CN115044543A - Method for improving activity of aged human body-derived muscle stem cells - Google Patents
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Abstract
The invention belongs to the field of anti-aging application of stem cell exosomes, and particularly relates to a method for improving activity of muscle stem cells derived from aging human bodies. The invention provides a method for improving the activity of aging human body-derived muscle stem cells by using high-activity muscle stem cell exosome vesicles, which comprises the following steps of firstly screening high-functional activity muscle stem cells to obtain exosome vesicles; the exosome vesicle can be rapidly fused with the muscle stem cell membrane of an aging individual, and the micro RNA, the growth factors, the heat shock protein and the like are sent into target cells in an endocytosis or pinocytosis mode, so that the effect is rapid; the muscle stem cell exosome vesicle is different from a chemically synthesized drug, can be automatically absorbed and degraded by cells, and does not have any damage to the cells.
Description
Technical Field
The invention belongs to the field of anti-aging application of stem cell exosomes, and particularly relates to a method for improving activity of aged human body-derived muscle stem cells.
Background
Compared with various stem cells, the research and clinical application of muscle stem cells at home and abroad are in a laggard state. The muscle tissue accounts for a great proportion of the human body, and the physical exercise and the muscle exercise can directly or indirectly improve the overall health state of the human body. Also, since a large amount of benign factors is secreted from muscle stem cells, muscle tissue directly affects other tissues in the body, such as bones, blood vessels, and skin, etc. Even at a later age when exercise is initiated, exercise can reverse the aging of people's muscles and other organs. Skeletal muscle is one of the most important muscles in the body and is responsible for providing support for a variety of body functions. Skeletal muscle musculature controls exclusively active movements, while the intense activity, exercise, and even daily activities of individuals are constantly creating physical micro-injuries to different degrees of musculature and cells. Repair of muscle damage itself must be accomplished through muscle stem cell activation and differentiation. A considerable number of muscle stem cells in the human body are constantly growing and differentiating to perform repair activities for repairing muscle micro-injuries. The muscle tissue of young individuals has a strong ability to repair. As the human body ages, a significant reduction in muscle function and repair capacity occurs. At present, progressive muscle weakness and regeneration disorders afflict a wide range of elderly people. Various muscular dystrophies, such as duchenne muscular dystrophy, also occur in young and young patients. Muscular dystrophy and dysgenesis are directly associated with the failure of the number and function of human muscle stem cells. Failure of muscle stem cells continues to occur with age or with the development of some diseases. The regenerative repair capacity of the muscle stem cells of an aging individual also gradually diminishes until it disappears as it ages. However, this process is not irreversible. In vitro culture and stimulation can promote the recovery of the regenerative capacity of aging muscle stem cells.
In the prior art, the transformation of adult cells into stem cells can be induced by the totipotenization induction technology (iPSC) of the adult cells, and the use of various anti-aging compounds can reverse the aging process of the cells to rejuvenate the cells. However, these are not natural biological processes and are associated with risks such as cytotoxicity of the transgenic components and the synthetic compounds.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for improving the activity of the aged human body-derived muscle stem cells by using high-activity muscle stem cell exosome vesicles, which comprises the steps of firstly screening high-functional activity muscle stem cells to obtain exosome vesicles; the exosome vesicle can be rapidly fused with the muscle stem cell membrane of an aging individual, and the micro RNA, the growth factors, the heat shock protein and the like are sent into target cells in an endocytosis or pinocytosis mode, so that the effect is rapid; the muscle stem cell exosome vesicle is different from a chemically synthesized drug, can be automatically absorbed and degraded by cells, and does not have any damage to the cells.
The technical scheme of the invention is as follows:
a method for increasing the activity of muscle stem cells derived from an aging human body, comprising the steps of:
(1) screening to obtain the muscle stem cells with high functional activity: extracting and culturing muscle stem cells (hMPC) from healthy young individuals, and screening cells ALDH + hMPC with higher ALDH1 enzyme activity;
(2) obtaining exosome vesicles of high-activity muscle stem cells: 2D culturing the high-activity cells screened in the step (1), introducing ALDH1 high-expression myocyte of 4 th to 6 th generations in logarithmic growth phase into a subculture 3D culture system for subculture, continuously culturing for 72h, and then carrying out low-temperature ultra-high-speed centrifugation to obtain exosome vesicles;
(3) the activity of the aged human muscle stem cells is improved: dissolving the exosome vesicle in the step (2) in a culture solution of fresh aged muscle stem cells; and replacing fresh exosomes and culture solution every 24-48 hours until the cells grow to the expected value.
Preferably, the process of screening for a cell with higher enzymatic activity of ALDH1 in step (1) is as follows: expanding primary culture of primary muscle stem cells to be more than 5x10 4 After the number of the cells is increased, the cells are separated and screened according to the activity of ALDH1, and the cells with high activity of ALDH1 of less than or equal to 15 percent are separated.
Preferably, the basic culture solution of the 3D culture system in the step (2) is a DMDM cell culture medium, and other additives comprise fetal calf serum with a final volume fraction of 10-30% and chicken embryo essence extract with a final volume fraction of 0.5-2%; the total volume of the cell culture medium is approximately 10mL culture mediumcm 3 A cytoskeleton; after the cells are continuously cultured for 48 hours, the gamma-interferon with the final concentration of 0.05 ng/ml-0.2 ng/ml is added, and after the reaction is carried out for 24 hours, the final concentration of the exosome in the cell culture solution can reach 2 mg/ml-5 mg/ml.
Preferably, the culture solution of the aged muscle stem cells in the step (3) is: the basic culture solution is a DMDM cell culture medium, the final volume fraction of other additive substances, namely fetal calf serum is 10-30%, and the final volume fraction of the chicken embryo essence extract is 0.5-2%; changing a new culture solution every 24-48 h, after culturing for 96h, centrifugally collecting muscle stem cells, and then suspending the muscle stem cells in a DMEM culture medium containing 10% fetal calf serum to detect the apoptosis rate, the aging rate and the differentiation efficiency, or continuing culturing; the final concentration of the exosome in the cell culture solution is 5-15 mg/ml.
The invention has the advantages of
The muscle stem cell exosome vesicle is taken as a cell source substance of a high-activity muscle stem cell of ALDH1 from a young individual, is rich in cell factors such as micro RNA (miRNA), growth factors, heat shock proteins and the like which are beneficial to cell apoptosis resistance, inflammation resistance, oxidation resistance, aging resistance and the like, and the enrichment liquid of the muscle stem cell exosome vesicle has very weak immunogenicity, foreign cells do not have any rejection effect on the cell, so that the muscle stem cell exosome vesicle can safely and effectively protect and improve the function of the muscle stem cell of an aging individual; the exosome vesicle can be rapidly fused with the muscle stem cell membrane of an aging individual, and the micro RNA, the growth factors, the heat shock protein and the like are sent into target cells in an endocytosis or pinocytosis mode, so that the effect is rapid; the muscle stem cell exosome vesicle is different from a chemically synthesized drug, can be automatically absorbed and degraded by cells, and does not have any damage to the cells.
The invention uses the mesenchymal stem cell exosome vesicle enrichment liquid to protect the T lymphocytes through the specific immune protection characteristic, thereby achieving good effect, the product prepared by the invention has no direct correlation with the muscle stem cells, has no any living cell component, is convenient for transportation and storage, can be stored in a refrigerator for a long time, is used for improving the activity and the applicability of the aging cells, and has excellent clinical experiment application value.
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FIG. 1 shows the screening and separation of ALDH + muscle stem cell and the extraction and identification of exosome. (A) Screening and separating the muscle stem cells by a flow cytometer according to the activity of ALDH1 enzyme; (B) exosomes produced by ALDH + muscle stem cells were collected, purified and size-characterized, with an average size of about 110 nm;
FIG. 2 shows that muscle stem cell exosomes enhance the growth and antioxidant capacity of senescent muscle stem cells. (A) Difference in growth rate of senescent myocyte stem cells in the state of exosome treatment (48h) (B) after hydrogen peroxide (200 nM) treatment of cells for 6 hours, culture was continued for 18 hours, and PI (propidium iodide) staining was performed to compare the difference in apoptosis rate with exosome treatment (48 h);
FIG. 3 is a graph showing the change in myogenic differentiation ability of muscle stem cells in vitro. The fluorescent staining result of MHC (myostatin latent chain) shows that the myogenic differentiation capacity of the aged myocyte is obviously enhanced after the exosome is treated for 48 hours;
FIG. 4 is a graph of changes in the expression of senescence-associated secretory phenotype (SASP) genes in senescent muscle stem cells. After exosome treatment, the expression of SASP genes (CXCL 1, MCP1, TNFR-1, IL1- α, IL1- β, IL-6 and TGF- β 1) and p16 decreased, while the expression of anti-aging genes (Klotho and IL-10) increased in senescent muscle stem cells.
The specific implementation mode is as follows:
the present invention will be described in detail with reference to specific embodiments, which are illustrative of the invention and are not to be construed as limiting the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1:
a method for improving activity of aging human body derived muscle stem cells comprises the following specific steps:
(1) screening to obtain the muscle stem cells with high functional activity: extracting and culturing muscle stem cells (hMPC) from healthy young individuals using flow cytometryCells with high ALDH1 enzyme activity (ALDH + mpc) were screened and cultured on a conventional 2D surface; primary culture expansion of muscle Stem cells to about 5X10 4 After the number of cells, the cells with the highest ALDH1 activity (about 1X 10) were isolated in about 15% by screening based on the ALDH1 activity 4 Cells);
(2) obtaining exosome vesicles of high-activity muscle stem cells: the high-expression muscle stem cells of ALDH1 of passage 4 in logarithmic growth phase were introduced into a tissue-engineered cytoskeleton made of collagen 3D (about 1X 10) 5 Cells/cm 3 ) (ii) a The basic culture solution is DMDM cell culture medium, and other additives including fetal calf serum with final volume fraction of 20% and chicken embryo essence extract (chicken embryo essential extracts/CEE) with final volume fraction of 1%; the total volume of the cell culture medium was about 10mL of the culture solution/cm 3 Cytoskeleton); after continuous culture for 48 hours, gamma-interferon (IFN-gamma) with the final concentration of 0.1ng/ml is added, and after 24 hours of action, the final concentration of the exosome in the cell culture solution is 3 mg/ml; carrying out low-temperature ultra-high-speed centrifugation to obtain exosome vesicles, and carrying out protein content determination on the obtained exosomes by using a BCA method and then freezing and storing the exosomes in a refrigerator at the temperature of-80 ℃;
(3) exosomes treat the activity of aging muscle stem cells: muscle stem cells were extracted from muscle tissue of aged individuals (skeletal muscle of 70 year old male individuals) and cultured by conventional 2D surface culture method to grow slowly to about 1X10 4 Adding cell culture fluid containing exosome; the basic culture solution is a DMDM cell culture medium, the final concentration of exosome in the cell culture solution is 10mg/ml, the final volume fraction of other additive substances, namely fetal calf serum is 20%, and the final volume fraction of chicken embryo essence extract (chicken embryo essential extracts/CEE) is 1%; after culturing for 96h with fresh exosomes and culture solution replaced every 24h, myocyte stem cells were collected by centrifugation at 4 ℃ for 8min at a centrifugal force of 300g, and then suspended in DMEM medium containing 10% fetal calf serum to measure apoptosis rate, senescence rate and differentiation efficiency.
Example 2:
a method for improving activity of aging human body derived muscle stem cells comprises the following specific steps:
(1) screening to obtain the muscle stem cells with high functional activity: extracting and culturing muscle stem cells (hMPC) from healthy young individuals, screening cells (ALDH + hMPC) with high ALDH1 enzyme activity by using a flow cytometer, and culturing on a conventional 2D surface; primary culture expansion of muscle Stem cells to approximately 5.5X 10 4 After the number of cells, about 15% of the cells with the highest ALDH1 activity (about 1X 10) were isolated by screening based on the ALDH1 activity 4 Cells);
(2) obtaining exosome vesicles of high-activity muscle stem cells: the ALDH1 high-expression myostem cells of generation 5 in the logarithmic growth phase were introduced into a tissue-engineered cytoskeleton made of 3D collagen (about 1X 10) 5 Cells/cm 3 ) (ii) a The basic culture solution is DMDM cell culture medium, and other additives including fetal calf serum with final volume fraction of 10% and chicken embryo essence extract (chicken embryo essential extracts/CEE) with final volume fraction of 0.5%; the total volume of the cell culture medium was about 10mL of culture medium/cm 3 Cytoskeleton); after continuous culture for 48h, adding gamma-interferon (IFN-gamma) with final concentration of 0.2ng/ml, and after 24h of action, the final concentration of the exosome in the cell culture solution is 5 mg/ml; carrying out low-temperature ultra-high-speed centrifugation to obtain exosome vesicles, and carrying out protein content determination on the obtained exosomes by using a BCA method and then freezing and storing the exosomes in a refrigerator at the temperature of-80 ℃;
(3) exosomes treat the activity of senescent muscle stem cells: muscle stem cells were extracted from muscle tissue of aged individuals (skeletal muscle of 70 year old male individuals) and cultured by conventional 2D surface culture method to grow slowly to about 1X10 4 Adding cell culture fluid containing exosome; the basic culture solution is a DMDM cell culture medium, the final concentration of the exosome in the cell culture solution is 5mg/ml, the final volume fraction of other additive fetal calf serum is 10%, and the final volume fraction of chicken embryo essence extract (chicken embryo essential extracts/CEE) is 0.5%; the exosomes and the culture solution were replaced with fresh exosomes every 24 hours, and after culturing for 96 hours, the muscle stem cells were collected by centrifugation at a centrifugal force of 300g for 8min at 4 ℃.
Example 3:
a method for improving the activity of muscle stem cells derived from an aged human body comprises the following specific steps:
(1) screening to obtain the muscle stem cells with high functional activity: extracting and culturing muscle stem cells (hMPC) from healthy young individuals, screening cells (ALDH + hMPC) with high ALDH1 enzyme activity by using a flow cytometer, and culturing on a conventional 2D surface; primary culture expansion of muscle Stem cells to about 6.0X 10 4 After the number of cells, the cells with the highest ALDH1 activity (about 1X 10) were isolated in about 15% by screening based on the ALDH1 activity 4 Cells);
(2) obtaining exosome vesicles of high-activity muscle stem cells: the ALDH1 high-expression myocyte of 5 th generation in logarithmic growth phase was introduced into a tissue-engineered cytoskeleton made of collagen 3D (about 1X 10) 5 Cells/cm 3 ) (ii) a The basic culture solution is DMDM cell culture medium, and other additives including fetal calf serum with final volume fraction of 30% and chicken embryo essence extract (chicken embryo essential extracts/CEE) with final volume fraction of 2%; the total volume of the cell culture medium was about 10mL of the culture solution/cm 3 Cytoskeleton); after continuous culture for 48h, adding gamma-interferon (IFN-gamma) with the final concentration of 0.05ng/ml, and after 24h of action, the final concentration of the exosome in the cell culture solution is 2 mg/ml; carrying out low-temperature ultra-high-speed centrifugation to obtain exosome vesicles, and carrying out protein content determination on the obtained exosomes by using a BCA method and then freezing and storing the exosomes in a refrigerator at the temperature of-80 ℃;
(3) exosomes treat the activity of senescent muscle stem cells: muscle stem cells were extracted from muscle tissue of aged individuals (skeletal muscle of 70 year old male individuals) and cultured by conventional 2D surface culture method to grow slowly to about 1X10 4 Adding cell culture fluid containing exosome; the basic culture solution is a DMDM cell culture medium, the final concentration of exosome in the cell culture solution is 15mg/ml, the final volume fraction of other additive substances, namely fetal calf serum is 30%, and the final volume fraction of chicken embryo essence extract (chicken embryo essential extracts/CEE) is 2%; replacing fresh exosome and culture solution every 48h, culturing for 96h, centrifuging at 4 deg.C for 8min with centrifugal force of 300g, and collectingCollecting muscle stem cells.
Examples of the effects of the invention
After muscle stem cells obtained from an aging individual are treated with the exosome product formulation obtained in example 1, the apoptosis rate, aging rate and differentiation efficiency of the cells are examined by methods such as flow cytometry. Muscle stem cells treated without the addition of the stem cell exosome vesicles served as control groups, and the results are shown in fig. 1 to 4. The results show that the cell culture solution added by the muscle stem cell exosome vesicle prepared by the invention can play a remarkable function improving role on the muscle stem cells from the aged individuals.
Claims (5)
1. A method for increasing the activity of aged human derived muscle stem cells, comprising the steps of:
(1) screening to obtain the muscle stem cells with high functional activity: extracting and culturing muscle stem cells hMPC from healthy young individuals, and screening cells ALDH + hMPC with higher ALDH1 enzyme activity;
(2) obtaining exosome vesicles of high-activity muscle stem cells: 2D culturing the high-activity cells screened in the step (1), introducing ALDH1 high-expression myocyte of 4 th to 6 th generations in logarithmic growth phase into a subculture 3D culture system for subculture, continuously culturing for 72h, and then carrying out low-temperature ultra-high-speed centrifugation to obtain exosome vesicles;
(3) the activity of the aged human muscle stem cells is improved: dissolving the exosome vesicle in the step (2) in a culture solution of fresh aged muscle stem cells; and replacing fresh exosomes and culture solution every 24-48 hours until the cells grow to the expected value.
2. The method for increasing the activity of aged human derived muscle stem cells according to claim 1, wherein the step (1) of screening for cells with higher activity of ALDH1 enzyme ALDH + hMPC comprises: expanding primary culture of primary muscle stem cells to be more than 5x10 4 After the number of the cells is increased, the cells are separated and screened according to the activity of ALDH1, and the cells with high activity of ALDH1 of less than or equal to 15 percent are separated.
3. The method for improving the activity of the aged human myogenic stem cells according to claim 1, wherein the basic culture fluid of the 3D culture system in the step (2) is a DMDM cell culture medium, and the other additives comprise fetal calf serum with a final volume fraction of 10% -30%, and chicken embryo essence extract with a final volume fraction of 0.5% -2%; the total volume of the cell culture medium was 10mL of the culture solution/cm 3 A cytoskeleton; after the cells are continuously cultured for 48 hours, the gamma-interferon with the final concentration of 0.05 ng/ml-0.2 ng/ml is added, and after the reaction is carried out for 24 hours, the final concentration of the exosome in the cell culture solution can reach 2 mg/ml-5 mg/ml.
4. The method for improving the activity of the aged human derived myostem cells as claimed in claim 1, wherein the culture solution of the aged myostem cells in step (3) is: the basic culture solution is a DMDM cell culture medium, the final volume fraction of other additive substances, namely fetal calf serum is 10-30%, and the final volume fraction of the chicken embryo essence extract is 0.5-2%; changing a new culture solution every 24-48 h, after culturing for 96h, centrifugally collecting muscle stem cells, and then suspending the muscle stem cells in a DMEM culture medium containing 10% fetal calf serum to detect the apoptosis rate, the aging rate and the differentiation efficiency, or continuing culturing; the final concentration of the exosome in the cell culture solution is 5-15 mg/ml.
5. The method for increasing the activity of senescent human-derived muscle stem cells according to claim 1, which is used to increase the activity of senescent human-derived muscle stem cells.
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