Preparation and application method of mesenchymal stem cell exosome incubated by GLP-1 medicine

Abstract

The invention provides a preparation method of a mesenchymal stem cell exosome incubated by a GLP-1 medicine. The preparation method comprises the following steps: S1, preparing materials; s2, separating the human adipose tissue-derived stromal cells; s3, cell surface marker protein detection; s4, carrying out GLP-1 incubation; and S5, extracting the exosome. According to the preparation method of the mesenchymal stem cell exosome incubated with the GLP-1 medicine, the human adipose tissue-derived stromal cell exosome is collected in a specific mode, the whole process is simple and convenient, the obtained human adipose tissue-derived stromal cell exosome is stable in effect, damage of myocardial cells after myocardial infarction can be effectively relieved, the heart function can be enhanced, and the human adipose tissue-derived stromal cell exosome can be used for treating myocardial infarction. The compensatory cardiac machine load is reduced, and the heart failure is relieved.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to a method for preparing and applying mesenchymal stem cell exosomes incubated with a GLP-1 drug. Background Art

[0002] Acute myocardial infarction is the cardiovascular disease with the highest mortality and disability rates worldwide. Early treatment is of great significance in preventing myocardial necrosis, reducing mortality and alleviating pain. At present, the treatment of acute myocardial infarction is mainly divided into a variety of programs such as drug therapy, interventional therapy and surgical operation. In terms of drug therapy, antiplatelet therapy, anticoagulation, blood pressure maintenance, and reduction of myocardial oxygen consumption can be used. Percutaneous coronary intervention (PCI) in interventional therapy is an effective treatment method with reliable efficacy and high surgical safety. It can restore myocardial blood supply in a short time, reduce the area of ​​myocardial necrosis, and achieve the effect of saving lives. However, the current treatment plan still has some potential defects. However, after myocardial infarction, , some myocardial cells die, and the remaining myocardial tissue may not be able to regenerate. Therefore, myocardial infarction causes relatively more complications, the most common of which is heart failure. The heart's contractile function decreases, the heart's blood discharge function weakens, the body's tissues do not get enough blood supply, and symptoms of systemic and pulmonary congestion appear, seriously affecting the patient's quality of life; secondly, myocardial death and tissue necrosis may cause arrhythmias, such as atrial fibrillation and ventricular tachycardia, and in severe cases may even lead to sudden death; thirdly, myocardial remodeling is also relatively common. After myocardial infarction, the area of ​​cell death will gradually be replaced by scar tissue. The elasticity of these scar tissues is very poor. Scar formation in myocardial remodeling may lead to dilation of the ventricular wall and decreased ventricular function.

[0003] Stem cell therapy, which has been developed in recent years, is considered a promising new method for treating myocardial infarction. Stem cells have the ability to differentiate into various types of cells, including cardiomyocytes, and can therefore be used to rebuild the myocardium. Studies have shown that stem cell therapy can cultivate cardiomyocytes to repair necrotic areas after myocardial infarction. However, the long-term effects of stem cell therapy for myocardial infarction have not yet been proven, and there may be safety and bioethical issues. Further large-scale clinical trials are needed to confirm the effectiveness and safety of stem cell therapy for myocardial infarction. In addition, it is difficult for stem cells to survive for a long time after being infused into the body. The protective effect of stem cells on the myocardium is mainly through paracrine effects, of which exosomes are the main carrier components of stem cell paracrine.

[0004] Exosomes are nano-scale vesicles wrapped in a double-layer phospholipid membrane. Stem cell exosomes contain biologically active components of cells, such as proteins, RNA, and small molecules. Their effects are somewhat similar to those of stem cells, which make them difficult to survive and have a high cure rate, but they avoid safety issues such as immune rejection and possible genetic infectious diseases when ordinary stem cells are implanted. In myocardial infarction, stem cell exosomes show more advantages than stem cell therapy. Stem cell exosomes can promote myocardial growth and repair, increase the number of myocardial cells in necrotic areas, and improve their metabolic activity. They play a role by strengthening the cell autophagy mechanism and promoting angiogenesis; secondly, myocardial Inflammatory response and fibrosis during infarction are the driving forces for myocardial damage and reconstruction. Stem cell exosomes can reduce the production of toxic immune cells, slow down inflammatory response and fibrosis through their hyaluronan hydrolases, apoptosis regulatory factors, MIRNA, etc. At the same time, stem cell exosomes have multiple protective effects such as antioxidant, anti-apoptotic, and anti-inflammatory, which can reduce myocardial cell damage and promote cell regeneration. In short, stem cell exosomes can promote myocardial regeneration and repair through multiple pathways, and are also safer and avoid the risk of possible genetic diseases. However, because the research on stem cell exosomes is still relatively superficial, its use in the treatment of myocardial infarction requires further research.

[0005] Adipose-derived mesenchymal stem cells are a type of stem cell with a wide range of sources and have many advantages: 1. Rich sources: ADSCs can be easily separated and extracted from adult adipose tissue, so it is easier to obtain ADSCs than to extract myeloid stem cells from bone marrow; 2. High stem cell concentration: The concentration of ADSCs is more than 100 times higher than that of other types of stem cells such as neural stem cells; 3. Multipotency: ADSCs can differentiate into any cell type except neural cells. Under appropriate culture conditions, they can differentiate into adipocytes, bone cells, chondrocytes, and cardiomyocytes, etc.; 4. Anti-inflammatory effect: ADSCs can inhibit excessive inflammation and can be used to treat inflammatory-related diseases; 5. Lack of immunogenicity: ADSCs lack matching Antigen, so it will not trigger an immune response; 6. High safety: ADSCs do not require surgical operations during extraction, which is very safe and very suitable for large-scale production and large-scale use; because of these advantages, the application of ADSCs in multiple medical fields continues to expand; in the treatment of cardiovascular diseases, ADSCs have been shown to have the effects of repairing blood vessels, accelerating the healing of damaged areas, improving vascular proliferation and myocardial remodeling, etc. ADSCs can survive for a long time after myocardial ischemia and reperfusion injury, and their sites in the body are far away from the injection site, so they have the advantage of a wider range of treatment types, because they are easy to obtain and highly safe, and can be studied and subsequently applied in multiple cutting-edge fields. Therefore, ADSCs are a very promising choice in the treatment of myocardial injury.

[0006] GLP-1 is a glucagon-like polypeptide-1, which plays an important role in regulating insulin secretion and participating in energy metabolism. Recent studies have shown that GLP-1 also has a protective effect on cardiomyocytes, and GLP-1 can promote the survival of stem cells after stem cell transplantation, so it is considered to be one of the potential therapeutic strategies for the treatment of myocardial infarction. Animal experiments have proved that GLP-1 incubation of stem cells to treat myocardial infarction is safe and effective. In short, the treatment of myocardial infarction by incubating stem cells with GLP-1 has produced quite excellent results in practice. The key is to combine the two active components. Biomaterial technology creates an ideal microenvironment to avoid immune response, so as to improve the survival rate and healing effect of stem cells. The application of GLP-1 incubated stem cell exosomes in myocardial ischemia has not been reported. Studies have shown that stem cell therapy can cultivate cardiomyocytes after myocardial infarction to repair necrotic areas, but the long-term effect of stem cell therapy for myocardial infarction has not been proven, and there may be safety and bioethical issues. Further large-scale clinical trials are needed to confirm the effect and safety of stem cell therapy for myocardial infarction.

[0007] Therefore, it is necessary to provide a method for preparing and applying mesenchymal stem cell exosomes incubated with GLP-1 drugs to solve the above technical problems. Summary of the invention

[0008] The present invention provides a method for preparing and applying mesenchymal stem cell exosomes incubated with a GLP-1 drug, which solves the problem that studies have shown that stem cell therapy can cultivate cardiomyocytes to repair necrotic areas after myocardial infarction, but the long-term effect of stem cell therapy for myocardial infarction has not yet been proven, and there may be safety and bioethical issues. Further large-scale clinical trials are needed to confirm the effectiveness and safety of stem cell therapy for myocardial infarction.

[0009] In order to solve the above technical problems, the present invention provides a method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug, comprising the following steps: S1: preparing materials, including normal human fat discarded after surgery, PBS, BSA, type IV collagenase, fetal bovine serum, and monoclonal antibodies of CD29+, CD44+, CD90+, CD34-, and CD45-;

[0010] S2: Isolation of human adipose-derived mesenchymal stem cells;

[0011] S21: About 15 mL of adipose tissue was collected under sterile conditions, and the connective tissue capsule and blood vessels on the surface of the adipose tissue were removed;

[0012] S22: Quickly chop into paste, add exosome-free culture medium, filter and collect the filtrate;

[0013] S23: centrifuging the obtained filtrate at 1500 r / min for 15 min, discarding the supernatant to obtain a precipitate;

[0014] S24: Wash the precipitate 2-3 times with 37℃ PBS, then add culture medium to the precipitate to resuspend it. After detecting cell activity, inoculate at a density of 2-6×104 cells / cm2 and culture in an incubator. After 12-14 hours of culture, change the medium for the first time. The change should be gentle to prevent the cells that have just attached to the wall from being replaced. After that, replace the fresh medium every 48 hours and observe the cell morphology under an electron microscope.

[0015] S3: Detection of cell surface marker proteins; observe the cell status and cell fusion under a microscope. When the cell status is good and the fusion degree exceeds 95%, digest the cells. When the cell edges begin to detach from the culture plate, add culture medium to stop digestion. After centrifugation, take the precipitate and resuspend the cells in fresh culture medium to adjust the cell concentration. Prepare multiple centrifuge tubes, add cell suspensions containing 1×106 cells, and then add adhesion molecule receptor expression markers CD29+ and CD44+, stem cell marker CD90+, and hematopoietic cell markers CD34- and CD45-, respectively. After incubation, test on the machine;

[0016] S4: GLP-1 incubation: human adipose-derived stem cells were incubated with 10 nmol / L GLP-1. After 8 hours, they were washed with PBS and the cell supernatant was collected.

[0017] S5: Extract exosomes; collect human adipose-derived mesenchymal stem cells, inoculate, and use serum culture medium without exosomes; after culturing for 12 hours, replace with serum culture medium without human adipose-derived mesenchymal stem cell exosomes, continue culturing for 36 hours, and collect cell culture fluid; the obtained cell culture fluid is subjected to differential centrifugation at 4°C, that is, centrifuged at 1500g for 10 minutes, and then centrifuged at 10000g for 10 minutes to remove cell debris and macromolecular proteins, and obtain supernatant; ultracentrifuge the supernatant, that is, ultracentrifuge at 100000g for 2 hours, and collect Collect the precipitate to obtain the target microspheres; add physiological saline to the microspheres for resuspending to obtain a resuspension, filter the resuspension with a 0.22 μm filter membrane to remove apoptotic bodies and microvesicles, purify the filtered target microspheres to obtain purified human adipose mesenchymal stem cell exosomes (EVs), sterilize the purified human adipose mesenchymal stem cell exosomes to obtain human adipose mesenchymal stem cell exosomes; identify the human adipose mesenchymal stem cell exosomes by electron microscopy; and identify the obtained human adipose mesenchymal stem cell exosomes by WB.

[0018] Preferably, 3 mL of the exosome-free culture medium is added, and the exosome-free culture medium contains type IV collagenase with a final concentration of 0.06-0.16% and 0.015-0.02% BSA, and the culture medium is kept at 37°C and shaken at 120 r / min for 30-35 min. The tissue digestion status is observed at any time, and the tissue is taken out and mixed every 5-8 min. After the digestion is terminated, it is filtered in sequence with 100 mesh and 200 mesh sieves to remove undigested tissue.

[0019] Preferably, the culture medium contains 10% by volume of exosome-free fetal bovine serum.

[0020] Preferably, the indoor conditions of the incubator are 37° C., 5% CO 2 , and 100% relative humidity.

[0021] Preferably, the 10 nmol / L GLP-1 is used to incubate human adipose stem cells for 4 hours.

[0022] Preferably, the exosome-free serum culture medium is cultured at 37° C. and 5% CO 2 .

[0023] Preferably, the serum culture medium without human adipose-derived mesenchymal stem cell exosomes contains 300 μmol / L dimethyloxalylglycine.

[0024] A method for applying mesenchymal stem cell exosomes incubated with a GLP-1 drug, comprising the following steps: S1: preparing a rat myocardial infarction model and exosome injection; randomly dividing SD rats into a Sham group, an AMI group, and an ExoBMSCs group, anesthetizing them with intraperitoneal injection, exposing the rat neck trachea, intubating the rats and connecting them to a ventilator for assisted breathing, making a 1 cm surgical incision between the 3rd and 4th ribs on the left side of the rat chest to expose the heart, ligating the starting part of the left anterior descending coronary artery with a 5-0 needle and thread, and after 30 minutes of ischemia treatment, cutting the silk thread to restore blood perfusion;

[0025] S2: When the ST segment on the electrocardiogram recorder was significantly elevated and the left ventricle was white due to ischemia, it indicated that the AMI model was successfully established. The rats in the Sham group were only threaded with the left anterior descending branch of the coronary artery, but not ligated. The rats in the ExoBMSCs group and the AMI group were injected with 25 μL of exosomes (750 mg / mL) at four sites in the myocardial infarction area. The Sham group and the AMI group were injected with the same amount of normal saline at the same site.

[0026] S3: One week after the group-based intervention, echocardiographic monitoring was performed to record the left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), left ventricular ejection fraction (LVEF) and fractional shortening (FS).

[0027] Preferably, sodium pentobarbital (30 mg / kg) is injected intraperitoneally during anesthesia.

[0028] Preferably, the breathing rate of the ventilator is 90 times / min, the tidal volume is 8 mL, and the breathing ratio is 1:2.

[0029] Compared with the related art, the preparation and application method of mesenchymal stem cell exosomes incubated with GLP-1 drug provided by the present invention has the following beneficial effects:

[0030] The present invention provides a method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug, wherein human adipose mesenchymal stem cell exosomes are collected in a specific manner, and the whole process is simple and convenient. The obtained human adipose mesenchymal stem cell exosomes have a stable effect, can effectively alleviate the damage of myocardial cells after myocardial infarction, enhance cardiac function, reduce compensatory cardiac load, and alleviate heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the structure of exosomes under a microscope in a method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug provided by the present invention;

[0032] Figure 2 This is a schematic diagram of the particle diameter distribution in the sample;

[0033] Figure 3 Schematic diagram of exosome white matter imprint detection. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0035] Please refer to Figure 1 , Figure 2 and Figure 3 ,in, Figure 1 A schematic diagram of the structure of exosomes under a microscope in a method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug provided by the present invention; Figure 2 for Figure 1 Schematic diagram of the structure of particle diameter distribution in the sample shown; Figure 3 for Figure 1 Schematic diagram of exosome white matter blot detection shown. A method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug, comprising the following steps: S1: preparing materials, including human fat, which is normal human fat discarded after surgery, PBS, BSA, type IV collagenase, fetal bovine serum, and monoclonal antibodies of CD29+, CD44+, CD90+, CD34-, and CD45-;

[0036] S2: Isolation of human adipose-derived mesenchymal stem cells;

[0037] S21: About 15 mL of adipose tissue was collected under sterile conditions, and the connective tissue capsule and blood vessels on the surface of the adipose tissue were removed;

[0038] S22: Quickly chop into paste, add exosome-free culture medium, filter and collect the filtrate;

[0039] S23: centrifuging the obtained filtrate at 1500 r / min for 15 min, discarding the supernatant to obtain a precipitate;

[0040] S24: Wash the precipitate 2-3 times with 37℃ PBS, then add culture medium to the precipitate to resuspend it. After detecting cell activity, inoculate at a density of 2-6×104 cells / cm2 and culture in an incubator. After 12-14 hours of culture, change the medium for the first time. The change should be gentle to prevent the cells that have just attached to the wall from being replaced. After that, replace the fresh medium every 48 hours and observe the cell morphology under an electron microscope.

[0041] S3: Detection of cell surface marker proteins; observe the cell status and cell fusion under a microscope. When the cell status is good and the fusion degree exceeds 95%, digest the cells. When the cell edges begin to detach from the culture plate, add culture medium to stop digestion. After centrifugation, take the precipitate and resuspend the cells in fresh culture medium to adjust the cell concentration. Prepare multiple centrifuge tubes, add cell suspensions containing 1×106 cells, and then add adhesion molecule receptor expression markers CD29+ and CD44+, stem cell marker CD90+, and hematopoietic cell markers CD34- and CD45-, respectively. After incubation, test on the machine;

[0042] S4: GLP-1 incubation: human adipose-derived stem cells were incubated with 10 nmol / L GLP-1. After 8 hours, they were washed with PBS and the cell supernatant was collected.

[0043] S5: Extract exosomes; collect human adipose-derived mesenchymal stem cells, inoculate, and use serum culture medium without exosomes; after culturing for 12 hours, replace with serum culture medium without human adipose-derived mesenchymal stem cell exosomes, continue culturing for 36 hours, and collect cell culture fluid; the obtained cell culture fluid is subjected to differential centrifugation at 4°C, that is, centrifuged at 1500g for 10 minutes, and then centrifuged at 10000g for 10 minutes to remove cell debris and macromolecular proteins, and obtain supernatant; ultracentrifuge the supernatant, that is, ultracentrifuge at 100000g for 2 hours, and collect Collect the precipitate to obtain the target microspheres; add physiological saline to the microspheres for resuspending to obtain a resuspension, filter the resuspension with a 0.22 μm filter membrane to remove apoptotic bodies and microvesicles, purify the filtered target microspheres to obtain purified human adipose mesenchymal stem cell exosomes (EVs), sterilize the purified human adipose mesenchymal stem cell exosomes to obtain human adipose mesenchymal stem cell exosomes; identify the human adipose mesenchymal stem cell exosomes by electron microscopy; and identify the obtained human adipose mesenchymal stem cell exosomes by WB.

[0044] 3 mL of the exosome-free culture medium was added, and the exosome-free culture medium contained type IV collagenase with a final concentration of 0.06-0.16% and 0.015-0.02% BSA. The culture medium was kept at 37°C and oscillated at 120 r / min for 30-35 min. The tissue digestion status was observed at any time. During this period, the tissue was taken out and mixed every 5-8 min. After the digestion was terminated, it was filtered in turn with 100 mesh and 200 mesh sieves to remove undigested tissue.

[0045] The culture medium contains 10% by volume of fetal bovine serum without exosomes.

[0046] The indoor conditions of the incubator were 37° C., 5% CO 2 , and 100% relative humidity.

[0047] The human adipose stem cells were incubated with 10 nmol / L GLP-1 for 4 hours.

[0048] The exosome-free serum medium was cultured at 37° C. and 5% CO 2 .

[0049] The serum culture medium of the human adipose-derived mesenchymal stem cell exosomes contains 300 μmol / L dimethyloxalylglycine.

[0050] The double-layered capsule structure is one of the important signs of exosomes. In this study, negative staining and transmission electron microscopy were used to observe the morphology of the samples. It can be observed that there is an obvious double-layered capsule structure in the samples, which is identified as exosomes. Figure 1 ;

[0051] Zetaview laser scattering microscope was used to quickly and accurately analyze the particle diameter distribution in the extracted samples. Most of the particles were around 100 nm in diameter, indicating that the extracted samples were exosomes. Figure 2 ;

[0052] Western blot (WB) detection technology found that the cell membrane surface protein markers CD9, TSG101 and HSC70 antibodies of the extracted samples were all positive, which proved that the extracted substances were exosomes. Figure 3 .

[0053] The working principle of the method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug provided by the present invention is as follows:

[0054] During operation, firstly, human adipose tissue discarded after surgery is collected, human adipose mesenchymal stem cells are digested and separated, human adipose mesenchymal stem cells are cultured, and cell culture fluid is collected; the cell culture fluid is differentially centrifuged, the supernatant is collected, the supernatant is ultracentrifuged to obtain the target microspheres, and then the target microspheres are filtered, purified and sterilized to obtain the aforementioned human adipose mesenchymal stem cell exosomes.

[0055] The human adipose mesenchymal stem cells are prepared by digesting human adipose tissue, inoculating and subculturing. The adipose tissue is digested by using 0.06-0.16% type IV collagenase and 0.015-0.02% BSA by mass.

[0056] Human adipose-derived mesenchymal stem cells were cultured for 2-10 generations, the digestion time was 25-40 minutes, the culture medium of human adipose-derived stem cells was 10% fetal bovine serum medium without exosomes, the inoculation density after digestion was 2-5×104cells / cm2, differential centrifugation included centrifugation at 1500g for 10 minutes and then at 10000g for 10 minutes, the ultracentrifugation conditions for extracting exosomes were centrifugation at 100000g for 2 hours, and the pore size of the filter membrane used was 0.22μm.

[0057] Compared with the related art, the preparation method of mesenchymal stem cell exosomes incubated with a GLP-1 drug provided by the present invention has the following beneficial effects:

[0058] The present invention provides a method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug, wherein human adipose mesenchymal stem cell exosomes are collected in a specific manner, and the whole process is simple and convenient. The obtained human adipose mesenchymal stem cell exosomes have a stable effect, can effectively alleviate the damage of myocardial cells after myocardial infarction, enhance cardiac function, reduce compensatory cardiac load, and alleviate heart failure.

[0059] Second embodiment

[0060] Based on the preparation and application method of mesenchymal stem cell exosomes incubated with a GLP-1 drug provided in the first embodiment of the present application, the second embodiment of the present application proposes another preparation and application method of mesenchymal stem cell exosomes incubated with a GLP-1 drug. The second embodiment is only a preferred embodiment of the first embodiment, and the implementation of the second embodiment will not affect the independent implementation of the first embodiment.

[0061] Specifically, the second embodiment of the present application provides a method for applying mesenchymal stem cell exosomes incubated with a GLP-1 drug, which is different in that the method for applying mesenchymal stem cell exosomes incubated with a GLP-1 drug comprises the following steps: S1: preparing a rat myocardial infarction model and exosome injection; randomly dividing SD rats into: Sham group, AMI group, ExoBMSCs group, anesthetizing by intraperitoneal injection, exposing the rat neck trachea, intubating the rat and connecting the ventilator to assist breathing, making a 1 cm surgical incision between the 3rd and 4th ribs on the left side of the rat chest, exposing the heart, and ligating the starting part of the left anterior descending coronary artery with a 5-0 needle and thread. After 30 minutes of ischemia treatment, the silk thread was cut to restore blood perfusion;

[0062] S2: When the ST segment on the electrocardiogram recorder was significantly elevated and the left ventricle was white due to ischemia, it indicated that the AMI model was successfully established. The rats in the Sham group were only threaded with the left anterior descending branch of the coronary artery, but not ligated. The rats in the ExoBMSCs group and the AMI group were injected with 25 μL of exosomes (750 mg / mL) at four sites in the myocardial infarction area. The Sham group and the AMI group were injected with the same amount of normal saline at the same site.

[0063] S3: One week after the group-based intervention, echocardiographic monitoring was performed to record the left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), left ventricular ejection fraction (LVEF) and fractional shortening (FS).

[0064] During the anesthesia, sodium pentobarbital (30 mg / kg) was injected intraperitoneally.

[0065] The breathing rate of the ventilator is 90 times / min, the tidal volume is 8 mL, and the respiratory ratio is 1:2.

[0066] Assessment of cardiac function in rats

[0067] Group LVDd(mm) LVDs(mm) LVEF (%) FS(%) Sham Group 5.69±0.35 2.76±0.22 77.92±5.57 50.33±3.41 AMI group 7.44±0.44* 5.38±0.26* 37.54±3.63* 25.49±2.17* MSC group 6.51±0.48# 4.64±0.32# 48.31±4.15# 33.52±3.22# ExoBMSCs group 6.06±0.43# 4.05±0.96# 60.62±4.32％# 45.37±6.91#

[0068] The working principle of the application method of mesenchymal stem cell exosomes incubated with GLP-1 drug provided by the present invention is as follows:

[0069] During the work, SD rats were first randomly divided into: Sham group, AMI group, ExoBMSCs group, and anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg). The rats' cervical trachea was exposed, and the endotracheal tube was connected to the ventilator for assisted breathing. The ventilator parameters were as follows: respiratory rate was 90 times / min, tidal volume was 8 mL, and respiratory ratio was 1:2. A 1 cm surgical incision was made between the 3rd and 4th ribs on the left side of the rat's chest to expose the heart. The left anterior descending coronary artery was ligated with 5-0 needle and thread at the beginning. After 30 minutes of ischemia, the silk thread was cut to restore blood perfusion. When the ST segment on the electrocardiogram recorder was significantly elevated and the left ventricle turned white due to ischemia, it indicated that the AMI model was successfully established. The rats in the Sham group were only threaded through the left anterior descending branch of the coronary artery, but not ligated. The rats in the ExoBMSCs group and the AMI group were injected with 25 μL of exosomes (750 mg / mL) at four sites in the myocardial infarction area, and the Sham group and the AMI group were injected with an equal amount of normal saline at the same site.

[0070] One week after the group-based intervention, echocardiography was performed to record the left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), left ventricular ejection fraction (LVEF) and fractional shortening (FS).

[0071] Compared with the related art, the application method of mesenchymal stem cell exosomes incubated with a GLP-1 drug provided by the present invention has the following beneficial effects:

[0072] The present invention provides an application method of mesenchymal stem cell exosomes incubated with GLP-1 drug, by successfully isolating stem cells from adipose tissue, the cell activity of stem cells is significantly improved after incubation with GLP-1, the proliferation rate is accelerated, the myocardial infarction area is reduced and the cardiac function is significantly improved after the secreted exosomes are injected into the rat myocardium. At present, there is no use of stem cell exosomes incubated with GLP-1 to treat myocardial infarction.

[0073] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug, characterized in that: The following steps are involved: S1: Prepare materials, including normal human fat discarded after surgery, PBS, BSA, type IV collagenase, fetal bovine serum, and monoclonal antibodies against CD29+, CD44+, CD90+, CD34-, and CD45-; S2: Isolation of human adipose-derived mesenchymal stem cells; S21: About 15 mL of adipose tissue was collected under sterile conditions, and the connective tissue capsule and blood vessels on the surface of the adipose tissue were removed; S22: Quickly chop into paste, add exosome-free culture medium, filter and collect the filtrate; S23: centrifuging the obtained filtrate at 1500 r / min for 15 min, discarding the supernatant to obtain a precipitate; S24: Wash the precipitate 2-3 times with 37℃ PBS, then add culture medium to the precipitate to resuspend it. After detecting cell activity, inoculate at a density of 2-6×104 cells / cm2 and culture in an incubator. After 12-14 hours of culture, change the medium for the first time. The change should be gentle to prevent the cells that have just attached to the wall from being replaced. After that, replace the fresh medium every 48 hours and observe the cell morphology under an electron microscope. S3: Detection of cell surface marker proteins; observe the cell status and cell fusion under a microscope. When the cell status is good and the fusion degree exceeds 95%, digest the cells. When the cell edges begin to detach from the culture plate, add culture medium to stop digestion. After centrifugation, take the precipitate and resuspend the cells in fresh culture medium to adjust the cell concentration. Prepare multiple centrifuge tubes, add cell suspensions containing 1×106 cells, and then add adhesion molecule receptor expression markers CD29+ and CD44+, stem cell marker CD90+, and hematopoietic cell markers CD34- and CD45-, respectively. After incubation, test on the machine; S4: GLP-1 incubation: human adipose-derived stem cells were incubated with 10 nmol / L GLP-1. After 8 hours, they were washed with PBS and the cell supernatant was collected. S5: Extract exosomes; collect human adipose-derived mesenchymal stem cells, inoculate, and use serum culture medium without exosomes; after culturing for 12 hours, replace with serum culture medium without human adipose-derived mesenchymal stem cell exosomes, continue culturing for 36 hours, and collect cell culture fluid; the obtained cell culture fluid is subjected to differential centrifugation at 4°C, that is, centrifuged at 1500g for 10 minutes, and then centrifuged at 10000g for 10 minutes to remove cell debris and macromolecular proteins, and obtain supernatant; ultracentrifuge the supernatant, that is, ultracentrifuge at 100000g for 2 hours, and collect Collect the precipitate to obtain the target microspheres; add physiological saline to the microspheres for resuspending to obtain a resuspension, filter the resuspension with a 0.22 μm filter membrane to remove apoptotic bodies and microvesicles, purify the filtered target microspheres to obtain purified human adipose mesenchymal stem cell exosomes (EVs), sterilize the purified human adipose mesenchymal stem cell exosomes to obtain human adipose mesenchymal stem cell exosomes; identify the human adipose mesenchymal stem cell exosomes by electron microscopy; and identify the obtained human adipose mesenchymal stem cell exosomes by WB.

2. The method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 1, characterized in that: 3 mL of the exosome-free culture medium was added, and the exosome-free culture medium contained type IV collagenase with a final concentration of 0.06-0.16% and 0.015-0.02% BSA. The culture medium was kept at 37°C and oscillated at 120 r / min for 30-35 min. The tissue digestion status was observed at any time. During this period, the tissue was taken out and mixed every 5-8 min. After the digestion was terminated, it was filtered in turn with 100 mesh and 200 mesh sieves to remove undigested tissue.

3. The method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 1, characterized in that: The culture medium contains 10% by volume of fetal bovine serum without exosomes.

4. The method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 1, characterized in that: The indoor conditions of the incubator were 37° C., 5% CO 2 , and 100% relative humidity.

5. The method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 1, characterized in that: The human adipose stem cells were incubated with 10 nmol / L GLP-1 for 4 hours.

6. The method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 1, characterized in that: The exosome-free serum medium was cultured at 37° C. and 5% CO 2 .

7. The method for preparing mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 1, characterized in that: The serum culture medium of the human adipose-derived mesenchymal stem cell exosomes contains 300 μmol / L dimethyloxalylglycine.

8. A method for using mesenchymal stem cell exosomes incubated with the GLP-1 drug according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1: Preparation of rat myocardial infarction model and exosome injection; SD rats were randomly divided into: Sham group, AMI group, ExoBMSCs group, intraperitoneal injection anesthesia, exposure of rat neck trachea, endotracheal intubation connected to ventilator assisted breathing, 1 cm surgical incision between the 3rd and 4th ribs on the left side of the rat chest, exposure of the heart, 5-0 needle and thread ligation at the beginning of the left anterior descending coronary artery, ischemia treatment for 30 minutes, cut the silk thread to restore blood perfusion; S2: When the ST segment on the electrocardiogram recorder was significantly elevated and the left ventricle was white due to ischemia, it indicated that the AMI model was successfully established. The rats in the Sham group were only threaded with the left anterior descending branch of the coronary artery, but not ligated. The rats in the ExoBMSCs group and the AMI group were injected with 25 μL of exosomes (750 mg / mL) at four sites in the myocardial infarction area. The Sham group and the AMI group were injected with the same amount of normal saline at the same site. S3: One week after the group-based intervention, echocardiographic monitoring was performed to record the left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), left ventricular ejection fraction (LVEF) and fractional shortening (FS).

9. The method for using mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 8, characterized in that: During the anesthesia, sodium pentobarbital (30 mg / kg) was injected intraperitoneally.

10. The method for using mesenchymal stem cell exosomes incubated with a GLP-1 drug according to claim 8, characterized in that: The breathing rate of the ventilator is 90 times / min, the tidal volume is 8 mL, and the respiratory ratio is 1:2.

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