Method for improving myocardial protection activity of human embryonic stem cell exosome and application thereof
By using green light irradiation and ultrahigh-speed centrifugation technology to increase the production and activity of embryonic stem cell exosomes, the problem of low biological activity of exosomes was solved, significant myocardial protection effects were achieved, and it has good application prospects.
Patent Information
- Application Number
- CN202510981242.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-17
AI Technical Summary
In the existing technology, the biological activity of embryonic stem cell exosomes is low, which limits their application in myocardial injury repair. In addition, the preparation cost is high and industrial production cannot be achieved.
Human embryonic stem cells are irradiated with green light. After light stimulation with a specific wavelength and brightness, exosomes are extracted using ultra-high-speed centrifugation technology to enhance their cardioprotective activity.
It significantly improves the production of exosomes and myocardial protective activity, promotes cardiomyocyte proliferation, inhibits apoptosis, reduces myocardial infarction area, restores cardiac function, is easy to operate, and has good production and application prospects.
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Figure CN120796174A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for improving the myocardial protection activity of human embryonic stem cell exosomes and application thereof. BACKGROUND
[0002] Myocardial infarction has a high incidence and mortality, and seriously threatens the health and quality of life of the people. When acute myocardial infarction occurs, local ischemia and hypoxia lead to a large number of functional cardiomyocytes to be necrotic and apoptotic. Since adult cardiomyocytes have exited the cell cycle, the missing cardiomyocytes are replaced by fibrous scar tissue, which in turn causes ventricular remodeling, a decline in heart function and eventually the development of heart failure. Although the development of drugs and percutaneous coronary intervention can improve the early survival rate of myocardial infarction patients. However, there is still a lack of effective prevention and treatment measures for myocardial ischemia-induced reduction in the number of cardiomyocytes in clinical practice.
[0003] Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the inner cell mass of human blastocysts, and have great prospects in heart regeneration due to their strong proliferation and differentiation capabilities. In animal models of myocardial infarction, embryonic stem cells have been shown to significantly improve heart regeneration and function. However, this stem cell therapy method has risks such as immune rejection, tumorigenicity and arrhythmia. In addition, poor survival and engraftment of transplanted cells are also major challenges faced by stem cell therapy. Exosomes (Exos) are a kind of natural nanoscale vesicles secreted by cells, with a diameter of 30-150 nm, containing various bioactive substances such as proteins, lipids and nucleic acids, and are important carriers for intercellular signal transmission. In recent years, a large number of studies have found that stem cell-derived exosomes have superior protective effects on myocardial injury and have advantages that previous stem cell therapy does not have, such as low immunogenicity, no cytotoxicity and the ability to penetrate biological barriers. Among them, exosomes derived from embryonic stem cells have been shown to enhance neovascularization, promote cardiomyocyte proliferation and survival, inhibit cardiomyocyte apoptosis and reduce myocardial fibrosis after infarction, bringing new hope for the repair treatment of myocardial infarction. Unfortunately, the in vitro culture of embryonic stem cells and the extraction of exosomes are costly, and the activity of exosomes released by embryonic stem cells in a natural state is low. If myocardial injury repair is to be achieved, higher requirements are placed on the biological activity and quantity of exosomes, making it impossible to industrialize the production of exosomes and apply them to the treatment of ischemic heart disease. Therefore, it is urgent to develop a safe and effective way to improve the yield and biological activity of human embryonic stem cell exosomes to fully exert the therapeutic effect of embryonic stem cell exosomes on injured myocardium. SUMMARY
[0004] In order to solve the problem of low biological activity of the embryonic stem cell exosome extracted by in-vitro culture, the application provides a method for improving the myocardial protection activity of human embryonic stem cell exosome and application thereof.
[0005] The technical scheme of the application is as follows:
[0006] The method for improving the myocardial protection activity of human embryonic stem cell exosome comprises the following steps: when human embryonic stem cells are cultured to a confluence degree of 70-80%, the human embryonic stem cells are vertically irradiated with green light as the only light source for a certain time; the human embryonic stem cells after light irradiation are transferred to a light-proof incubator for continuous culture; and then the exosome produced by the human embryonic stem cells is extracted by centrifugation.
[0007] Further, the central wavelength of the green light is 520 nm, and the radiation brightness of the green light is 1-5 mW / cm 2 .
[0008] Further, the radiation brightness of the green light is 2.5 mW / cm 2 .
[0009] Further, the irradiation time of the green light is 20-40 min.
[0010] Further, the culture condition of the human embryonic stem cells is 37 DEG C and 5% CO2.
[0011] Further, the culture time of the human embryonic stem cells after light irradiation is 48 h.
[0012] Further, the centrifugation is ultrahigh-speed centrifugation, the human embryonic stem cell culture solution is first centrifuged at 4 DEG C and 3000 g for 20 min, then the supernatant obtained by centrifugation is transferred to a new centrifuge tube, and the centrifuge tube is again centrifuged at 4 DEG C and 10000 g for 60 min, then the supernatant obtained by centrifugation is filtered by a 0.22 mu m microporous filter, the filtrate is collected and again placed in 4 DEG C 100000 g for 70 min, and the precipitate obtained by centrifugation is resuspended in PBS buffer to obtain the human embryonic stem cell exosome.
[0013] The application of the human embryonic stem cell exosome extracted by the method for improving the myocardial protection activity of human embryonic stem cell exosome in the preparation of a drug for treating myocardial infarction.
[0014] Further, the drug for treating myocardial infarction is injected into the tissue of the marginal zone of myocardial infarction in the form of myocardial in-situ injection.
[0015] Further, the exosome for treating myocardial infarction reduces the myocardial infarction area by promoting the proliferation of myocardial cells and inhibiting the myocardial cell apoptosis caused by hypoxia.
[0016] The human embryonic stem cell in the present application is not a human embryonic stem cell isolated or obtained from a human embryo developed in vivo, nor is it a stem cell isolated or obtained from a human embryo fertilized for more than 14 days without in vivo development, but is an established human embryonic stem cell line.
[0017] Advantages of the present application:
[0018] The present application uses green light to irradiate human embryonic stem cells, which not only improves the yield of human embryonic stem cell exosomes, but also significantly improves the myocardial protection activity. Compared with normal cultured human embryonic stem cells without treatment, the exosomes produced by the human embryonic stem cells irradiated by green light can significantly promote the proliferation of primary rat myocardial cells, inhibit the apoptosis of primary rat myocardial cells caused by hypoxia, significantly activate adult mouse myocardial cell regeneration, inhibit myocardial cell apoptosis, reduce myocardial infarction area and restore infarct heart function.
[0019] The present application is simple to operate, does not require complex equipment and processes, and therefore has good production and application prospects, and is expected to become an important technology in the field of cardiovascular disease treatment in the future. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 A comparison chart of cell viability of rat myocardial cells in different treatment groups in Example 4;
[0021] Figure 2 A transmission electron micrograph of human embryonic stem cell exosomes obtained by green light irradiation culture and conventional culture in Example 5;
[0022] Figure 3 A comparison chart of particle density of human embryonic stem cell exosomes obtained by green light irradiation culture and conventional culture in Example 5;
[0023] Figure 4 and Figure 5 The Western blot detection results and expression level comparison chart of marker proteins TSG101, CD63 and Flotillin1 of human embryonic stem cell exosomes obtained by green light irradiation culture and conventional culture in Example 5, respectively;
[0024] Figure 6 An immunofluorescence staining chart of myocardial cells in different treatment groups in the immunofluorescence experiment of Example 6;
[0025] Figure 7 and Figure 8 Comparison charts of expression of proliferation markers Ki67 and pH3 of myocardial cells in different treatment groups in the immunofluorescence experiment of Example 6, respectively;
[0026] Figure 9 A fluorescence staining chart of myocardial cells in different treatment groups in the TUNEL detection of Example 6;
[0027] Figure 10 This is a comparison of the number of apoptotic cardiomyocytes in different treatment groups in TUNEL assay in Example 6;
[0028] Figure 11 The echocardiograms of mice in different treatment groups in the cardiac ultrasound examination of Example 7 are shown;
[0029] Figure 12 and Figure 13 These are comparative diagrams of ejection fraction and fractional shortening of mice in different treatment groups during cardiac ultrasound examination in Example 7;
[0030] Figure 14 The stained images of the hearts of mice in different treatment groups in the Masson staining experiment of Example 7 are shown;
[0031] Figure 15 This is a comparison of myocardial infarction areas in mice of different treatment groups in the Masson staining experiment of Example 7;
[0032] Figure 16 This is the staining image of the cardiomyocytes of mice in different treatment groups in the immunofluorescence experiment of Example 7;
[0033] Figure 17 and Figure 18 These are comparative diagrams of the expression of cardiomyocyte proliferation markers Ki67 and pH3 in mice treated in different groups in the immunofluorescence experiment of Example 7;
[0034] Figure 19 This is a fluorescence staining image of cardiomyocytes of mice in different treatment groups in TUNEL assay in Example 7;
[0035] Figure 20 This is a comparison of the number of apoptotic cardiomyocytes in mice in different treatment groups in TUNEL assay in Example 7. DETAILED DESCRIPTION
[0036] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.
[0037] Example 1
[0038] This embodiment provides a method for improving the cardioprotective activity of human embryonic stem cell exosomes.
[0039] The human embryonic stem cells used in this embodiment are not human embryonic stem cells isolated or obtained from human embryos developed in vivo and are not stem cells isolated or obtained from human embryos fertilized for more than 14 days without in vivo development, but are established human embryonic stem cell lines. The human embryonic stem cells used in this embodiment are purchased from the National Stem Cell Resource Library of the Institute of Animal Sciences, Chinese Academy of Sciences, and the cell number is CB0003.
[0040] This embodiment provides a subculture method of human embryonic stem cells, and the steps are as follows:
[0041] The human embryonic stem cells are observed under a microscope, and when the confluence reaches 70-80%, they can be subcultured. Before subculture, the PSCeasy® human pluripotent stem cell complete medium (Beijing Saibei Biotechnology Co., Ltd.) is balanced to room temperature in advance, the remaining working solution of the Matrigel working solution (Beijing Saibei Biotechnology Co., Ltd.) pre-coated in the six-well plate is sucked out with a pipette, 2 mL of PSCeasy® human pluripotent stem cell complete medium is added to each well, and then it is transferred to a 37 ℃, 5% CO2 incubator for preheating. The original culture solution of the cells to be subcultured is discarded, the cells are rinsed with an appropriate amount of PBS buffer solution containing no calcium and magnesium ions and then discarded, 1 mL of PSCeasy® human pluripotent stem cell digestion solution (Beijing Saibei Biotechnology Co., Ltd.) is added and immediately transferred to a 37 ℃, 5% CO2 incubator for separation and digestion, 3 min later, the cell state is observed under a microscope, when the cell gap increases and there is slight shedding, the digestion solution is discarded in a clean bench, an appropriate amount of PSCeasy® human pluripotent stem cell complete medium is added according to the cell density, the bottom of the well is gently blown 3-4 times with a pipette, it is transferred to a cell culture plate at a ratio of 1:3, and it is horizontally shaken and placed in a clean bench for 3-5 min, 4-10 cells are observed under an optical microscope as a cell mass, and the distribution is uniform.
[0042] The method for improving the myocardial protective activity of human embryonic stem cell exosomes in this embodiment, the specific steps are as follows:
[0043] The human embryonic stem cells with a confluence of 70-80% are taken and the fresh PSCeasy® human pluripotent stem cell complete medium is replaced. The human embryonic stem cell culture plate is placed under a light-proof cover with a hole at the top, and a green light source is placed at the top of the cover. The green light can be vertically irradiated to the human embryonic stem cell culture plate through the hole at the top.
[0044] The central wavelength of the green light source in this embodiment is 520 nm, and the irradiance is 1 mW / cm 2, and irradiate the human embryonic stem cells for 30 min. After the light stimulation condition, the human embryonic stem cells are placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0045] The exosomes produced by the human embryonic stem cells are extracted by ultrahigh-speed centrifugation. The culture solution of the human embryonic stem cells is centrifuged at 3000 g at 4℃ for 20 min, and then the supernatant obtained by centrifugation is transferred to a new centrifuge tube and centrifuged at 10000 g at 4℃ for 60 min. The supernatant obtained by centrifugation is filtered by a 0.22 μm microporous filter, and the filtrate is placed in a new centrifuge tube and centrifuged at 100000 g at 4℃ for 70 min. The precipitate obtained by centrifugation is resuspended in PBS buffer to obtain the exosomes of the human embryonic stem cells.
[0046] Example 2
[0047] The method for improving the myocardial protection activity of the exosomes of the human embryonic stem cells provided in this example is different from that of Example 1 only in that the central wavelength of the green light source in this example is 520 nm, and the irradiation intensity is 2.5 mW / cm 2 , and irradiate the human embryonic stem cells for 30 min. After the light stimulation condition, the human embryonic stem cells are placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0048] Example 3
[0049] The method for improving the myocardial protection activity of the exosomes of the human embryonic stem cells provided in this example is different from that of Example 1 only in that the central wavelength of the green light source in this example is 520 nm, and the irradiation intensity is 5 mW / cm 2 , and irradiate the human embryonic stem cells for 30 min. After the light stimulation condition, the human embryonic stem cells are placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0050] Comparative Example 1
[0051] The comparative example is different from Example 1 only in that a red light source is used to irradiate the human embryonic stem cells in the comparative example, the central wavelength of the red light source is 630 nm, and the irradiation intensity is 1 mW / cm 2 , and irradiate the human embryonic stem cells for 30 min. After the light stimulation condition, the human embryonic stem cells are placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0052] Comparative Example 2
[0053] The comparative example is different from Example 1 only in that a red light source is used to irradiate the human embryonic stem cells in the comparative example, the central wavelength of the red light source is 630 nm, and the irradiation intensity is 2.5 mW / cm 2The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were put back into the 37 °C, 5% CO2 incubator for continuous culture for 48 h.
[0054] Comparative Example 3
[0055] The difference between this comparative example and Example 1 is that this comparative example uses a red light source to irradiate the human embryonic stem cells, the central wavelength of the red light source is 630 nm, and the irradiation intensity is 5 mW / cm 2 The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were put back into the 37 °C, 5% CO2 incubator for continuous culture for 48 h.
[0056] Comparative Example 4
[0057] The difference between this comparative example and Example 1 is that this comparative example uses a blue light source to irradiate the human embryonic stem cells, the central wavelength of the blue light source is 470 nm, and the irradiation intensity is 1 mW / cm 2 The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were put back into the 37 °C, 5% CO2 incubator for continuous culture for 48 h.
[0058] Comparative Example 5
[0059] The difference between this comparative example and Example 1 is that this comparative example uses a blue light source to irradiate the human embryonic stem cells, the central wavelength of the blue light source is 470 nm, and the irradiation intensity is 2.5 mW / cm 2 The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were put back into the 37 °C, 5% CO2 incubator for continuous culture for 48 h.
[0060] Comparative Example 6
[0061] The difference between this comparative example and Example 1 is that this comparative example uses a blue light source to irradiate the human embryonic stem cells, the central wavelength of the blue light source is 470 nm, and the irradiation intensity is 5 mW / cm 2 The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were put back into the 37 °C, 5% CO2 incubator for continuous culture for 48 h.
[0062] Comparative Example 7
[0063] The difference between this comparative example and Example 1 is that this comparative example uses a yellow light source to irradiate the human embryonic stem cells, the central wavelength of the yellow light source is 590 nm, and the irradiation intensity is 1 mW / cm 2The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0064] Comparative Example 8
[0065] The difference between this comparative example and Example 1 is that the human embryonic stem cells in this comparative example were irradiated by a yellow light source, the central wavelength of the yellow light source was 590 nm, and the irradiation intensity was 2.5 mW / cm 2 The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0066] Comparative Example 9
[0067] The difference between this comparative example and Example 1 is that the human embryonic stem cells in this comparative example were irradiated by a yellow light source, the central wavelength of the yellow light source was 590 nm, and the irradiation intensity was 5 mW / cm 2 The human embryonic stem cells were irradiated for 30 min. After the light stimulation condition, the human embryonic stem cells were placed back into the 37℃, 5% CO2 incubator for continuous culture for 48 h.
[0068] Comparative Example 10
[0069] The difference between this comparative example and Example 1 is that the human embryonic stem cells in this comparative example were not irradiated, and the human embryonic stem cells were always cultured in a 37℃, 5% CO2 incubator, and the culture time was the same as that in Example 1.
[0070] Example 4
[0071] This example detected the effect of the human embryonic stem cell exosomes extracted in Examples 1-3 and Comparative Examples 1-10 on the activity of milk rat myocardial cells by CCK8 experiment.
[0072] CCK8 experiment detection:
[0073] The milk rat primary myocardial cells were inoculated in a 96-well plate. After the cells adhered, equal amounts of exosomes produced by the human embryonic stem cells in Comparative Example 10 without light treatment and the exosomes produced by the human embryonic stem cells in Examples 1-3 and Comparative Examples 1-9 after light irradiation (3×10 3 particles / myocardial cell) were added to the primary cultured milk rat myocardial cells, and after 24 h, the myocardial cell activity after exosome treatment was detected by CCK8 experiment. 10 μL CCK-8 solution (Bi Yun Tian Biotechnology Co., Ltd.) was added to each well of the myocardial cells, and then placed into a 37℃ incubator for 1 h, and then the absorbance was measured at 450 nm using an enzyme label instrument.
[0074] The results are as follows:Figure 1 As shown in FIG. 2B, the green light irradiation at an irradiation intensity of 2.5 mW / cm 2 The human embryonic stem cell exosomes can significantly improve the viability of cardiomyocytes after green light irradiation for 30 min at an irradiation intensity of 2.5 mW / cm
[0075] Example 5
[0076] This example investigates the effect of green light irradiation on the yield of human embryonic stem cell exosomes.
[0077] The microstructure of the human embryonic stem cell exosomes obtained by green light irradiation culture of Example 2 and conventional culture of Comparative Example 10 was characterized by transmission electron microscopy (TEM), and the results are shown in FIG. 3. Figure 2 As shown in FIG. 3, the extracted exosomes all have a typical cup-shaped vesicle structure.
[0078] The particle size and particle density of the human embryonic stem cell exosomes obtained by green light irradiation culture of Example 2 and conventional culture of Comparative Example 10 were evaluated by nanoparticle tracking analysis (NTA), and the results are shown in FIG. 4. Figure 3 As shown in FIG. 4, the exosome particle size is unimodal distribution, and the green light treatment does not affect the size of the human embryonic stem cell exosomes secreted by the human embryonic stem cells, but the number of exosomes increases after green light treatment.
[0079] The expression levels of the marker proteins TSG101, CD63, and Flotillin1 of the human embryonic stem cell exosomes obtained by green light irradiation culture of Example 2 and conventional culture of Comparative Example 10 were detected by Western blotting, and the results are shown in FIG. 5 and FIG. 6. Figure 4 Figure 5 As shown in FIG. 5 and FIG. 6, compared with the conventional exosome group, the expression levels of the exosome marker proteins of the green light exosome group are significantly increased, indicating that green light treatment can increase the yield of human embryonic stem cell exosomes.
[0080] Example 6
[0081] This example investigates the effect of green light irradiation of human embryonic stem cells on the proliferation and apoptosis of primary cultured milk rat cardiomyocytes.
[0082] I. The method for culturing primary milk rat cardiomyocytes is as follows:
[0083] The heart of a 1-3-day-old mouse was taken out under sterile conditions, washed to remove blood, and the aorta and atrium were cut off. The heart was then cut into small pieces and completely digested with 0.25% trypsin (Beijing Solabio Technology Co., Ltd.). The mixture was then centrifuged at 1000 rpm for 5 min. The suspended cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin (Thermo Fisher Scientific) for 2 h. The non-adherent cardiomyocytes were then transferred to a new culture plate and cultured under normoxic conditions at 37°C for 48 h.
[0084] II. The effect of the exosomes produced by the green light irradiated human embryonic stem cells on the proliferation ability of cardiomyocytes was detected by immunofluorescence experiment, and the specific experimental method was as follows:
[0085] The primary rat cardiomyocytes inoculated in the 24-well plate were added with equal amounts of the conventional exosomes obtained in Comparative Example 10 and the exosomes obtained by culturing the human embryonic stem cells under green light irradiation in Example 2, and the amount of exosomes added was 3 x 10 3 After 24 h of incubation, the cell culture medium was discarded. The cells were washed 3 times with PBS, fixed with 4% paraformaldehyde (Beijing Solabio Technology Co., Ltd.) for 20 min, and then permeabilized with 0.4% Triton X-100 (Shanghai Blueyuntian Biotechnology Co., Ltd.) for 15 min at room temperature. After washing with PBS, the samples were blocked with goat serum (Wuhan Biodee Biological Engineering Co., Ltd.) at 37°C for 30 min to reduce non-specific binding. Next, the samples were incubated with antigen antibodies against pH3 (Abcam (Shanghai) Trading Co., Ltd.), Ki67 (Abcam (Shanghai) Trading Co., Ltd.), and a-Actinin (Abcam (Shanghai) Trading Co., Ltd.) overnight at 4°C, and then incubated with secondary antibodies at 37°C for 1 h in the dark. The cell nuclei were stained with DAPI (Beijing Solabio Technology Co., Ltd.) for 10 min, and the expression of the cardiomyocyte proliferation markers Ki67 and pH3 was observed using a confocal laser scanning microscope.
[0086] The results are shown in Table 1, and the control group in the figure is the cardiomyocytes without exosome treatment. Compared with the conventional human embryonic stem cell-derived exosomes, the same amount of green light exosomes can significantly promote the proliferation of cardiomyocytes. Figure 6-8
[0087] III. The effect of the exosomes produced by the green light irradiated human embryonic stem cells on the apoptosis of the primary cultured rat cardiomyocytes was detected by TUNEL, and the specific experimental method was as follows:
[0088] Take the primary milk rat myocardial cells inoculated in the 24-hole plate, and add equal amounts of the conventional exosomes obtained from Comparative Example 10 and the exosomes obtained from the green light irradiation culture of human embryonic stem cells in Example 2 to the primary cultured milk rat myocardial cells, respectively. The myocardial cells are transferred to an anoxic incubator composed of 94% N2, 5% CO2 and 1% O2, and incubated for 24 h. The cell culture medium is discarded. Wash 3 times with PBS, add 500 μL 4% paraformaldehyde (Beijing Solabio Technology Co., Ltd.) for fixation for 30 min, add 500 μL Triton X-100 (Beijing Solabio Technology Co., Ltd.), 1% BSA (Dr. De Biological Engineering Co., Ltd.) for incubation at room temperature for 1 h, 500 μL goat serum (Dr. De Biological Engineering Co., Ltd.) for blocking for 1 h, and use the TUNEL cell apoptosis assay kit (Bi Yun Tian Biological Technology Co., Ltd.) to stain the myocardial cells. Incubate DAPI (Dr. De Biological Engineering Co., Ltd.) at room temperature for 10 min, and use a fluorescence microscope to evaluate the myocardial cell apoptosis.
[0089] The results are shown in Figure 9 and Figure 10 The control group in the figure is the myocardial cells that are not treated with exosomes and cultured in an anoxic environment. Compared with the untreated human embryonic stem cell-derived exosomes, the same amount of green light exosomes can significantly inhibit the myocardial cell apoptosis caused by anoxia.
[0090] Example 7
[0091] This example investigates the effect of the exosomes extracted from the green light irradiated human embryonic stem cells on the heart regeneration and heart repair of myocardial infarction mice.
[0092] I. The myocardial infarction model is constructed as follows:
[0093] C57BL / J male mice, weighing about 25 g, are anesthetized with 2-4% isoflurane and fixed in a supine position. PE-90 number cannula is used for tracheal intubation and connected to a mouse respirator. The tidal volume of the mouse is adjusted to 250-300 mL / min, and the respiratory rate is controlled at 110-130 times / min. The intercostal muscle is bluntly separated at the 4th or 5th intercostal space, and a self-made hook is used to ligate the left anterior descending branch (LAD) of the coronary artery about 2 mm below the left auricle. The electrocardiogram shows that the ST segment is arch-shaped upward, and the corresponding area of the heart surface changes from original color to pale, indicating successful ischemia. The sham operation group is not ligated, and the other operations are the same as those of the myocardial infarction model group.
[0094] II. The administration method of the treatment group is as follows:
[0095] An equal amount of conventional exosomes obtained from Comparative Example 10 and exosomes obtained from Example 2 green light treated human embryonic stem cells were injected into the myocardial infarction edge zone tissue of mice by myocardial in-situ injection, and the injection amount was 5 x 10 8 The ribs, muscles and skin of the mice were sequentially sutured.
[0096] III. Echocardiography detection method as follows:
[0097] Four weeks after injection, the mice were anesthetized, the limbs were fixed, the chest was shaved and coated with an appropriate amount of coupling agent. The left lateral position was taken, and a probe with a frequency of 12 Mhz was used for cardiac ultrasound examination (U.S. GE Company). The long axis section of the left ventricle was taken, and the left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured by M-mode ultrasound. The left ventricular short axis shortening rate (FS) and ejection fraction (EF) were calculated. The measured value was the average value measured for 5 consecutive cardiac cycles.
[0098] The results are shown in Figure 11-13 Compared with the myocardial infarction model group and the myocardial infarction model + conventional exosome group, the green light exosomes can significantly improve the cardiac function of the myocardial infarction mice.
[0099] IV. Masson staining technique was used to observe the changes in myocardial infarction area, and the specific experimental method was as follows:
[0100] The heart of the myocardial infarction model mouse was fixed with 4% paraformaldehyde for 24 h, paraffin-embedded to prepare 4 μm sections, and after deparaffinization and hydration, Weigert iron hematoxylin staining was performed for 5-10 min, and then acidified ethanol was differentiated and washed with water, and then Masson blue solution was returned to blue and washed with water; After washing with distilled water for 1 min, it was stained with ponceau red for 5-10 min, then washed with pre-prepared weak acid working solution (distilled water: weak acid solution = 2:1) for 1 min, molybdenum phosphoric acid solution for 1-2 min, and weak acid working solution for 1 min; Directly transfer to aniline blue for 1-2 min, and then wash with weak acid working solution, then dehydrate with 95% ethanol for 3 times (5-10 s each time), xylene for 3 times (1-2 min each time), and finally seal with neutral resin.
[0101] The results are shown in Figure 14 and Figure 15 Compared with the untreated human embryonic stem cell-derived exosomes, the exosomes produced by the green light treated human embryonic stem cells can significantly reduce the myocardial infarction area.
[0102] V. The effect of exosomes produced by green light irradiated human embryonic stem cells on adult mouse myocardial cell regeneration was detected by immunofluorescence experiment, and the specific experimental method was as follows:
[0103] At 14 days after operation, the heart tissues of mice in each group were taken, embedded with embedding agent, and then frozen section was performed by using a freezing microtome, with each slice being 5 μm thick. The samples were fixed with 4% paraformaldehyde (Beijing Solabio Science and Technology Co., Ltd.) for 20 min, and then permeabilized with 0.4% Triton X-100 (Shanghai Biyun Tian Biological Technology Co., Ltd.) for 15 min at room temperature. After washing with PBS, the samples were blocked with goat serum (Wuhan Biodee Biological Engineering Co., Ltd.) for 30 min at 37°C to reduce non-specific binding. Next, the samples were incubated with antigen antibodies against pH3 (Abcam (Shanghai) Trading Co., Ltd.), Ki67 (Abcam (Shanghai) Trading Co., Ltd.) and a-actinin (Abcam (Shanghai) Trading Co., Ltd.) overnight at 4°C, and then incubated with secondary antibodies for 1 h at 37°C in the dark. The nuclei were stained with DAPI (Beijing Solabio Science and Technology Co., Ltd.) for 10 min, and the expression of the proliferation markers Ki67 and pH3 in the myocardial tissue slices was observed using a confocal laser scanning microscope.
[0104] The results are shown in Figure 16-18 Compared with the conventional exosomes, the green light exosomes can significantly promote the proliferation and regeneration ability of adult mouse myocardial cells.
[0105] Six, the influence of exosomes produced after green light irradiation of human embryonic stem cells on the apoptosis of adult mouse myocardial cells was detected by TUNEL, and the specific experimental method was as follows:
[0106] At 7 days after operation, the heart tissues of mice in each group were taken, embedded with embedding agent, and then immediately stored in liquid nitrogen for freezing, and then frozen section was performed by using a freezing microtome, and 4% paraformaldehyde (Beijing Solabio Science and Technology Co., Ltd.) was added for fixation for 20 min, and then permeabilized with 0.4% Triton X-100 (Shanghai Biyun Tian Biological Technology Co., Ltd.) for 15 min at room temperature. After washing with PBS, the samples were blocked with goat serum (Wuhan Biodee Biological Engineering Co., Ltd.) for 30 min at 37°C to reduce non-specific binding. The heart tissue slices were stained using a TUNEL cell apoptosis assay kit (Biyuntian Biological Technology Co., Ltd.), DAPI (Beijing Solabio Science and Technology Co., Ltd.) was incubated at room temperature for 10 min, and the apoptosis of myocardial cells was evaluated using a fluorescence microscope.
[0107] The results are shown in Figure 19 and Figure 20 Compared with the conventional exosomes, the exosomes produced after green light irradiation of human embryonic stem cells can significantly inhibit the apoptosis of myocardial cells caused by myocardial infarction.
[0108] Seven, statistics and analysis:
[0109] Data were analyzed and processed using GraphPad Prism 9.0 statistical software. Data were expressed as mean ± SEM. Comparison between two groups was performed using student t-test and Mann Whitney U test. Comparison among multiple groups was performed using one way ANOVA. P < 0.05 was considered statistically significant.
Claims
1. A method for improving the cardioprotective activity of human embryonic stem cell exosomes, characterized in that: When human embryonic stem cells are subcultured to a confluence of 70-80%, they are vertically irradiated with green light as the sole light source for a certain period of time. The light-stimulated human embryonic stem cells are transferred to a light-tight incubator for further culture, and then the exosomes produced by the human embryonic stem cells are extracted by centrifugation.
2. A method for improving the cardioprotective activity of human embryonic stem cell exosomes according to claim 1, characterized in that: The central wavelength of the green light is 520 nm, and the radiant brightness of the green light is 1-5 mW / cm 2 .
3. A method for improving the cardioprotective activity of human embryonic stem cell exosomes according to claim 1 or 2, characterized in that: The radiant brightness of the green light is 2.5 mW / cm 2 .
4. A method for improving the cardioprotective activity of human embryonic stem cell exosomes according to claim 3, characterized in that: The green light irradiation time is 20 to 40 minutes.
5. A method for improving the cardioprotective activity of human embryonic stem cell exosomes according to claim 4, characterized in that: Human embryonic stem cells were cultured at 37°C and 5% CO2.
6. A method for improving the cardioprotective activity of human embryonic stem cell exosomes according to claim 5, characterized in that: The culture time of the human embryonic stem cells after light stimulation is 48 hours.
7. A method for improving the cardioprotective activity of human embryonic stem cell exosomes according to claim 6, characterized in that: The centrifugation is ultrahigh-speed centrifugation. First, human embryonic stem cell culture medium is centrifuged at 4°C, 3000g for 20 minutes. The supernatant obtained by centrifugation is then transferred to a new centrifuge tube and centrifuged again at 4°C, 10,000g for 60 minutes. The supernatant obtained by centrifugation is then filtered through a 0.22 μm microporous filter membrane, and the collected filtrate is again centrifuged at 4°C, 100,000g for 70 minutes. The precipitate obtained by centrifugation is resuspended in PBS buffer to obtain human embryonic stem cell exosomes.
8. Use of human embryonic stem cell exosomes extracted by the method for improving the cardioprotective activity of human embryonic stem cell exosomes according to any one of claims 1 to 7 in the preparation of a drug for treating myocardial infarction.
9. The use of human embryonic stem cell exosomes in the preparation of a medicament for treating myocardial infarction according to claim 8, characterized in that: The medicine for treating myocardial infarction is injected into the myocardial infarction marginal zone tissue in a manner of in situ myocardial injection.
10. Use of human embryonic stem cell exosomes in the preparation of a medicament for treating myocardial infarction according to claim 8 or 9, characterized in that: The exosomes for treating myocardial infarction reduce the area of myocardial infarction by promoting myocardial cell proliferation and inhibiting myocardial cell apoptosis caused by hypoxia.