[0011] In some embodiments, the concentration of resveratrol in the medium is 0.03125 μmol / L-0.5 μmol / L.

[0012] In some embodiments, the concentration of resveratrol in the medium is 0.03125 μmol / L-0.5 μmol / L.

[0013] In some embodiments, the concentration of resveratrol in the medium is 0.125 μmol / L-0.5 μmol / L.

[0014] In some embodiments, the concentration of resveratrol in the medium is 0.2 μmol / L-0.3 μmol / L.

[0015] In some embodiments, the culturing time is greater than 10 hours.

[0016] In some embodiments, the culturing time is 20 hours-72 hours.

[0017] In some embodiments, the culturing conditions include 35-40°C, 5% CO2.

[0018] In some embodiments, the differential centrifugation includes centrifugation at 400-600 g for 30-90 min, discarding the precipitate, centrifugation of the supernatant at 2000-3000 g for 30-90 min, discarding the precipitate, and centrifugation of the supernatant at 15000-25000 g for 30-90 min.

[0019] In some embodiments, the differential centrifugation includes centrifugation at 450-550 g for 50-70 min, discarding the precipitate, centrifugation of the supernatant at 2300-2800 g for 50-70 min, discarding the precipitate, and centrifugation of the supernatant at 18000-22000 g for 50-70 min.

[0020] The application also provides the activated stem cell microcapsules prepared by the preparation method. Compared with the non-activated stem cell microcapsules, the activated stem cell microcapsules have a significantly increased content of the pro-angiogenic factor, a better effect of improving the mitochondrial potential of the damaged vascular endothelial cells, and a better effect of improving the function of the vascular endothelial cells.

[0021] The application also provides the activated stem cell microcapsules loaded with a nitric oxide donor. The activated stem cell microcapsules loaded with the nitric oxide donor have a strong ability to induce the vascular endothelial cells to produce endogenous nitric oxide, and can induce the damaged vascular endothelial cells to release a large amount of endogenous nitric oxide. The activated stem cell microcapsules loaded with the nitric oxide donor can promote the formation of blood vessels in ischemic sites, can significantly improve the angiogenic ability of the damaged endothelial cells, and can effectively treat diseases such as peripheral vascular diseases.

[0022] Specifically, the application includes the following technical solutions.

[0023] The application also provides the activated stem cell microcapsules loaded with a nitric oxide donor. The activated stem cell microcapsules loaded with the nitric oxide donor have a strong ability to induce the vascular endothelial cells to produce endogenous nitric oxide, and can induce the damaged vascular endothelial cells to release a large amount of endogenous nitric oxide. The activated stem cell microcapsules loaded with the nitric oxide donor can promote the formation of blood vessels in ischemic sites, can significantly improve the angiogenic ability of the damaged endothelial cells, and can effectively treat diseases such as peripheral vascular diseases.

[0024] In some embodiments, the nitric oxide donor is L-arginine and / or nitroglycerin.

[0025] In some embodiments, the mass ratio of the activated stem cell microcapsules to the nitric oxide donor is 1-3:1.

[0026] In some embodiments, the mass ratio of the activated stem cell microcapsules to the nitric oxide donor is 1-3:1.

[0027] The application also provides a preparation method of the activated stem cell microcapsules loaded with the nitric oxide donor.

[0028] The application also provides a preparation method of the activated stem cell microcapsules loaded with the nitric oxide donor.

[0029] The application also provides a preparation method of the activated stem cell microcapsules loaded with the nitric oxide donor.

[0030] The application also provides the application of the activated stem cell microcapsules and the activated stem cell microcapsules loaded with the nitric oxide donor.

[0031] The application also provides the application of the activated stem cell microcapsules and the activated stem cell microcapsules loaded with the nitric oxide donor.

[0032] The present application proposes to use cell microvesicles autonomously secreted by stem cells to replace stem cells to play a role in repairing blood vessels. The cell microvesicle is one of the extracellular vesicles autonomously secreted by cells, which inherits the regenerative function of the parent cell. And unlike the high cost required for exosome extraction, cell microvesicles can be obtained under conventional high-speed centrifugation conditions. In order to increase the content of growth factors carried in the cell microvesicles and improve the autonomous secretion amount of stem cells, the present application uses resveratrol as an activator to stimulate stem cells to secrete more cell microvesicles carrying growth factors, and then extracts the cell microvesicles by gradient centrifugation. The nitric oxide donor, which has the effect of expanding blood vessels but no effect of promoting angiogenesis, is loaded into the stem cell microvesicles that improve the ischemic environment and improve the function of angiogenesis. Finally, an activated stem cell microvesicle loaded with a nitric oxide donor is constructed for treating peripheral vascular lesions, promoting angiogenesis and repairing damaged tissues. The activated stem cell microvesicle loaded with a nitric oxide donor provided by the present application brings a new design of drugs for treating peripheral vascular lesions, overcomes the hidden danger of canceration of stem cells, and improves the treatment effectiveness and stability of stem cell therapy.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present inventors found that a small amount of resveratrol can promote the regenerative function of stem cells. By activating stem cells with a certain concentration of resveratrol, more cell microvesicles carrying growth factors can be secreted. The activated stem cell microvesicles obtained by the present application have a significantly higher content of pro-angiogenic growth factors than unactivated stem cell microvesicles, have a better effect on improving the mitochondrial potential of damaged vascular endothelial cells, have a better effect on improving the function of vascular endothelial cells, have a better vascular repair effect, and can be used for preparing drugs for treating peripheral vascular lesions.

[0035] Further, the present application incubates the activated stem cell microvesicles obtained with a nitric oxide donor that produces nitric oxide or stimulates endogenous nitric oxide production to obtain an activated stem cell microvesicle loaded with a nitric oxide donor. The activated stem cell microvesicle loaded with a nitric oxide donor repairs damaged blood vessels, regulates inflammation through stem cell microvesicles, and delivers a nitric oxide donor to the lesion site to expand blood vessels and recruit vascular smooth muscle cells. The two work together to significantly promote vascular tissue regeneration, promote angiogenesis in ischemic areas, significantly improve the angiogenic ability of hypoxic damaged endothelial cells, and improve the effect of treating peripheral vascular lesions. The activated stem cell microvesicle loaded with a nitric oxide donor has a significantly better treatment effect on severe limb ischemia than the simple stem cell microvesicle and nitric oxide donor group, and is better than the resveratrol-activated stem cell microvesicle of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 For the stem cells of Example 1.

[0037] Figure 2 Effect of Resveratrol on Stem Cell Viability for Example 2.

[0038] Figure 3 Effect of Resveratrol on Stem Cell ATP Production for Example 3.

[0039] Figure 4 Effect of Resveratrol on Vascular Endothelial Cell Viability for Example 4.

[0040] Figure 5 Effect of Resveratrol on Ischemic Limb Repair for Example 5.

[0041] Figure 6 Effect of Resveratrol on Stem Cell Secreted Cell Microvesicles for Example 6.

[0042] Figure 7 Results of Activated Stem Cell Microvesicle (AMS) Angiogenic Factor Assay for Example 7.

[0043] Figure 8 Effect of Activated Stem Cell Microvesicle (AMS) on Hypoxically Damaged Vascular Endothelial Cell Mitochondrial Viability for Example 8.

[0044] Figure 9 Effect of Activated Stem Cell Microvesicle (AMS) on Hypoxically Damaged Vascular Endothelial Cell Viability for Example 9.

[0045] Figure 10 Ability of Nitric Oxide Donor Loaded Activated Stem Cell Microvesicle (AMS-NO) to Induce Endogenous Nitric Oxide Production in Hypoxically Damaged Vascular Endothelial Cells for Example 10.

[0046] Figure 11 Ability of Nitric Oxide Donor Loaded Activated Stem Cell Microvesicle (AMS-NO) to Induce Endogenous Nitric Oxide Release in Hypoxically Damaged Vascular Endothelial Cells for Example 11.

[0047] Figure 12 Effect of Nitric Oxide Donor Loaded Activated Stem Cell Microvesicle (AMS-NO) on Promoting Angiogenesis in Damaged Blood Vessels for Example 12.

[0048] Figure 13 Pharmacodynamic Results of Nitric Oxide Donor Loaded Activated Stem Cell Microvesicle (AMS-NO) for Treating Critical Limb Ischemia for Example 13. DETAILED DESCRIPTION

[0049] The experimental methods in the following examples of the present application, unless otherwise specified, are generally carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers. The various common chemical reagents used in the examples are commercially available products.

[0050] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0051] The terms "comprising" and "having" and any variations thereof used in the present application are intended to cover a non-exclusive inclusion. For example, a process, method, device, product or apparatus that comprises a list of steps or modules is not limited to the listed steps or modules, but can optionally further include other steps or modules not listed, or can optionally further include other steps inherent to such processes, methods, products or apparatus.

[0052] In the present application, "a plurality of" means two or more. "And / or", which describes the association relationship of the associated objects, means that there can be three relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. The character " / " generally represents that the associated objects before and after it are in an "or" relationship.

[0053] The following are specific examples.

[0054] Example 1 Preparation of activated stem cell microcapsules loaded with nitric oxide donors (AMS-NO)

[0055] I. Preparation of Resveratrol Drug Stock Solution

[0056] 69.6 mg of resveratrol powder was weighed and dissolved in 6.1 mL of DMSO to prepare a drug solution with a final concentration of 50 mmol / L.

[0057] II. Isolation, culture and identification of mouse bone marrow mesenchymal stem cells

[0058] (1) Adherent culture method was used to isolate mouse bone marrow mesenchymal stem cells. 8-10 weeks old female BALB / c mice were prepared, and the femur and tibia of the mice were isolated, the surrounding connective tissue was stripped, and then soaked in the prepared culture medium and transferred to a sterile environment in a clean bench for subsequent operation. The bone was cut at both ends to make it open, a 10 mL syringe was filled with culture medium, a 1 mL syringe needle was replaced, the needle was inserted into one end of the bone, and the bone marrow cavity was repeatedly flushed until the bone turned white. The obtained bone marrow cells were collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, 1 mL of red blood cell lysis solution was added, and after blowing evenly, it was left to stand for 3 min. 10 mL of culture medium was added to terminate the reaction, and centrifuged at 1500 rpm for 5 min.

[0059] (2) The cell pellet was resuspended in DMEM / F12 medium containing 10% serum and 1% double antibody, then inoculated in a culture dish, and cultured in a cell incubator at 37°C, 5% CO2. After 72 h, the supernatant was aspirated, the non-adherent cells were washed away, fresh culture medium was added, and the culture was continued, and the medium was changed every 2 days. When the cell confluence reached 80%, the cells were treated with 0.25% trypsin containing EDTA and passaged. Mesenchymal stem cells of 5-20 passages were used for subsequent experiments.

[0060] (3) The obtained mouse bone marrow mesenchymal stem cells were phenotypically identified by flow cytometry. Well-grown mesenchymal stem cells in the logarithmic growth phase were collected, washed with an appropriate amount of cell staining buffer, and centrifuged at 1500 rpm for 5 min. The cell pellet was resuspended with 100 μL of cell staining buffer, and the antibodies Sca-1 and CD29 for labeling mouse bone marrow mesenchymal stem cells, and the antibodies CD45 and CD11b for labeling blood cells in bone marrow were added. Incubate at room temperature for 30 min, wash the cells 3 times with cell staining buffer, and then detect on the machine. Undyed MSC cells were used as a blank control. The results of stem cell identification are shown in Figure 1

[0061] III. Preparation of activated stem cell microcapsules loaded with nitric oxide donors (AMS-NO)

[0062] (1) Collect mesenchymal stem cells MSC in good growth state and in logarithmic growth phase, inoculate at a density of 4 x 10 6 Inoculate at a density of 4 x 10

[0063] ​(2) Lipid-soluble resveratrol was dissolved in cell-grade DMSO to prepare a solution with a concentration of 50 mM. Resveratrol drug stock solution was prepared with DMEM / F12 medium containing 10% serum and 1% double antibodies to have a concentration of 0.25 μmol / L. After the cells adhered, the original culture medium in the culture dish was aspirated, and 0.25 μmol / L resveratrol drug solution was added to stimulate stem cells. The culture was incubated at 37°C in a 5% CO2 incubator.

[0064] (3) After 24 hours of drug action, the supernatant in the culture dish was collected.

[0065] (4) The collected supernatant was centrifuged at 4°C and 500g for 1 hour to remove cells and cell debris. Then the supernatant was transferred to a new centrifuge tube and centrifuged at 4°C and 2500g for 60 minutes to remove the precipitate. The supernatant was transferred to a new centrifuge tube and centrifuged at 20000g for 60 minutes. The precipitate was collected as activated stem cell microcapsules (AMS) and resuspended in sterile PBS for storage in a 4°C refrigerator.

[0066] (5) The activated stem cell microcapsules were mixed with a nitric oxide donor (L-arginine or nitrate compounds) at a mass ratio of 2:1 and incubated at 4°C for 12 hours to obtain activated stem cell microcapsules loaded with a nitric oxide donor (AMS-NO).

[0067] Example 2 Effect of Resveratrol on Stem Cell Activity

[0068] The effect of resveratrol on stem cell activity was investigated by MTT method. The specific steps are as follows:

[0069] (1) Mesenchymal stem cells in the logarithmic growth phase were digested with trypsin to prepare a single cell suspension, which was mixed uniformly. The cells were inoculated into a 96-well plate at a density of 5×10 3 cells / well, 150 μL of medium was added to each well, and the culture was incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0070] (2) Lipid-soluble resveratrol was dissolved in cell-grade DMSO to prepare a solution with a concentration of 50 mM. Resveratrol drug stock solution was prepared with DMEM / F12 medium containing 10% serum and 1% double antibodies to have a concentration of 0.25 μmol / L. After the cells adhered, the original culture medium in the culture dish was aspirated, and 0.25 μmol / L resveratrol drug solution was added to stimulate stem cells. The culture was incubated at 37°C in a 5% CO2 incubator.

[0071] (3) Add 20 μL of 5 mg / mL MTT solution to each well, and continue to incubate at 37°C in a 5% CO2 incubator for 4 h.

[0072] (4) Discard the original culture medium, and then add 150 μL of DMSO to each well, and shake on a shaker for 10 min to fully dissolve the methylene blue crystals. Detect the absorbance values at 570 nm and 630 nm by a microplate reader, and calculate the cell viability according to the following formula:

[0073]

[0074] The results are shown in Table 1. Figure 2 As can be seen from Table 1, the stem cell viability can be improved under the stimulation of low-dose resveratrol (RSV). Figure 2 It can also be seen from the results that resveratrol only has a promoting effect on the cell viability of stem cells within a certain concentration range, and the effect is not better with a larger dose. The optimal concentration that can significantly improve the stem cell viability is 0.25 μmol / L.

[0075] Example 3: Effect of resveratrol on ATP production of stem cells

[0076] After the stem cells are treated with resveratrol solutions of different concentrations for 24 h or 48 h, the ATP level in the cells is detected to investigate the ATP production of the stem cells. The specific operation is as follows:

[0077] (1) Collect mesenchymal stem cells in the logarithmic growth phase, and inoculate 5 × 10 5 cells / well into a 6-well plate, add 2 mL of culture medium to each well, and incubate at 37°C in a 5% CO2 incubator for 24 h.

[0078] (2) Dissolve liposoluble resveratrol in cell-grade DMSO to prepare a solution with a concentration of 50 mmol / L. Use the dilution method to prepare resveratrol solutions with concentrations of 4 μmol / L, 2 μmol / L, 1 μmol / L, 0.5 μmol / L, 0.25 μmol / L, 0.125 μmol / L, 0.0625 μmol / L, and 0.03125 μmol / L in DMEM / F12 culture medium containing 10% serum and 1% double antibody. Discard the original culture medium in the culture plate, and add 2 mL of the above-mentioned drug solution of different concentrations to each well. The blank group is added with culture medium without resveratrol. Continue to incubate at 37°C in a 5% CO2 incubator for 24 h or 48 h.

[0079] (3) Aspirate the culture medium and add 200 μL of lysis buffer to each well to lyse the cells. Use a pipette to repeatedly pipette to ensure that the lysis buffer fully contacts and lyses the cells. Collect the cell lysate and centrifuge at 12,000 g for 5 minutes at 4°C. Remove the supernatant and use it for subsequent measurements.

[0080] (4) Dissolve the reagents to be used in an ice bath and dilute the ATP standard solution with ATP detection lysis buffer to an appropriate concentration gradient. The concentrations are 0 μmol / L, 0.3125 μmol / L, 0.625 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, and 10 μmol / L.

[0081] (5) Add 100 μL of ATP assay working solution to each assay well. After standing at room temperature for 3–5 min, add 100 μL of sample or standard to the assay well. Mix thoroughly and immediately measure the relative light unit (RLU) value using a multifunctional microplate reader. Calculate the ATP content in the sample based on the standard curve.

[0082] Test results such as Figure 3 As shown: The stimulating effect of resveratrol (RSV) on stem cells can increase the ATP production capacity of stem cells. At a dose of 0.25μmol / L, the ATP production capacity of stem cells can be increased by 2 times.

[0083] Example 4 Effect of resveratrol on vascular endothelial cell viability

[0084] The MTT assay was used to investigate the effect of resveratrol on vascular endothelial cell viability. The specific steps are as follows:

[0085] (1) The vascular endothelial cells in the logarithmic growth phase were digested with trypsin to prepare a single cell suspension and mixed evenly. 3 Cells were seeded into 96-well plates at a density of 10 cells / well, 150 μL of culture medium was added to each well, and the cells were cultured in 5% CO2 and 37°C. After the cells attached, 500 μmol / L cobalt chloride was added and cultured for 12 hours to induce cell hypoxia.

[0086] (2) Resveratrol was dissolved in cell grade DMSO to make a solution with a concentration of 50 mmol / L. The solution was diluted by a factor of two to make solutions with concentrations of 1000 μmol / L, 900 μmol / L, 800 μmol / L, 700 μmol / L, 600 μmol / L, 500 μmol / L, 400 μmol / L, 300 μmol / L, 200 μmol / L, and 100 μmol / L in DMEM / F12 medium containing 10% serum and 1% double-antibiotic. The original medium in the culture plate was removed, and 200 μL of the above solution was added to each well. The blank group was added with medium without resveratrol. The plate was incubated in a 37°C, 5% CO2 incubator for 24 h or 48 h.

[0087] (3) 20 μL of 5 mg / mL MTT solution was added to each well, and the plate was incubated in a 37°C, 5% CO2 incubator for 4 h.

[0088] (4) The original medium was removed, and 150 μL of DMSO was added to each well. The plate was shaken on a shaker for 10 min to dissolve the MTT crystals. The absorbance values at 570 nm and 630 nm were detected by a microplate reader, and the cell viability was calculated according to the following formula:

[0089]

[0090] The results are shown in Table 1. Figure 4 Free resveratrol had no repairing effect on the vascular endothelial cells after hypoxic injury, and even at a high dose, it aggravated the injury of the vascular endothelial cells.

[0091] Example 5: Repairing effect of resveratrol on ischemic lower limbs

[0092] BALB / c mice of 5-8 weeks were anesthetized by intraperitoneal injection of 40 mg / kg ketamine hydrochloride and 10 mg / kg thiopental. When the mice were muscle relaxed and the limbs were immobile, the fur on the abdomen and limbs was removed with depilatory cream, and the skin was disinfected with 75% ethanol. The mice were placed in a supine position and fixed on a surgical board. The left lower limb was the surgical area. The skin was gently lifted with an ophthalmic forceps, and a longitudinal incision about 5 mm long was made from the groin to the medial thigh along the blood vessels with an ophthalmic scissors. The inguinal fat pad was separated to expose the femoral artery, femoral vein, and their branches. Then the femoral artery was separated, and the femoral vein and femoral nerve were carefully protected. A segment of the femoral artery was ligated and disconnected at the proximal end of the starting point of the femoral artery with a No. 7 surgical suture, to make a femoral artery limb ischemia model.

[0093] After modeling, 50 μL of free resveratrol drug with a concentration of 2 μmol / mL was injected into the thigh muscle, and the treatment of the ischemic leg was observed. The results are shown in Figure 5 Table 1: Free resveratrol has no significant repair effect on ischemic lower limbs, and ischemic limbs are still necrotic after 14 days of treatment.

[0094] Example 6: Effect of resveratrol on the amount of cell microcapsules secreted by stem cells

[0095] The stem cells in the logarithmic growth phase were collected and inoculated in 150 mm culture dishes at a seeding density of 4 x 10 6 After the cells adhered, the DMEM / F12 complete culture medium containing 0.25 μmol / L resveratrol was replaced, and the cell supernatant was collected after 24 h for gradient centrifugation. First, centrifuge at 4°C, 500g for 60 min to remove cells and cell debris. Then transfer the supernatant to a new centrifuge tube and centrifuge at 4°C, 2500g for 60 min to remove the precipitate. Transfer the supernatant to a new centrifuge tube and continue to centrifuge at 20000g for 60 min. Collect the precipitate, which is the activated stem cell microcapsule (AMS), resuspend it in sterile PBS, and store it in the refrigerator at 4°C. The content of the extracted microcapsules was detected by BCA protein detection method. The preparation process of ordinary stem cell microcapsules (MS) does not add resveratrol, and the other steps are consistent with the above method.

[0096] The detection results are shown in Table 2: Figure 6 Compared with the amount of microcapsules secreted by ordinary stem cells (MS), the amount of cell microcapsules secreted by activated stem cells under the stimulation of resveratrol (AMS) is more, which is increased by nearly 1.5 times.

[0097] Example 7: Determination of the amount of activated stem cell growth factors secreted

[0098] The content of growth factors in the cell microcapsules was detected by western blotting, including basic fibroblast growth factor (bFGF); hepatocyte growth factor (HGF); transforming growth factor-β (TGF-β); vascular endothelial growth factor (VEGF); and insulin-like growth factor-1 (IGF-1). The specific steps are as follows:

[0099] (1) Transfer 50 μg of protein of activated stem cell microcapsules (preparation method same as Example 1) or unactivated stem cell microcapsules (preparation method different from Example 1 only in that no resveratrol is added) to a clean centrifuge tube, and use RIPA lysis buffer to correct each protein sample to the same concentration. Then add an equal volume of 5x SDS-PAGE protein loading buffer to each tube, mix well, and denature in a 95°C water bath for 10 min, and then quickly cool on ice, and centrifuge for 1 min.

[0100] (2) Select two electrophoresis glass plates with a size of 1.5, assemble them into a complete device with a matching clamp, add ultrapure water and stand for 30 min, and check whether there is any leakage. Prepare 10 mL of 10% separation gel according to the SDS-PAGE gel preparation kit instructions. Mix well immediately after adding TEMED, pour the gel to the appropriate height, seal with ultrapure water, and stand at room temperature. Then prepare 4 mL of concentrated gel. After a clear interface appears between the gel and the ultrapure water (about 30 min), pour out the water, add the concentrated gel to the top, and immediately insert a comb with a size of 1.5. Avoid air bubbles during the process. Stand at room temperature until the gel solidifies (about 30 min).

[0101] (3) Place the assembled glass plate device vertically in the electrophoresis tank, add 1x electrophoresis liquid to the tank, and stand for about 10 min without any leakage. Slowly add the sample to the sample well with a micro-sampler, fill the tank with 1x electrophoresis liquid, cover the tank, and adjust the voltage to 60 V. After about 1 h, the sample migrates through the concentrated gel, adjust the voltage to 100 V, and stop electrophoresis when the sample migrates to 1 cm from the gel bottom.

[0102] (4) Cut the PVDF membrane to the appropriate size, mark the front and back clearly, and activate it in methanol for 2 min. Place the black side of the clamp down, arrange the sponge, filter paper, gel, membrane, filter paper, and sponge in order from bottom to top, and close the clamp after completely removing the air bubbles. Place it in the transfer tank and run at a constant current of 250 mA at 4°C for 2 h.

[0103] 5. Western blot detection

[0104] After the transfer is completed, carefully remove the PVDF membrane and wash it with TBST buffer for 10 min each time for three times. Place the membrane in a solution containing 5% BSA and incubate at room temperature for 1 h. Place the PVDF membrane in a clean antibody box with a certain proportion of primary antibody solution and incubate at 4°C overnight. The next day, remove the membrane combined with the primary antibody, wash it with TBST for 7 times, each for 6 min, and then incubate it with a certain concentration of secondary antibody solution at room temperature for 1 h. After removing it, wash it with TBST buffer for 7 times, each for 6 min. Then develop it using the Tanon chemiluminescence imaging system.

[0105] The detection results are shown in Table 1: Figure 7 Compared with the unactivated stem cell microcapsules (MS), the growth factor content of the activated stem cell microcapsules (AMS) is generally increased by 2-4 times.

[0106] Example 8: Effect of activated stem cell microcapsules (AMS) on mitochondrial activity of hypoxia-damaged vascular endothelial cells

[0107] The logarithmic growth phase cell line SVEC4-10 (mouse lymph node endothelial cells) was collected at a concentration of 1 x 10 5 The cells were seeded in a 24-well plate at a density of 1 x 10 3 The cells were incubated at 37°C in 5% CO2 for 12 h. 500 μM CoCl2 solution was added to simulate a hypoxic environment, and the cells were incubated for another 12 h. Then, 20 μg / mL activated stem cell microcapsules (AMS) prepared according to Example 1 or non-activated stem cell microcapsules (MS) prepared according to Example 1 except that resveratrol was not added were added to the cells, respectively, and the same volume of medium was added to the control group. After 6 h, the old medium was removed, and 0.5 mL of cell culture solution and 0.5 mL of JC-1 staining solution were added. The cells were incubated at 37°C in a cell incubator for 20 min. After the incubation, the supernatant was removed, and the cells were washed twice with JC-1 staining buffer (1 x). Then, 1 mL of cell culture solution was added, and the fluorescence intensity of each well was determined using a microplate reader. The excitation wavelength was set to 490 nm, and the emission wavelength was set to 530 nm for detecting JC-1 monomers. The excitation wavelength was set to 525 nm, and the emission wavelength was set to 590 nm for detecting JC-1 polymers. Then, the cells were collected and analyzed for changes in mitochondrial membrane potential in hypoxia-damaged endothelial cells using a flow cytometer.

[0108] The results are shown in Table 1. Figure 8 After ischemic injury, the content of mitochondria with high potential in the vascular endothelial cells (control) was only 12.6%. After treatment with non-activated stem cell microcapsules (MS), the content of mitochondria with high potential was only increased by 4.4%. However, after treatment with activated stem cell microcapsules (AMS), the content of mitochondria with high potential was increased by 12.2%, and the improvement was more obvious.

[0109] Example 9: Effect of activated stem cell microcapsules (AMS) on the survival rate of hypoxia-damaged vascular endothelial cells

[0110] 1. The logarithmic growth phase vascular endothelial cells SVEC4-10 were digested with trypsin to prepare a single cell suspension, which was mixed uniformly. The cells were seeded in a 96-well plate at a density of 5 x 10 3 The cells were incubated at 37°C in 5% CO2.

[0111] 2. When the cells were completely adherent and the confluence was about 80%, CoCl2 was added to a concentration of 500 μM to simulate a hypoxic environment, and the cells were incubated for another 12 h.

[0112] 3. Then, the stem cell microcapsules (AMS) of different concentrations (activated stem cell microcapsules (AMS) and non-activated stem cell microcapsules (MS) prepared according to the method of Example 1 except that resveratrol was not added) were added respectively, and a control group was added with a culture medium without stem cell microcapsules, and then cultured in a cell incubator at 37°C and 5% CO2.

[0113] 4. After 6 hours of drug action, 20 μL of 5 mg / mL MTT solution was added to each well, and then incubated in a culture box at 37°C and 5% CO2 for 4 hours.

[0114] 5. The original culture medium was removed, and then 150 μL of DMSO was added to each well, and then shaken on a shaker for 10 minutes to fully dissolve the MTT crystals. The absorbance values at 570 nm and 630 nm were detected by an enzyme label instrument, and the cell viability was calculated according to the following formula:

[0115]

[0116] The detection results are shown in Table 1. Figure 9 For severely damaged vascular endothelial cells, non-activated stem cell microcapsules (MS) failed to show a repairing effect within a certain dose range, while activated stem cell microcapsules (AMS) can effectively improve the survival rate of hypoxia-damaged endothelial cells.

[0117] Example 10 Investigation of the ability of activated stem cell microcapsules loaded with a nitric oxide donor (AMS-NO) to induce endogenous nitric oxide production in hypoxia-damaged vascular endothelial cells

[0118] SVEC4-10 in the logarithmic growth phase was collected, and then inoculated in a 12-well plate at a cell inoculation density of 2 x 10 5 After the cells were induced by 500 μM CoCl2 and cultured at 37°C and 5% CO2, 12 hours later, the same concentration of L-arginine and AMS-NO loaded with L-arginine (prepared according to the method of Example 1) was added. DAF-FM DA (a nitric oxide fluorescent probe) was used to detect nitric oxide, and the fluorescent probe DAF-FM DA was diluted according to a ratio of 1:1000. The cells after 0, 1, 2 and 4 hours of administration were collected, and then resuspended with the diluted DAF-FM DA, and then incubated in a cell incubator at 37°C for 20 minutes. The cells were washed with sterile PBS for three times to sufficiently remove the DAF-FM DA that did not enter the cells. Then, the level of nitric oxide in the cells was detected by a flow cytometer.

[0119] This example demonstrates the ability of activated stem cell microcapsules (AMS-NO) to induce the release of endogenous nitric oxide from hypoxia-damaged vascular endothelial cells. The results show that (Table 1) compared to the nitric oxide donor L-arginine (L-Arg), activated stem cell microcapsules loaded with L-arginine (AMS-NO) induced the release of more endogenous nitric oxide from damaged vascular endothelial cells. Figure 10

[0120] Example 11 Ability of activated stem cell microcapsules loaded with a nitric oxide donor (AMS-NO) to induce the release of nitric oxide from hypoxia-damaged vascular endothelial cells

[0121] SVEC4-10 cells were seeded into 96-well plates and, when the cells were fully adherent and had reached approximately 80% confluency, CoCl2was added to a concentration of 500 μM and the cells were incubated for a further 12 h. Subsequently, the same concentration of nitroglycerin (GTN) and AMS-NO loaded with nitroglycerin (prepared according to the method of Example 1) were added, respectively, and the control group was added with the same volume of medium and incubated in a cell incubator at 37°C and 5% CO2. The cell culture supernatant was collected at 0, 1, 2, 4, 6, 8, 12 and 24 h after administration, respectively. 50 μL of each group of collected supernatant was taken and added to a 96-well plate, and 50 μL of Griess Reagent I and Griess Reagent II from a nitric oxide detection kit were added, respectively. After 10 min at room temperature, the absorbance was measured at 540 nm. The content of released nitric oxide was calculated according to the standard curve.

[0122] This example demonstrates the ability of activated stem cell microcapsules (AMS-NO) to induce the release of endogenous nitric oxide from hypoxia-damaged vascular endothelial cells. The results show that (Table 1) compared to the nitric oxide donor L-arginine (L-Arg), activated stem cell microcapsules loaded with L-arginine (AMS-NO) induced the release of more endogenous nitric oxide from damaged vascular endothelial cells. Figure 11

[0123] Example 12 Role of activated stem cell microcapsules loaded with a nitric oxide donor (AMS-NO) in promoting angiogenesis in damaged blood vessels

[0124] ​​BALB / c mice of 5-8 weeks were anesthetized by intraperitoneal injection of ketamine hydrochloride (40 mg / kg) and xylazine (10 mg / kg). When the mice were relaxed and the limbs were immobile, the hair on the abdomen and limbs was removed with depilatory cream, and the skin was disinfected with 75% ethanol. The mice were placed in a supine position and fixed on a surgical board. The left lower limb was the surgical area. The skin was gently lifted with an ophthalmic forceps, and a longitudinal incision about 5 mm long was made from the groin to the medial thigh along the blood vessels with an ophthalmic scissors. The inguinal fat pad was separated to expose the femoral artery, femoral vein and their branches. Then the femoral artery was separated, and the femoral vein and femoral nerve were protected. A segment of the femoral artery was ligated at the proximal end of the starting point and then disconnected to prepare the high ligation and disconnection of the femoral artery limb ischemia model. Subsequently, the thigh muscle was injected with AMS (prepared according to the method of Example 1) (100 μg per mouse), GTN (50 μg per mouse), AMS-NO loaded with GTN (prepared according to the method of Example 1) (100 μg AMS + 50 μg GTN per mouse) and PBS solution. On the 3rd day, the blood vessel regeneration at the surgical site was observed by a Doppler flow detector.

[0125] The results are shown in Table 1. Figure 12 The blood vessel regeneration of the ischemic lower limb of the mice was detected by a Doppler flow detector. The brighter the color in the image, the more abundant the blood flow, and vice versa. In the mouse arterial ischemia model, the lower limbs of the mice in the non-drug group had completely fallen off. The activated stem cell microcapsules alone had a certain repair effect, which could delay the necrosis and shedding of the limbs, but the image of the ischemic lower limb observed by the Doppler flow detector was black, indicating that there was no new blood vessel formation in the lower limb, and the blood flow transportation was blocked. The lower limbs of the mice administered with the nitrate compound alone could be seen in a lighter blue color, indicating that the nitrate compound alone could dilate the terminal blood vessels to provide blood flow to the ischemic lower limb, but the effect was limited. The Doppler image of the activated stem cell microcapsules loaded with the nitrate compound group could be clearly observed to be yellow and blue in the lower limb, indicating that the activated stem cell microcapsules loaded with the nitrate compound promoted the angiogenesis of the ischemic lower limb, and the blood flow recovery of the ischemic lower limb was significantly better than that of the other groups.

[0126] Example 13 Treatment of severe limb ischemia by activated stem cell microcapsules loaded with a nitric oxide donor (AMS-NO)

[0127] 1. Select 5-8 weeks of female BALB / c mice, anesthetize the mice by intraperitoneal injection of 40 mg / kg ketamine hydrochloride and 10 mg / kg thiopental, and when the muscles of the mice are relaxed and the limbs are not active, remove the hair on the abdomen and limbs with depilatory cream, clean and disinfect the skin with 75% ethanol. The mouse is placed in a supine position and fixed on the operating board, the left lower limb is the surgical area, the skin is gently lifted with an ophthalmic forceps, and an ophthalmic scissors is used to cut a longitudinal incision from the groin to the medial thigh along the blood vessels, about 5 mm long. Isolate the inguinal fat pad, expose the femoral artery, femoral vein and their branches. Then isolate the femoral artery, pay attention to damage the femoral vein and femoral nerve, and make a high ligation and disconnection of a segment of the femoral artery at the proximal end of the starting point of the femoral artery. The method of making a high ligation and disconnection of a segment of the femoral artery limb ischemia model.

[0128] 2. On the day of modeling, the mice were randomly divided into 4 groups, 5 mice in each group, and intramuscularly administered. The groups include Control group, L-Arginine group (50 μg per mouse), AMS group (preparation method same as Example 1) (100 μg per mouse), and L-arginine-loaded AMS-NO group (preparation method same as Example 1) (100 μg AMS + 50 μg L-arginine per mouse). The recovery of ischemic limb tissue was scored on the 1st, 3rd, 7th, and 14th days after operation. Complete recovery is scored as 6 points, slight necrosis or nail loss is scored as 5 points, partial toe amputation is scored as 4 points, total toe amputation is scored as 3 points, partial or total foot amputation is scored as 2 points, and partial or total limb amputation is scored as 1 point.

[0129] This example constructs a mouse severe limb ischemia model, and is administered with normal saline (Control), L-arginine (L-Arginine), activated stem cell microcapsules (AMS), and L-arginine-loaded activated stem cell microcapsules (AMS-NO), respectively, and the recovery effect of each group of mice after treatment is scored. The results show that Figure 13 ) the score of the AMS group on the 1st and 3rd days after administration is higher than that of the control group, indicating that AMS can relieve the necrosis of the limbs to a certain extent, and the score on the 7th day is 0, indicating that AMS can have some effect, but still cannot provide blood flow to the ischemic lower limbs. After loading the AMS with a nitric oxide donor, the therapeutic effect can be greatly improved, and compared with L-arginine and activated stem cell microcapsules (AMS), L-arginine-loaded activated stem cell microcapsules (AMS-NO) significantly improves the therapeutic effect, and even achieves complete repair.

[0130] Any combination of the technical features in the above-described embodiments can be made, and for the sake of brevity, not all possible combinations are described, however, as long as there is no conflict, any combination of the technical features should be considered within the scope of the present disclosure.

[0131] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the scope of protection of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

Claims

1. An activated stem cell microcapsule loaded with a nitric oxide donor, characterized in that: The activated stem cell microcapsules are prepared by incubating a mixture of activated stem cell microcapsules and a nitric oxide donor at a mass ratio of 1-3:1; the nitric oxide donor is L-arginine and / or nitroglycerin; The method for preparing the activated stem cell microcapsule comprises the following steps: Mesenchymal stem cells are cultured in a medium containing resveratrol, and then the supernatant is collected and centrifuged using differential centrifugation to obtain activated stem cell microcapsules; the resveratrol is the only activator used to activate the mesenchymal stem cells; The concentration of resveratrol in the culture medium is 0.2 µmol / L-0.3 µmol / L; The culturing time is 20 hours to 72 hours; The differential centrifugation method includes: centrifuging at 400 g-600 g for 30 min-90 min; discarding the precipitate, taking the supernatant and centrifuging at 2000 g-3000 g for 30 min-90 min; discarding the precipitate, taking the supernatant and centrifuging at 15000 g-25000 g for 30 min-90 min.

2. The activated stem cell microcapsule loaded with nitric oxide donor according to claim 1, characterized in that: The concentration of resveratrol in the culture medium is 0.25µmol / L.

3. The activated stem cell microcapsule loaded with nitric oxide donor according to claim 1, characterized in that: The culture time is 24 hours or 48 hours.

4. The activated stem cell microcapsule loaded with nitric oxide donor according to claim 1, characterized in that: The differential centrifugation method includes: centrifuging at 450 g-550 g for 50 min-70 min; discarding the precipitate, taking the supernatant and centrifuging at 2300 g-2800 g for 50 min-70 min; discarding the precipitate, taking the supernatant and centrifuging at 18000 g-22000 g for 50 min-70 min.

5. The activated stem cell microcapsule loaded with nitric oxide donor according to claim 4, characterized in that: The differential centrifugation method includes: centrifuging at 500g for 60 min; discarding the precipitate, taking the supernatant and centrifuging at 2500g for 60 min; discarding the precipitate, taking the supernatant and centrifuging at 20000g for 60 min.

6. The activated stem cell microcapsule loaded with nitric oxide donor according to claim 1, characterized in that: The mass ratio of the activated stem cell microcapsules to the nitric oxide donor is 1.8-2.2:

1.

7. The activated stem cell microcapsule loaded with nitric oxide donor according to claim 6, characterized in that: The mass ratio of the activated stem cell microcapsules to the nitric oxide donor is 2:

1.

8. A method for preparing activated stem cell microcapsules loaded with nitric oxide donors according to any one of claims 1 to 7, characterized in that: The steps include: The activated stem cell microcapsules are mixed with the nitric oxide donor and incubated at a constant temperature of 0° C. to 8° C. to obtain the activated stem cell microcapsules loaded with the nitric oxide donor.

9. Use of the activated stem cell microcapsules loaded with nitric oxide donors according to any one of claims 1 to 7 in the preparation of a drug for promoting regeneration of damaged blood vessels.

10. Use of the activated stem cell microcapsules loaded with nitric oxide donors according to any one of claims 1 to 7 in the preparation of a medicament for treating critical limb ischemia.

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