A method for inducing stem cells to secrete specific factors

Abstract

The present invention relates to the field of stem cell technology. The present invention discloses a method for inducing stem cells to secrete specific factors, which includes stimulating stem cells with high-frequency waves, emitting sound waves of a specific frequency to stem cells, regulating stem cells in a hypoxic environment, culturing stem cells in a hypoxic environment, stimulating stem cells with oxidative stress, and subjecting stem cells to oxidative stimulation treatment. The present invention can significantly enhance the proliferation ability of stem cells, promote the secretion of multiple active factors, and improve cell function and adaptability. As a non-invasive physical method, high-frequency wave stimulation can activate internal cell signal pathways without introducing exogenous substances, significantly improving the proliferation ability and tissue repair potential of stem cells. Hypoxic environment regulation simulates the naturally existing microenvironmental conditions in the body, greatly promoting the secretion of active factors such as vascular endothelial growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor for tissue repair and immune regulation.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, and in particular to a method for inducing stem cells to secrete specific factors. Background Art

[0002] Stem cells have unique self-renewal and differentiation properties. They are divided into embryonic stem cells and adult stem cells according to their developmental stage. Adult stem cells include hematopoietic stem cells and stem cells. Stem cells have the characteristics of multi-lineage differentiation potential, promoting angiogenesis, immunomodulation, strong in vitro expansion ability and easy acquisition from adult tissues. They are the most commonly studied non-hematopoietic adult stem cells. Although there are many clinical trials using stem cells, stem cell-based therapies have safety issues that cannot be ignored, such as the possibility of differentiation into undesirable cell types after transplantation, excessive immunosuppression, and the risk of viral transmission.

[0003] It is generally recognized that the therapeutic effect of stem cell infusion is not through the replacement of damaged tissues by stem cells, but through its paracrine effect. That is, the infused stem cells release extracellular vesicles such as exosomes or cytokines, which act on target cells, change the function of target cells, and then promote the regenerative activity of target cells. This new treatment scheme using the secretome has become a hot topic in the field of regenerative medicine. However, due to the heterogeneity and complexity of the stem cell secretome, the road from laboratory to clinic is still long. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present invention discloses a technology for inducing stem cells to secrete specific components in vitro, and a method for inducing stem cells to secrete specific factors to solve the problem of heterogeneity and complexity of the stem cell secretome.

[0005] The present invention discloses a method for inducing stem cells to secrete specific factors, which comprises:

[0006] High-frequency waves stimulate stem cells, emitting sound waves of a specific frequency to the stem cells;

[0007] Hypoxic environment regulates stem cells, and the stem cells are cultured in a hypoxic environment;

[0008] Oxidative stress stimulates stem cells, and the stem cells are subjected to oxidative stimulation.

[0009] Furthermore, the specific method of stimulating stem cells with high-frequency waves is to use a device that can emit a spectrum of waves to emit sound waves with a frequency of 1-20 MHz to the stem cells in the culture bottle.

[0010] Furthermore, the specific method of regulating stem cells in a hypoxic environment is to place the stem cells in a saturated humidity incubator with a volume fraction of 0.5%-15% O2, a volume fraction of 5% CO2, and a volume fraction of 80%-94.5% N2 and culture them for 24 hours.

[0011] Furthermore, the specific method of stimulating stem cells with oxidative stress is to inoculate the stem cells in a well plate, and then add H2O2 at a concentration of 30-120 μmol / L for treatment after the stem cells adhere to the wall.

[0012] Furthermore, the specific ways in which high-frequency waves stimulate stem cells are:

[0013] Ensure that all experimental equipment has been sterilized and is ready for use. Seed the stem cells into appropriate culture flasks and pre-culture them to an appropriate cell density under standard conditions at 37°C and 5% CO2.

[0014] Using an acoustic wave emitting device for biological sample processing, adjusting the emission frequency range of the acoustic wave to be 1-20 MHz, turning on the acoustic wave emitting device, and performing acoustic wave irradiation treatment on the stem cells;

[0015] On the first, third and fifth days after the sonication device was turned on, the original culture medium was aspirated and then an appropriate amount of fresh culture medium was added. After the fresh culture medium was replaced, the sonication was stopped to allow the cells to recover and continue to grow under normal conditions.

[0016] After turning on the acoustic wave emission device, the cell morphology changes and growth conditions were observed regularly, and the cell supernatant was collected regularly to detect the secretion of specific factors. Finally, the data was analyzed to evaluate the changes in cell response and secretion patterns at different time points.

[0017] Furthermore, the specific ways in which hypoxic environment regulates stem cells are:

[0018] Ensure that all experimental equipment is sterilized and ready for use, prepare stem cell suspension, and adjust cell density to a concentration suitable for inoculation;

[0019] Adjust the gas ratio of the saturated humidity incubator to 0.5%-15% O2, 5% CO2, and 80%-94.5% N2 by volume, confirm that the incubator temperature is set to 37°C, and maintain saturated humidity; use sterile techniques to inoculate the stem cell suspension into a 96-well plate, add 80-120 μL of cell suspension to each well, and gently shake the 96-well plate to evenly distribute the cells;

[0020] The inoculated 96-well plate was placed under standard culture conditions at 37°C and 5% CO2 to allow the cells to completely adhere to the plate under normal oxygen conditions for 2-4 hours;

[0021] After the cells are completely attached, transfer the 96-well plate from the standard incubator to an incubator with a low oxygen environment set up. Confirm that the gas ratio of the low oxygen incubator is 1% O2, 5% CO2, and 94% N2 by volume, and maintain saturated humidity and a temperature of 37°C.

[0022] At different time points, 0 h, 12 h, 24 h, and 48 h, at each predetermined culture time point, an appropriate amount of culture medium was drawn for subsequent analysis, an equal amount of fresh culture medium was added to the wells, cell morphology changes were checked, and cell survival rate and other relevant indicators were recorded.

[0023] Furthermore, the specific ways in which oxidative stress stimulates stem cells are:

[0024] Ensure that all experimental equipment has been sterilized and is ready for use, prepare umbilical cord stem cell suspension within 10 generations with good growth status, and adjust the cell density to a concentration suitable for inoculation;

[0025] Prepare H2O2 stock solutions of different concentrations using aseptic techniques and prepare fresh complete culture medium in advance for subsequent replacement of culture medium;

[0026] Using sterile techniques, inoculate the umbilical cord stem cell suspension into a 6-well plate, add 2 mL of cell suspension to each well, and gently shake the 6-well plate to ensure that the cells are evenly distributed;

[0027] The inoculated 6-well plate was placed under standard culture conditions at 37°C and 5% CO2 to allow the cells to completely adhere to the plate under normal oxygen conditions for 2-4 hours;

[0028] After the cells are completely attached, the original culture medium is aspirated, and 2 mL of fresh complete culture medium containing a final concentration of 30-120 μmol / L H2O2 is added to each well. The 6-well plate is returned to a 37°C, 5% CO2 saturated humidity incubator and cultured for 24 hours.

[0029] Beneficial effects of the present invention:

[0030] A method of inducing stem cells to secrete specific factors of the present invention can significantly enhance the growth capacity of stem cells, promote the secretion of multiple active factors, and improve cell function and adaptability by combining high-frequency wave stimulation, hypoxic environment regulation, and oxidative stress stimulation. As a non-invasive physical method, high-frequency wave stimulation can activate the internal signaling pathway of cells without introducing exogenous substances, significantly improving the growth capacity and tissue repair potential of stem cells. Hypoxic environment regulation simulates the microenvironment conditions naturally existing in the body, greatly promoting the secretion of active factors such as vascular endothelial growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor that are crucial for tissue repair and immune regulation, while improving the survival rate and adaptability of cells after transplantation. Moderate oxidative stress treatment helps reduce inflammatory responses by upregulating the expression of anti-inflammatory factors, such as interleukin 10, which is beneficial to patient recovery and may also bring anti-aging effects. In addition, these methods all maintain the original characteristics of cells, reduce potential safety risks, are easy to control, and are simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of a cytokine in an embodiment of the present application.

[0032] Figure 2 Another schematic diagram of a cytokine in an embodiment of the present application. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the specific implementation modes of the present invention will be clearly and completely described below.

[0034] The present invention discloses a method for inducing stem cells to secrete specific factors, which includes stimulating stem cells with high-frequency waves, emitting sound waves of specific frequencies to stem cells; regulating stem cells in a hypoxic environment, placing stem cells in a hypoxic environment for culture; stimulating stem cells with oxidative stress, and subjecting stem cells to oxidative stimulation. Cultivating stem cells in a hypoxic environment simulates the microenvironment conditions naturally existing in the body. A hypoxic environment can greatly promote the secretion of a variety of active factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor. These factors are crucial for promoting angiogenesis, supporting neuronal survival, and regulating immune responses, making the treatment effect more significant. Many tissues are in a relatively hypoxic state, so this method is closer to the actual human environment, improving the relevance and success rate of treatment. Cells pretreated with hypoxia will have better adaptability and survival rate when transplanted to ischemic injury sites. Applying oxidative stress to stem cells means exposing cells to a higher level of reactive oxygen in a certain way. Moderate oxidative stress can act as a signaling molecule to participate in cell fate determination and upregulate the expression of certain genes, such as bFGF, VEGF, BDNF, stromal cell-derived factor 1α, interleukin 10, and tumor necrosis factor-stimulated gene 6. These factors are not only important for tissue repair, but some also have anti-inflammatory properties, such as IL-10, which can help reduce inflammatory responses and facilitate patient recovery. Cells treated with oxidative stress acquire stronger antioxidant capacity and tolerance to adverse environments. And moderate oxidative stress triggers the cell's self-protection mechanism and delays aging-related changes.

[0035] As an implementation method, the specific method of high-frequency wave stimulation of stem cells is to use a device that can emit a spectrum of waves to emit sound waves with a frequency of 1-20MHz to the stem cells in the culture bottle. Within this frequency range, sound waves can activate mechanically sensitive ion channels inside the cells or affect the cytoskeleton structure, thereby promoting cell cycle progression and DNA synthesis, and significantly improving the growth capacity of stem cells. As a physical stimulation method, high-frequency waves do not require the introduction of any chemicals or genetic modifications, reducing interference with the cells themselves and potential safety risks, and maintaining the original characteristics of the cells. Sound waves of appropriate frequencies can affect the fate of stem cells and guide them to differentiate in a specific direction, which is crucial for tissue engineering and regenerative medicine applications. High-frequency wave stimulation changes the properties of the extracellular matrix or intercellular signal transduction, optimizing the microenvironmental conditions required for cell growth and functional performance. The enhanced stem cell population not only increases in number, but also has higher activity, which helps to respond to damage signals faster and participate in the repair process, especially for tissues that require a large number of cells to participate in repair, such as the heart and bones. Using specially designed equipment to apply sound waves of precise frequencies can be easily regulated in a laboratory environment to ensure consistency and repeatability of each experiment.

[0036] As an implementation method, the specific method of regulating stem cells in a hypoxic environment is to place the stem cells in a saturated humidity incubator with a volume fraction of 0.5%-15% O2, a volume fraction of 5% CO2, and a volume fraction of 80%-94.5% N2 for 24 hours. Hypoxic environment regulation significantly improves the level of key growth factors: the hypoxic environment greatly promotes the secretion of active factors such as vascular endothelial growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor. These factors play an important role in angiogenesis, cell proliferation and differentiation, and neuroprotection. Many tissues are in a relatively hypoxic state, so this culture condition is closer to the actual human environment and increases the possibility of cell survival in vivo after transplantation. Cells pretreated with hypoxia gain stronger tolerance and the ability to resist adverse environments, which helps them survive and function better when transplanted to ischemic injury sites. The hypoxic environment can induce stem cells to secrete more anti-inflammatory factors, such as interleukin 10, to help reduce inflammatory responses and facilitate patient recovery. By regulating the immune system, cells pretreated with hypoxia reduce immune rejection after transplantation and improve the therapeutic effect. A hypoxic environment can change metabolic pathways within cells, such as switching from aerobic respiration to anaerobic glycolysis, which will affect the cell's energy supply and overall metabolic state, thereby affecting its functional performance. Cells under hypoxic stimulation show higher repair and regeneration potential, which is particularly important for tissue engineering and regenerative medicine applications. Using an incubator with precisely controlled gas ratios and humidity conditions, highly consistent operating procedures can be achieved between different laboratories, ensuring the reliability and reproducibility of experimental results. By regulating the behavior of stem cells, this method can significantly improve the effectiveness of stem cell-based therapies, especially in the fields of cardiovascular disease and neurological diseases. Adjusting hypoxic conditions according to individual differences in patients provides the possibility of developing personalized treatment plans.

[0037] As an implementation method, the specific method of oxidative stress stimulation of stem cells is to inoculate stem cells in a well plate, and after the stem cells adhere to the wall, add H2O2 at a concentration of 30-120 μmol / L for treatment. Cells pretreated with oxidative stress will gain stronger antioxidant capacity and tolerance to adverse environments, which helps them survive better when transplanted into ischemic injury sites. Under certain pathological conditions, such as ischemia-reperfusion injury, high levels of reactive oxygen species are naturally produced in the body, and pre-exposure to oxidative stress can help cells better adapt to this environment. Moderate oxidative stress participates in cell fate determination as a signal molecule, affects the differentiation path of stem cells, and guides them to differentiate in a specific direction, which is very important for regenerative medicine applications. The upregulation of anti-inflammatory factors such as IL-10 helps to reduce inflammatory responses, is beneficial to patient recovery, and reduces the risk of immune rejection after transplantation. By changing the cell surface markers or secretion factor spectrum, low-concentration H2O2 treatment gives stem cells better immunoregulatory properties. Using a known concentration of H2O2 treatment is a relatively simple method that can achieve highly consistent operating procedures between different laboratories to ensure the reliability and repeatability of experimental results. The concentration of H2O2 can be adjusted according to the specific research purpose to explore the effects of different levels of oxidative stress on cells and find the optimal treatment conditions. By regulating the behavior of stem cells, this method can significantly improve the effectiveness of stem cell-based treatments, especially in the fields of cardiovascular disease, neurological diseases, etc.

[0038] As an implementation method, the specific method of high-frequency wave stimulation of stem cells is as follows: ensure that all experimental equipment has been sterilized and is ready for use, inoculate stem cells into appropriate culture bottles, and pre-culture to a suitable cell density under standard conditions of 37°C and 5% CO2;

[0039] Using an acoustic wave emitting device for biological sample processing, adjusting the emission frequency range of the acoustic wave to be 1-20 MHz, turning on the acoustic wave emitting device, and performing acoustic wave irradiation treatment on the stem cells;

[0040] On the first, third and fifth days after the sonication device was turned on, the original culture medium was aspirated and then an appropriate amount of fresh culture medium was added. After the fresh culture medium was replaced, the sonication was stopped to allow the cells to recover and continue to grow under normal conditions.

[0041] After turning on the acoustic wave emission device, the cell morphology changes and growth conditions were observed regularly, and the cell supernatant was collected regularly to detect the secretion of specific factors. Finally, the data was analyzed to evaluate the changes in cell response and secretion patterns at different time points.

[0042] Sound waves in the frequency range of 1-20MHz can activate mechanically sensitive ion channels inside cells or affect the cytoskeleton structure, thereby promoting cell cycle progression and DNA synthesis, and significantly improving the proliferation capacity of stem cells. Appropriate sound wave stimulation changes the properties of the extracellular matrix or intercellular signal transduction, optimizing the microenvironmental conditions required for cell growth and functional performance. On the first, third and fifth days after the sound wave transmitter is turned on, fresh culture medium is replaced and sound wave irradiation is stopped to allow cells to recover and continue to grow. This allows researchers to monitor cell morphological changes and growth in real time at different time points and adjust experimental conditions in a timely manner. By regularly collecting cell supernatants to detect the secretion of specific factors, and analyzing data to evaluate changes in cell response and secretion patterns at different time points, it helps to deeply understand the mechanism of the effect of sound wave stimulation on cell behavior. As a physical stimulation method, high-frequency waves do not require the introduction of any chemicals or genetic modifications, reducing interference with the cells themselves and potential safety risks, and maintaining the original characteristics of the cells. After high-frequency wave stimulation, the stem cell population not only increases in number, but also has higher activity, which helps to respond to damage signals faster and participate in the repair process, especially for tissues that require a large number of cells to participate in repair, such as the heart and bones. This method provides a tool for developing new stem cell-based treatments, especially in the fields of tissue engineering and regenerative medicine, and has important application prospects. By systematically collecting and analyzing data at different time points, valuable information about changes in cell responses and secretion patterns can be obtained, providing a solid scientific basis for subsequent research. Long-term tracking of changes in cell behavior can help verify whether high-frequency wave stimulation can effectively enhance the effectiveness of stem cell therapy and provide direction for further optimization of the technology. This method is suitable for different types of stem cell research, including basic biological research, drug screening, and preclinical trials, and has broad application potential.

[0043] As an implementation method, the specific method of regulating stem cells in a hypoxic environment is as follows: ensuring that all experimental equipment has been sterilized and is ready for use, preparing a stem cell suspension, and adjusting the cell density to a concentration suitable for inoculation;

[0044] Adjust the gas ratio of the saturated humidity incubator to 0.5%-15% O2, 5% CO2, and 80%-94.5% N2 by volume, confirm that the incubator temperature is set to 37°C, and maintain saturated humidity; use sterile techniques to inoculate the stem cell suspension into a 96-well plate, add 80-120 μL of cell suspension to each well, and gently shake the 96-well plate to evenly distribute the cells;

[0045] The inoculated 96-well plate was placed under standard culture conditions at 37°C and 5% CO2 to allow the cells to completely adhere to the plate under normal oxygen conditions for 2-4 hours;

[0046] After the cells are completely attached, transfer the 96-well plate from the standard incubator to an incubator with a low oxygen environment set up. Confirm that the gas ratio of the low oxygen incubator is 1% O2, 5% CO2, and 94% N2 by volume, and maintain saturated humidity and a temperature of 37°C.

[0047] At different time points, 0 h, 12 h, 24 h, and 48 h, at each predetermined culture time point, an appropriate amount of culture medium was drawn for subsequent analysis, an equal amount of fresh culture medium was added to the wells, cell morphology changes were checked, and cell survival rate and other relevant indicators were recorded.

[0048] Many tissues are in a relatively low oxygen state, so this culture condition is closer to the actual human environment and increases the possibility of cell survival in vivo after transplantation. Cells pretreated with hypoxia gain stronger tolerance and ability to resist adverse environments, which helps them survive better when transplanted into ischemic injury sites. The hypoxic environment greatly promotes the secretion of active factors such as vascular endothelial growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor. These factors play an important role in angiogenesis, cell proliferation and differentiation, and neuroprotection. At different time points, an appropriate amount of culture medium is drawn for subsequent analysis, and an equal amount of fresh culture medium is added to the wells to check cell morphological changes, record cell survival rate and other related indicators. This allows researchers to monitor cell behavior changes and growth in real time at different time points and adjust experimental conditions in a timely manner. By regularly collecting culture fluid to detect the secretion of specific factors and analyzing the data to evaluate changes in cell response and secretion patterns at different time points, it helps to deeply understand the mechanism of the influence of hypoxic environment on cell behavior. As a physical regulation method, the hypoxic environment does not require the introduction of any chemicals or genetic modification, which reduces interference with the cells themselves and potential safety risks, and maintains the original characteristics of the cells. Using an incubator with precisely controlled gas ratios and humidity conditions, highly consistent operating procedures can be achieved between different laboratories, ensuring the reliability and reproducibility of experimental results. After hypoxic pretreatment, the stem cell population not only increases in number, but also has higher activity, which helps to respond to damage signals faster and participate in the repair process, especially for tissues that require a large number of cells to participate in repair, such as the heart and bones.

[0049] As an implementation method, the specific method of oxidative stress stimulation of stem cells is: ensuring that all experimental equipment has been sterilized and ready for use, preparing a suspension of umbilical cord stem cells within 10 generations in good growth state, and adjusting the cell density to a concentration suitable for inoculation;

[0050] Prepare H2O2 stock solutions of different concentrations using aseptic techniques and prepare fresh complete culture medium in advance for subsequent replacement of culture medium;

[0051] Using sterile techniques, inoculate the umbilical cord stem cell suspension into a 6-well plate, add 2 mL of cell suspension to each well, and gently shake the 6-well plate to ensure that the cells are evenly distributed;

[0052] The inoculated 6-well plate was placed under standard culture conditions at 37°C and 5% CO2 to allow the cells to completely adhere to the plate under normal oxygen conditions for 2-4 hours;

[0053] After the cells are completely attached to the wall, the original culture medium is aspirated, and 2 mL of fresh complete culture medium containing a final concentration of 30-120 μmol / L H2O2 is added to each well. The 6-well plate is returned to a 37°C, 5% CO2 saturated humidity incubator for further culture for 24 hours. Under certain pathological conditions, such as ischemia-reperfusion injury, high levels of reactive oxygen species are naturally produced in the body. Pre-exposure to oxidative stress can help cells better adapt to this environment and enhance their survival rate and functional performance after transplantation. Cells pretreated with oxidative stress will acquire stronger antioxidant capacity and tolerance to adverse environments, which helps them survive better when transplanted into ischemic injury sites. Moderate oxidative stress can significantly increase the expression levels of factors such as basic fibroblast growth factor, vascular endothelial growth factor, brain-derived neurotrophic factor, stromal cell-derived factor 1α, interleukin 10, and tumor necrosis factor-stimulated gene 6 in stem cells. These factors are essential for tissue repair, angiogenesis, neuroprotection, and immune regulation. After the cells were seeded into 6-well plates and adhered under standard conditions, oxidative stress was induced by adding fresh complete medium containing H2O2 and continued to be cultured for 24 hours. This allows researchers to monitor cell morphological changes and growth in real time at different time points and adjust experimental conditions in a timely manner. By regularly collecting cell supernatants to detect the secretion of specific factors and analyzing the data to evaluate changes in cell responses and secretion patterns at different concentrations, it helps to deeply understand the mechanism of the impact of oxidative stress on cell behavior. As a physicochemical method, H2O2 treatment does not require the introduction of genetic modification, reduces interference with the cells themselves and potential safety risks, and maintains the original characteristics of the cells. Using a known concentration of H2O2 treatment is a relatively simple method. The H2O2 concentration can be adjusted according to the specific research purpose to explore the effects of different levels of oxidative stress on cells and find the optimal treatment conditions.

[0054] Embodiment 1:

[0055] The stem cells were irradiated with 10 MHz frequency sound waves, and the original culture medium was sucked out and new culture medium was added on the 1st, 3rd and 5th days respectively, and the spectrum was turned off for normal culture.

[0056] Embodiment 2:

[0057] The stem cells were irradiated with 15 MHz frequency sound waves, and the original culture medium was sucked out and new culture medium was added on the 1st, 3rd and 5th days respectively, and the spectrum was turned off for normal culture.

[0058] Comparative Example 1:

[0059] No spectral irradiation was used, and other culture conditions were the same as those of the experimental group.

[0060] in conclusion:

[0061] Before spectral irradiation, the inoculation density of the two groups of cells was basically the same, and both grew in short spindle or triangular shapes. After 1 day of spectral irradiation, the irradiated cells gradually became dense, while the cells in comparative example 1 were very sparse. After 3 days of spectral irradiation, the irradiated cells were long spindle-shaped, the cell colonies were dense, and they grew in clusters, while the cell colonies in comparative example 1 were scattered and in local clusters. After 5 days of spectral irradiation, a large number of typical BMSC clustered hyperplasia foci were formed in the irradiation group, which were closely arranged in a vortex shape, and the cell density was significantly higher than that in comparative example 1. Using flow cytometry, the stem cell proliferation index of the 10MHz irradiation group can exceed that of comparative example 1 by 148%, and the stem cell proliferation index of the 15MHz irradiation group exceeds that of comparative example 1 by 161%.

[0062] Embodiment 3:

[0063] The stem cells were seeded in a 96-well plate, 100 μL per well. After the cells were completely attached to the wall, the cells were placed in a saturated humidity incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 0, 12, 24, and 48 hours.

[0064] Comparative Example 2: The stem cells were cultured normally in a saturated humidity incubator at 37° C., 5% CO 2 , and 21% O 2 for 0, 12, 24, and 48 h.

[0065] Finally, the concentration of active factors was determined by enzyme-linked immunosorbent assay.

[0066] in conclusion:

[0067] In Example 3, the concentration of basic fibroblast growth factor was increased by 180% compared with that in Comparative Example 2, the concentration of vascular endothelial growth factor was increased by 98% compared with that in Comparative Example 2, the concentration of brain-derived neurotrophic factor was increased by 77% compared with that in Comparative Example 2, and the concentration of stromal cell-derived factor 1α was increased by 48% compared with that in Comparative Example 2.

[0068] Embodiment 4:

[0069] Umbilical cord stem cells within 10 generations with good growth status were selected and inoculated into 6-well plates. After the cells were completely attached to the wall, H2O2 with a final concentration of 50 μmol / L was added and cultured in a 37°C, 5% CO2 saturated humidity incubator for 24 h.

[0070] Embodiment 5:

[0071] Umbilical cord stem cells within 10 generations with good growth status were selected and inoculated into 6-well plates. After the cells were completely attached to the wall, H2O2 with a final concentration of 100 μmol / L was added and cultured in a 37°C, 5% CO2 saturated humidity incubator for 24 h.

[0072] Comparative Example 3: Umbilical cord stem cells within 10 generations with good growth status were selected and inoculated into 6-well plates. After the cells were completely attached to the plate, they were cultured in a 37° C., 5% CO 2 saturated humidity incubator for 24 h.

[0073] in conclusion:

[0074] Under the condition of 50 μmol / L H2O2, the concentration of basic fibroblast growth factor increased by 108% compared with that in comparison example 3, the concentration of vascular endothelial growth factor increased by 99% compared with that in comparison example 3, the concentration of brain-derived neurotrophic factor increased by 131% compared with that in comparison example 3, the concentration of interleukin 10 increased by 236% compared with that in comparison example 3, and the concentration of tumor necrosis factor-stimulating factor 6 increased by 105% compared with that in comparison example 3.

[0075] Under the condition of 100 μmol / L H2O2, the concentration of basic fibroblast growth factor increased by 106% compared with that in comparison example 3, the concentration of vascular endothelial growth factor increased by 245% compared with that in comparison example 3, the concentration of brain-derived neurotrophic factor increased by 141% compared with that in comparison example 3, the concentration of interleukin 10 increased by 242% compared with that in comparison example 3, and the concentration of tumor necrosis factor-stimulating factor 6 increased by 127% compared with that in comparison example 3.

[0076] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for inducing stem cells to secrete specific factors, characterized in that: include: High-frequency waves stimulate stem cells, emitting sound waves of a specific frequency to the stem cells; Hypoxic environment regulates stem cells, and the stem cells are cultured in a hypoxic environment; Oxidative stress stimulates stem cells, and the stem cells are subjected to oxidative stimulation.

2. A method for inducing stem cells to secrete specific factors according to claim 1, characterized in that: The specific method of high-frequency wave stimulation of stem cells is to use a device that can emit a spectrum of waves to emit sound waves with a frequency of 1-20MHz to the stem cells in the culture bottle.

3. A method for inducing stem cells to secrete specific factors according to claim 1, characterized in that: The specific method of regulating stem cells in a hypoxic environment is to place the stem cells in a saturated humidity incubator with a volume fraction of 0.5%-15% O2, a volume fraction of 5% CO2, and a volume fraction of 80%-94.5% N2 and culture them for 24 hours.

4. The method of inducing stem cells to secrete specific factors according to claim 1, characterized in that: The specific method of stimulating stem cells with oxidative stress is to inoculate the stem cells in a well plate, and then add H2O2 at a concentration of 30-120 μmol / L for treatment after the stem cells adhere to the wall.

5. The method of inducing stem cells to secrete specific factors according to claim 2, characterized in that: The specific way high-frequency waves stimulate stem cells is: Ensure that all experimental equipment has been sterilized and is ready for use. Seed the stem cells into appropriate culture flasks and pre-culture them to an appropriate cell density under standard conditions at 37°C and 5% CO2. Using an acoustic wave emitting device for biological sample processing, adjusting the emission frequency range of the acoustic wave to be 1-20 MHz, turning on the acoustic wave emitting device, and performing acoustic wave irradiation treatment on the stem cells; On the first, third and fifth days after the sonication device was turned on, the original culture medium was aspirated and then an appropriate amount of fresh culture medium was added. After the fresh culture medium was replaced, the sonication was stopped to allow the cells to recover and continue to grow under normal conditions. After turning on the acoustic wave emission device, the cell morphology changes and growth conditions were observed regularly, and the cell supernatant was collected regularly to detect the secretion of specific factors. Finally, the data was analyzed to evaluate the changes in cell response and secretion patterns at different time points.

6. The method of inducing stem cells to secrete specific factors according to claim 3, characterized in that: The specific ways in which hypoxic environment regulates stem cells are: Ensure that all experimental equipment is sterilized and ready for use, prepare stem cell suspension, and adjust cell density to a concentration suitable for inoculation; Adjust the gas ratio of the saturated humidity incubator to 0.5%-15% O2, 5% CO2, and 80%-94.5% N2 by volume, confirm that the incubator temperature is set to 37°C, and maintain saturated humidity; use sterile techniques to inoculate the stem cell suspension into a 96-well plate, add 80-120 μL of cell suspension to each well, and gently shake the 96-well plate to evenly distribute the cells; The inoculated 96-well plate was placed under standard culture conditions at 37°C and 5% CO2 to allow the cells to completely adhere to the plate under normal oxygen conditions for 2-4 hours; After the cells are completely attached, transfer the 96-well plate from the standard incubator to an incubator with a low oxygen environment set up. Confirm that the gas ratio of the low oxygen incubator is 1% O2, 5% CO2, and 94% N2 by volume, and maintain saturated humidity and a temperature of 37°C. At different time points, 0 h, 12 h, 24 h, and 48 h, at each predetermined culture time point, an appropriate amount of culture medium was drawn for subsequent analysis, an equal amount of fresh culture medium was added to the wells, cell morphology changes were checked, and cell survival rate and other relevant indicators were recorded.

7. The method of inducing stem cells to secrete specific factors according to claim 4, characterized in that: The specific ways in which oxidative stress stimulates stem cells are: Ensure that all experimental equipment has been sterilized and is ready for use, prepare umbilical cord stem cell suspension within 10 generations with good growth status, and adjust the cell density to a concentration suitable for inoculation; Prepare H2O2 stock solutions of different concentrations using aseptic techniques and prepare fresh complete culture medium in advance for subsequent replacement of culture medium; Using sterile techniques, inoculate the umbilical cord stem cell suspension into a 6-well plate, add 2 mL of cell suspension to each well, and gently shake the 6-well plate to ensure that the cells are evenly distributed; The inoculated 6-well plate was placed under standard culture conditions at 37°C and 5% CO2 to allow the cells to completely adhere to the plate under normal oxygen conditions for 2-4 hours; After the cells are completely attached, the original culture medium is aspirated, and 2 mL of fresh complete culture medium containing a final concentration of 30-120 μmol / L H2O2 is added to each well. The 6-well plate is returned to a 37°C, 5% CO2 saturated humidity incubator and cultured for 24 hours.

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