Use of mesenchymal stem cells in the preparation of anti-aging drugs

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of mesenchymal stem cells in the preparation of anti-aging drugs. The mesenchymal stem cells are human adipose-derived mesenchymal stem cells (hAMSCs) or mouse bone marrow mesenchymal stem cells (BM-MSCs). This invention utilizes mesenchymal stem cells to regulate senescent stromal cells and the spleen's ECM, improving the thinning stromal state in the tissue microenvironment through multiple targets. Specifically, it improves the aging-related secretory phenotype of stromal cells, enhances mitochondrial activity, strengthens their proliferation capacity, and delays the reduction of collagen secretion. This, in turn, delays the disordered distribution and functional decline of immune cells caused by aging and enhances tissue regeneration capacity, indirectly affecting various other cells in the tissue microenvironment, thus exhibiting an antagonistic effect against systemic organ aging.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, specifically relating to the application of mesenchymal stem cells in the preparation of anti-aging drugs. Background Technology

[0002] As we age, the accumulation of external and internal stresses gradually impairs the body's physiological integrity, while the ability of tissues to recover from stress weakens, leading to impaired bodily function and an increased risk of death. How to slow down aging, reduce the incidence of age-related diseases, and thus achieve healthy longevity is a long-standing problem in aging research. Studies have shown that organ structure changes during aging, leading to a decline in organ function.

[0003] Stem cells possess regenerative capabilities, can regulate the microenvironment, and can even reverse aging, making them a comprehensive and effective choice in anti-aging strategies. Compared to embryonic stem cells, adult stem cells have advantages in availability and safety, making their clinical application more feasible. Patent CN115678838A discloses a method for obtaining and applying stem cell preparations for anti-aging treatment using autologous stem cells. The cell source is the patient's own cells, including hematopoietic stem cells, mesenchymal stem cells, monocytes, and other cells contained in the bone marrow microenvironment. This patented technology uses stem cell preparations to treat tissue aging damage, cell aging, and delay aging, with the aging tissue being an aging brain tissue model. Patent CN109864964A discloses an anti-aging composition containing stem cells and its application. The composition includes stem cells, immune cells involved in immune responses, iPS cell extracts, and a protective agent, and describes the use of this composition in the preparation of anti-aging preparations. However, current research on the mechanisms by which mesenchymal stem cells delay cellular and tissue aging mainly focuses on the regulation of overall tissue aging markers and the levels of age-related secretory phenotypes (SASPs). This application, on the other hand, focuses on the anti-aging effect of stem cell preparations on stromal cells in the tissue microenvironment. It verifies that stem cells can improve the aging state of the thin matrix in the tissue microenvironment through multiple targets, indirectly affecting various other cells in the tissue microenvironment, thereby exhibiting an antagonistic effect against systemic organ aging, and its therapeutic significance and scope are broader. In addition, based on in vitro studies, the results of in vivo stem cell injection therapy in mice have been added, making the credibility and safety more convincing.

[0004] The extracellular matrix (ECM) is a crucial component of the tissue microenvironment, primarily secreted by mesenchymal cells. It is a network of collagen fibers mainly composed of type I and type III collagen, various proteoglycans, elastin, and fibronectin. ECM components not only provide dynamic tissue integrity, but the ECM itself also acts as a signaling molecule, participating in and driving many biological responses. Its dysregulation is a direct or indirect cause of most chronic diseases. Aging not only leads to decreased immune tissue mechanical compliance but may also alter the mechanical properties of the ECM perceived by the immune system to some extent, reducing mechanosensors that downregulate pro-inflammatory pathways. Furthermore, changes in organ structure are closely related to the maintenance of ECM homeostasis within the tissue microenvironment. Therefore, exploring the complex connections between MSCs and surrounding cells during anti-aging by acting on organ structural ECM is of great significance for treating diseases and maintaining healthy aging. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by proposing the application of mesenchymal stem cells in the preparation of anti-aging drugs.

[0006] Specifically, this is achieved through the following technical solutions:

[0007] This invention provides the application of mesenchymal stem cells in the preparation of anti-aging drugs, wherein the mesenchymal stem cells are human adipose-derived mesenchymal stem cells or mouse bone marrow-derived mesenchymal stem cells.

[0008] Furthermore, the anti-aging drug is a drug used to improve the aging phenotype of aging stromal cells.

[0009] Furthermore, the anti-aging drug is a drug used to promote collagen secretion by aging stromal cells.

[0010] Furthermore, the anti-aging drug is a drug used to regulate the expression of mitochondria in aging stromal cells.

[0011] Furthermore, the anti-aging drug is a drug used to downregulate the content of reactive oxygen free radicals in aging stromal cells.

[0012] Furthermore, the anti-aging drug is a drug that upregulates ATP in aging stromal cells.

[0013] Furthermore, the anti-aging drug is a drug used to increase the content of Col1a1 in the spleen ECM.

[0014] The methods of application include: co-culturing mesenchymal stem cells and senescent stromal cells in vitro, and transplanting mesenchymal stem cells in vivo.

[0015] Beneficial effects:

[0016] This invention utilizes mesenchymal stem cells to regulate senescent stromal cells and spleen ECM, improving the thin stromal state of the tissue microenvironment through multiple targets. Specifically, it inhibits the senescence-related secretory phenotype of stromal cells, enhances mitochondrial activity of stromal cells, strengthens their proliferation capacity, and delays the reduction of collagen secretion. This, in turn, delays the structural and functional aging of immune cells and enhances the regenerative capacity of tissues, indirectly affecting other cells in the tissue microenvironment, thus exhibiting an antagonistic effect against systemic organ aging.

[0017] This invention can effectively exert anti-T cell aging effects, laying a theoretical foundation for the development of treatments for age-related diseases. Attached Figure Description

[0018] Figure 1 In Example 1, co-culturing hAMSCs can improve the senescent phenotype of senescent stromal cells;

[0019] Figure 2 In Example 1, co-culturing hAMSCs can promote collagen secretion from senescent stromal cells;

[0020] Figure 3 In Example 1, co-culturing hAMSCs can regulate mitochondrial expression in senescent stromal cells;

[0021] Figure 4 In Example 1, co-culturing hAMSCs can reduce the content of reactive oxygen species in senescent stromal cells;

[0022] Figure 5 In Example 1, co-culturing hAMSCs can increase the ATP content of senescent stromal cells;

[0023] Figure 6 This demonstrates how stem cell transplantation in Example 1 improves the aging phenotype of stromal cells in aging mice.

[0024] Figure 7 In Example 1, stem cell transplantation can promote collagen secretion from stromal cells in aging mice;

[0025] Figure 8 In Example 1, stem cell transplantation was able to regulate mitochondrial expression in stromal cells of aging mice.

[0026] Figure 9 In Example 1, stem cell transplantation reduced the content of reactive oxygen species in the stromal cells of aging mice.

[0027] Figure 10 In Example 1, stem cell transplantation increased the ATP content of stromal cells in aging mice;

[0028] Figure 11 This is the result of the effect of in vivo stem cell transplantation in Example 1 on increasing collagen content in the ECM environment and improving the aging of immune T cells;

[0029] Figure 12 This is the result of the effect of increasing the collagen 1 content of the spleen ECM in vitro and alleviating the aging of immune T cells in Example 1. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in further detail below, but the present invention is not limited to these embodiments. Any improvements or substitutions based on the basic spirit of these embodiments shall still fall within the scope of protection claimed by the claims of the present invention.

[0031] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the art.

[0032] All qRT-PCR and Western blotting experiments used the universal housekeeping gene / protein GAPDH as an internal control gene / protein. All statistical analyses were performed using GraphPad Prism 9 (GraphPad Prism, San Diego, CA) software. Statistical comparisons between two groups were performed using t-tests. One-way ANOVA was used for comparisons among multiple groups. Statistically significant differences were defined as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0033] Example 1

[0034] Culture and induction of human adipose-derived mesenchymal stem cells:

[0035] Primary human adipose-derived mesenchymal stem cells were cultured in stem cell culture medium (DMEM / F12 + 2% fetal bovine serum + penicillin 100 IU / mL + streptomycin 100 μg / mL) at 37°C in a 5% CO2 incubator. The culture medium was changed every 2 days. When the cell growth density reached 80%-90%, the cells were passaged and used in various experiments.

[0036] Example 2

[0037] Results of co-culturing hAMSCs in improving the senescent phenotype of senescent stromal cells:

[0038] An in vitro replicative stromal cell senescence model was established by seeding stromal cells in 10 cm dishes at a culture volume of 10-12 mL and a cell density of 4.5-10 × 10⁻⁶ cells / mL. 6Cells were cultured in a cell culture incubator (37℃, 5% CO2). The complete culture medium (DMEM / F12 + 2% fetal bovine serum + penicillin 100 IU / mL + streptomycin 100 μg / mL) was changed every 48 hours. Cells were passaged when the cell density reached 80%-90%, and this process was repeated 15 times to establish a replicative senescence model for subsequent experiments. Aseptically extracted primary hAMSCs and senescent stromal cells were co-cultured in a transwell. After 48 hours, the differences in senescence phenotypes among the young stromal cell control group (Y-Ctrl), the non-co-cultured control group (RS-Ctrl), and the hAMSCs co-cultured experimental group (RS-MSCs) were observed. Stromal cells were identified by SA-β-gal staining, and proteins and RNA were extracted from the stromal cells for Western blotting and qRT-PCR experiments according to standard procedures.

[0039] Experimental results are as follows Figure 1 As shown. Among them. Figure 1 A shows the SA-β-gal staining results of stromal cells; Figure 1 B represents the relative expression of aging markers in stromal cells detected by qRT-PCR and WB (Note: scale bar is 200 μm).

[0040] Depend on Figure 1 As shown in Figure A, the positive rate of SA-β-gal staining in the RS-Ctrl group was significantly higher than that in the Y-Ctrl group, while the positive rate of staining in the RS-hMSCs group recovered somewhat, showing a significant difference compared to the RS-Ctrl group, suggesting that cell senescence was alleviated after co-culturing with stem cells; Figure 1 B shows that the expression levels of the RS-Ctrl and RS-hMSCs groups were significantly different from those of the Y-Ctrl group. Western blot images showed the protein expression of GAPDH (as an internal control), P16, P21, and Lamb1. The expression of each protein was displayed in the Y-Ctrl, RS-Ctrl, and RS-hMSCs groups, and changes in the intensity of the protein bands could be observed. The RS-Ctrl group showed significant differences compared to both the RS-hMSCs and Y-Ctrl groups. The results indicate that the RS-hMSCs group had significantly higher proportions of SA-β-gal positive cells and significantly higher expression levels of p16, p21, and Lamb1 genes than the other two groups. Therefore, hAMSCs can effectively improve the senescent phenotype of senescent stromal cells.

[0041] Example 3

[0042] Results of co-culturing hAMSCs promoting collagen secretion in senescent stromal cells:

[0043] Aseptically extracted primary hAMSCs and senescent stromal cells were co-cultured in a transwell. After 48 hours, the differences in the expression of collagen synthesis-related genes were observed among the young stromal cell control group (Y-Ctrl group), the unco-cultured control group (RS-Ctrl group), and the experimental RS-MSCs group co-cultured with hAMSCs. Proteins and RNA were extracted from the stromal cells, and Western blotting and qRT-PCR experiments were performed according to standard procedures.

[0044] Experimental results are as follows Figure 2 As shown. Figure 2 The relative expression of collagen synthesis-related genes in stromal cells was detected by qRT-PCR and WB.

[0045] Depend on Figure 2 It was found that, at the mRNA level, compared with the young control Y-Ctrl group, the expression of COL1A1 and Col3A1 in the senescent cell RS-Ctrl group was significantly reduced. After stem cell co-culture, the expression of these two collagens increased in the RS-MSCs group. The expression of Mmp1, Mmp2, and Mmp12 in the RS-Ctrl group was significantly higher than that in the Y-Ctrl group, but decreased in the RS-hMSCs group. Western blot images showed that the expression of each protein was present in the Y-Ctrl, RS-Ctrl, and RS-hMSCs groups, and the changes in protein band intensity were similar to those at the mRNA level. The results indicate that the expression of Col1a1 and Col3a1 was significantly increased in the RS-hMSCs group, while the expression of Mmp1, Mmp2, and Mmp12 was significantly decreased. Stem cell co-culture can improve the secretory capacity of senescent stromal cells.

[0046] Example 4

[0047] Results of hAMSCs co-culture regulating the expression of genes related to mitochondrial development in senescent stromal cells:

[0048] Aseptically extracted primary hAMSCs and senescent stromal cells were co-cultured in a transwell. After 48 hours, the differences in mitochondrial genes expression were observed between the young stromal cell control group (Y-Ctrl group), the unco-cultured control group (RS-Ctrl group), and the experimental RS-MSCs group (co-cultured with hAMSCs). Proteins and RNA were extracted from the stromal cells, and Western blotting and qRT-PCR experiments were performed according to standard procedures.

[0049] Experimental results are as follows Figure 3 As shown. Figure 3The relative expression of mitochondrial genes in stromal cells was detected by qRT-PCR and Western blotting. Sirt1 and Pgc-1α are typically associated with mitochondrial function and energy metabolism, while Nrf1 and Tfam are involved in oxidative stress resistance and mitochondrial DNA maintenance.

[0050] Depend on Figure 3 It was found that, at the mRNA level, compared with the young control Y-Ctrl group, the expression of Sirt1, Pgc-1α, Nrf1, and Tfam was significantly reduced in the senescent cell RS-Ctrl group. After stem cell co-culture, the expression of these four genes increased in the RS-MSCs group. Western blot images showed that the expression of each protein was present in the Y-Ctrl, RS-Ctrl, and RS-hMSCs groups, and the changes in protein band intensity were similar to those at the mRNA level. The results indicate that the expression levels of Sirt1, Pgc-1α, Nrf1, and Tfam genes were significantly increased in the RS-hMSCs group, and the same trend was observed at the protein level.

[0051] Example 5

[0052] Results of co-culturing hAMSCs reducing the content of reactive oxygen species in senescent stromal cells:

[0053] Aseptically extracted primary hAMSCs and senescent stromal cells were co-cultured in transwells, with cell density in six-well plates less than 5 × 10⁶ cells / well. 5 After 72 hours, the differences in reactive oxygen species (ROS) content were detected between the young stromal cell control Y-Ctrl group (non-co-cultured), the experimental RS-Ctrl group (co-cultured with hAMSCs), and the experimental RS-MSCs group (co-cultured with hAMSCs).

[0054] Remove the cell culture medium, load the ROS probe, and add an appropriate volume of diluted DCFH-DA working solution (dilute DCFH-DA 1:1000 with serum-free culture medium to a final concentration of 10 μM). The added volume should fully cover the cells. Incubate at 37°C in the dark. After 30 min, wash the cells 1-2 times with serum-free culture medium to thoroughly remove any DCFH-DA that has not entered the cells. Finally, digest with trypsin to prepare a single-cell suspension and analyze using flow cytometry.

[0055] Experimental results are as follows Figure 4 As shown. Among them. Figure 4 This shows the changes in the content of reactive oxygen species in senescent stromal cells before and after co-culture.

[0056] Depend on Figure 4The results showed that the ROS level in the RS-Ctrl group was significantly higher than that in the Y-Ctrl group, while the ROS level in the RS-hMSCs group was reduced. This indicates that the RS-hMSCs group significantly reduced intracellular ROS levels compared to the RS-Ctrl group. This suggests that hMSCs treatment may have an antioxidant effect, effectively reducing intracellular reactive oxygen species levels and oxidative stress.

[0057] Example 6

[0058] Results of co-culturing hAMSCs increasing ATP content in senescent stromal cells:

[0059] Aseptically extracted primary hAMSCs and senescent stromal cells were co-cultured in transwells, with cell density in six-well plates less than 5 × 10⁶ cells / well. 5 After 72 hours, the difference in intracellular ATP content between the young stromal cell control Y-Ctrl group, the unco-cultured control RS-Ctrl group, and the experimental RS-MSCs group co-cultured with hAMSCs was detected:

[0060] Luciferase requires ATP to catalyze the production of fluorescence from luciferin. Within a certain concentration range, the production of fluorescence is directly proportional to the concentration of ATP. Follow the instructions on the ATP assay kit and use a fluorescence spectrophotometer to detect the results.

[0061] Experimental results are as follows Figure 5 As shown. Among them. Figure 5 This shows the changes in ATP content in senescent stromal cells before and after co-culture.

[0062] Depend on Figure 5 It was found that the ATP level in the RS-Ctrl group was significantly lower than that in the Y-Ctrl group. Compared to the RS-Ctrl group, the ATP level in the RS-hMSCs group increased after treatment. ATP is the main carrier of cellular energy, and changes in its level can reflect changes in cellular energy status. The RS-Ctrl group showed lower ATP levels in senescent cells, while the RS-hMSCs group showed a certain degree of recovery in ATP levels, which may indicate that hMSCs treatment has a positive effect on cellular energy metabolism.

[0063] Example 7

[0064] Results of stem cell transplantation improving the aging phenotype of stromal cells in aging mice:

[0065] Female C57 mice were used as the research subjects. One-month-old mice were injected intravenously with PBS every 15 days for a total of five injections. Alternatively, 24-month-old mice were injected intravenously with PBS or young-derived BM-MSCs using the same protocol. This simulated a treatment regimen of periodic MSC transplantation to alleviate aging in middle-aged and elderly individuals. Stromal cells were collected on day 7 after injection for relevant analysis. Three-month-old mice injected with PBS served as the young control group (Y-Ctrl), 26-month-old mice injected with PBS served as the aging control group (A-Ctrl), and 26-month-old mice injected with BM-MSCs served as the aging control with MSCs group (A-MSCs). Stromal cells were identified by SA-β-gal staining, and proteins and RNA were extracted from the stromal cells for Western blotting and qRT-PCR experiments according to standard procedures. The results were consistent with in vitro experiments.

[0066] Experimental results are as follows Figure 6 As shown. Among them. Figure 6 A shows the SA-β-gal staining results of stromal cells; Figure 6 B represents the relative expression of aging markers in stromal cells detected by qRT-PCR and WB (Note: scale bar is 200 μm).

[0067] Depend on Figure 6 As shown in Figure A, the positive rate of SA-β-gal staining in cells of the A-Ctrl group was significantly higher than that in the Y-Ctrl group, while the positive rate of staining in the A-MSCs group was reduced compared to the A-Ctrl group, suggesting that aging was alleviated after stem cell injection therapy; Figure 6 B shows that the expression levels of the A-Ctrl and A-MSCs groups were significantly different from those of the Y-Ctrl group. Western blot images showed the protein expression of GAPDH (as an internal control), P16, P21, and Lamb1. The expression of each protein was displayed in the Y-Ctrl, A-Ctrl, and A-MSCs groups, and changes in the intensity of the protein bands could be observed. The A-Ctrl group showed significant differences compared to both the A-MSCs and Y-Ctrl groups. The results indicate that the A-MSCs group had significantly higher proportions of SA-β-gal positive cells and significantly higher expression levels of p16, p21, and Lamb1 genes than the other two groups. Therefore, BM-MSCs can effectively improve the senescent phenotype of senescent stromal cells.

[0068] Example 8

[0069] The results of stem cell transplantation promoting collagen secretion from stromal cells in aging mice:

[0070] The mouse stem cell transplantation procedure was exactly the same as in Example 7. Proteins and RNA were extracted from stromal cells, and Western blotting and qRT-PCR experiments were performed according to standard procedures. The results were consistent with the in vitro experiments.

[0071] Experimental results are as follows Figure 7 As shown. Figure 7 The relative expression of collagen synthesis-related genes in stromal cells was detected by qRT-PCR and WB.

[0072] Depend on Figure 7 It was found that, at the mRNA level, compared with the young control Y-Ctrl group, COL1A1 expression was significantly reduced in the senescent cell A-Ctrl group. After stem cell co-culture, collagen expression increased in the A-MSCs group. Mmp9 expression was significantly higher in the A-Ctrl group than in the Y-Ctrl group, but decreased in the A-MSCs group. Western blot images showed that the expression of each protein was present in the Y-Ctrl, A-Ctrl, and A-MSCs groups, and the changes in protein band intensity were similar to those at the mRNA level. The results indicate that Col1A1 expression was significantly increased and Mmp9 expression was significantly decreased in the A-MSCs group. This suggests that BM-MSCs treatment has a significant effect on the expression of these genes, potentially affecting the secretory capacity of senescent stromal cells.

[0073] Example 9

[0074] Stem cell transplantation can regulate mitochondrial expression in stromal cells of aging mice.

[0075] The mouse stem cell transplantation procedure was exactly the same as in Example 7. Proteins and RNA were extracted from stromal cells, and Western blotting and qRT-PCR experiments were performed according to standard procedures. The results were consistent with the in vitro experiments.

[0076] Experimental results are as follows Figure 8 As shown. Figure 8 The relative expression of mitochondrial genes in stromal cells was detected by qRT-PCR and Western blotting.

[0077] Depend on Figure 8It was found that Sirt1 expression was significantly lower in the A-Ctrl group than in the Y-Ctrl group, but significantly higher in the A-MSCs group than in the Y-Ctrl group; Nrf1 expression was significantly lower in the A-Ctrl group than in the Y-Ctrl group, but rebounded in the A-MSCs group compared to the A-Ctrl group; Tfam expression was significantly lower in the A-Ctrl group than in the Y-Ctrl group, but significantly higher in the A-MSCs group than in the A-Ctrl group. Western blot images showed that NRF1 and TFAM protein expression was observed in all three groups, with changes in protein band intensity. Sirt1 and Tfam are typically associated with mitochondrial function and energy metabolism, while Nrf1 is associated with oxidative stress. The results indicate that the expression levels of Sirt1, Nrf1, and Tfam genes were significantly increased in the A-MSCs group, and the same trend was observed at the protein level. This suggests that BM-MSCs treatment significantly promotes the expression of these genes, potentially affecting cellular mitoogenesis and energy metabolism.

[0078] Example 10

[0079] The results showed that stem cell transplantation reduced the content of reactive oxygen species in stromal cells of aging mice.

[0080] The mouse stem cell transplantation procedure was exactly the same as in Example 7, with a cell density of less than 5 × 10⁻⁶ cells in the six-well plate. 5 After the stromal cells adhered, the cell culture medium was removed, ROS probes were loaded, and an appropriate volume of diluted DCFH-DA working solution (DCFH-DA diluted 1:1000 with serum-free culture medium to a final concentration of 10 μM) was added. The added volume should completely cover the cells, and the cells were incubated at 37°C in the dark. After 30 minutes, the cells were washed 1-2 times with serum-free culture medium to thoroughly remove any DCFH-DA that had not entered the cells. Finally, the cells were digested with trypsin to prepare a single-cell suspension, which was then analyzed using flow cytometry. The results were consistent with those of the in vitro experiments.

[0081] Experimental results are as follows Figure 9 As shown. Among them. Figure 9 This describes the changes in the content of reactive oxygen species in senescent stromal cells.

[0082] Depend on Figure 9The results showed that the ROS level in the A-Ctrl group was significantly higher than that in the Y-Ctrl group, exhibiting a relative ROS level approximately 1.5 times higher. The ROS level in the A-MSCs group fell between the two, showing a relative ROS level approximately 1.2 times higher. Therefore, there were significant differences between the Y-Ctrl and A-Ctrl groups, and also significant differences between the Y-Ctrl and A-MSCs groups. These results indicate that the intracellular ROS level in the A-MSCs group was significantly lower compared to the A-Ctrl and Y-Ctrl groups. This suggests that BM-MSCs may have antioxidant effects, effectively reducing intracellular reactive oxygen species levels.

[0083] Example 11

[0084] The result that stem cell transplantation can increase the ATP content of stromal cells in aging mice:

[0085] The mouse stem cell transplantation process was completely consistent with that in Example 7 and the detection steps were the same as those in Example 6. After the stromal cells adhered, the procedure was performed according to the ATP detection kit steps, and the results were detected using a fluorescence spectrophotometer.

[0086] Experimental results are as follows Figure 10 As shown. Among them. Figure 10 This shows the changes in ATP content in senescent stromal cells.

[0087] Depend on Figure 10 It was found that the ATP level in the A-Ctrl group was significantly lower than that in the Y-Ctrl group. Compared to the A-Ctrl group, the ATP level in the A-MSCs group increased after treatment. ATP is the main carrier of cellular energy, and changes in its level can reflect changes in cellular energy status. The ATP level in senescent cells of the A-Ctrl group was low, while BM-MSCs treatment restored ATP levels to some extent, which may indicate that BM-MSCs treatment has a positive effect on cellular energy metabolism.

[0088] Example 12

[0089] In vivo stem cell transplantation increases collagen content in the ECM environment and improves the aging of immune T cells.

[0090] The mouse stem cell transplantation procedure was identical to that in Example 7. Subsequently, mouse spleen sections were stained for CD8 immunofluorescence staining to locate and detect the number and distribution of CD8+ T cells in the spleen of mice injected with BM-MSCs. Simultaneously, collagen 1 was stained in the spleen to detect the distribution of T cells and the expression of aging markers in the transplanted stem cell group (A-MSCs). The results showed that BM-MSCs increased the collagen 1 content in the microenvironment and maintained the distribution of CD8+ T cells, thus alleviating aging. (Note:) Figure 12A scale bar is 200 μm; Figure 12 (B scale is 50 μm.)

[0091] Experimental results are as follows Figure 11 As shown. Among them. Figure 11 A shows the number and distribution of CD8+ T cells in the spleen of mice after injection of BM-MSCs, as determined by CD8 immunofluorescence staining. Figure 11 B represents the expression of P21 in CD8+ T cells after BM-MSC treatment. Figure 11 C represents the staining of collagen 1 in the spleen of mice after injection of BM-MSC.

[0092] Depend on Figure 11 It can be seen that, Figure 11 A indicates that CD8+ T cells in the spleen of young control group Y-Ctrl showed aggregated distribution, while the number of CD8+ T cells in the spleen of elderly control group A-Ctrl was significantly reduced and scattered, without obvious white pulp structure. In contrast, the number of CD8+ T cells in the spleen of mice transplanted with stem cells A-MSCs was increased compared with that of elderly control group and showed aggregated distribution, similar to Y-Ctrl group. Figure 11 B. Immunofluorescence double staining of CD8 and p21 showed that p21 expression was upregulated in CD8+ T cells in the spleen compared with that in younger mice, while p21 expression was significantly downregulated in the A-MSCs group. Figure 11 Immunofluorescence staining of mouse spleens by C revealed that as Col1a1 was lost from the spleens of aging mice, the expression level of Col1a1 in the A-MSCs group was significantly increased, and Col1a1 was distributed around CD8+ T cells. This indicates that stem cell therapy in the A-MSCs group restored the chemotactic and localization abilities of CD8+ T cells, improved the aging phenotype of the spleen, and also improved the secretion and ECM regulation characteristics, thus alleviating the loss of Col1a1 in the aging spleen.

[0093] Example 13

[0094] In vitro, increasing collagen 1 content in the spleen's ECM can alleviate the aging of immune T cells.

[0095] The mouse stem cell transplantation procedure was identical to that in Example 7, with the addition of an exogenous collange1 (A-Col1 group) supplementation. The distribution of T cells and the expression of aging markers were examined in both the transplanted stem cell group (A-MSCs) and the A-Col1 group with exogenous collagen supplementation. The results showed that BM-MSCs maintain the distribution of CD8+ T cells by increasing collange1 levels in the microenvironment, thereby alleviating aging.

[0096] Experimental results are as follows Figure 12 As shown. Figure 12 A represents the distribution of T cells. In the stem cell transplantation group, or with the addition of exogenous collange1, T cells can maintain a colony-like distribution. Figure 12 B represents the immunofluorescence results of aging marker genes. Exogenous collagen supplementation significantly decreased the expression of aging markers in CD8+ T cells. (Note:) Figure 12 A scale bar is 200 μm; Figure 12 (B scale is 50 μm.)

[0097] Depend on Figure 12 It was observed that T cells in the A-Ctrl group were scattered, while lymphocyte colonies could be observed in both the A-Col1 and A-MSCs groups. Simultaneously, exogenous supplementation with Col1a1 improved the p21 upregulation associated with CD8+ T cell senescence, achieving a similar effect to the A-MSCs group. These results indicate that the A-MSCs group alleviated spleen senescence by increasing the content of type I collagen in the mouse spleen to maintain the concentrated distribution of CD8+ T cells.

Claims

1. The application of mesenchymal stem cells in the preparation of anti-aging drugs, characterized in that, The mesenchymal stem cells are human adipose-derived mesenchymal stem cells or mouse bone marrow-derived mesenchymal stem cells.

2. The application of mesenchymal stem cells as described in claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is a drug used to improve the aging phenotype of aging stromal cells.

3. The application of mesenchymal stem cells as described in claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is a drug used to promote collagen secretion in aging stromal cells.

4. The application of mesenchymal stem cells as described in claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is a drug used to regulate the expression of mitochondria in aging stromal cells.

5. The application of mesenchymal stem cells as described in claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is used to downregulate the content of reactive oxygen free radicals in aging stromal cells.

6. The application of mesenchymal stem cells as described in claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is one that upregulates ATP in aging stromal cells.

7. The application of mesenchymal stem cells as described in claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drug is used to increase the content of Col1a1 in the spleen's ECM.

8. The use of mesenchymal stem cells as described in any one of claims 1-7 in the preparation of anti-aging drugs, characterized in that, The methods of application include: co-culturing mesenchymal stem cells and senescent stromal cells in vitro, and transplanting mesenchymal stem cells in vivo.

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