Mesenchymal stem cell exosome preparation for delaying senescence and preparation method thereof

Abstract

The invention belongs to the technical field of stem cell exosome preparation. The present invention relates to the field of stem cells, and more particularly, to adipose-derived stem cells (ADSCs), conditioned media (CMs) thereof, a preparation method thereof, and uses thereof in cosmetics and drugs, and more particularly, to adipose-derived stem cells (ADSCs), conditioned media (CMs) thereof, and a preparation method thereof, and uses thereof in cosmetics and drugs. The invention provides a mesenchymal stem cell exosome preparation for delaying senescence. The mesenchymal stem cell exosome preparation is a conditioned medium obtained by culturing mesenchymal stem cells in an environment of hypotonic induction combined with erpicotide stimulation. Tests show that the conditioned media can significantly enhance the proliferation and migration ability of senescent HSFs cells; the expression of cell beta-galactosidase is reduced; the expression of p16 and p53 of cell-related senescence genes is reduced, the generation of ROS (reactive oxygen species) in cells is reduced, and a remarkable anti-senescence effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell exosome preparation. More specifically, it relates to adipose-derived stem cells (ADSCs), their conditioned medium (CM), their preparation methods, and their uses in cosmetics and pharmaceuticals. Background Art

[0002] Mesenchymal stem cell (MSC)-derived exosomes (MSCs-Exos) can play an important role in treating skin aging and tissue repair through various mechanisms of action such as improving the state of human dermal fibroblasts (HDFs), promoting collagen synthesis, and reducing ROS production. Wang et al. [1] found that MSCs-Exos reduced the production of ROS, DNA damage, and abnormal calcium signaling in H2O2-stimulated keratinocytes or UV-irradiated mouse skin through the Nrf2 signaling pathway, and alleviated oxidative stress-induced skin damage. Similarly, ADSCs-Exos promoted the proliferation, migration of HDFs, and optimized collagen deposition by stimulating the PI3K / Akt pathway, upregulating the gene and protein expression of MMP-1, bFGF, and TGF-β1 [2] , and simultaneously promoted the gene expression of type I and III collagens and cyclin-1. In addition, pretreatment with MSCs-Exos significantly promoted the proliferation of HDFs, increased the expression of GPX-1 and Col-1, and inhibited the production of ROS and the expression of MMP-1 induced by UVB, thereby protecting cells from UVB-induced photoaging [3] . In short, MSCs-Exos promote collagen synthesis through various mechanisms of action, downregulate the levels of ROS and MMPs, and have important application prospects in treating skin aging.

[0003] Adipose mesenchymal stem cells are derived from adipose tissue and are a type of adult stem cell that has been widely studied in the field of stem cells after bone marrow mesenchymal stem cells. They are rich in sources and easy to extract, making them ideal seed cells. Currently, Ding et al. [4] have shown that hepatocyte growth factor and basic fibroblast growth factor secreted by human adipose-derived stem cells can improve ovarian function during natural aging by activating the SIRT1 / FOXO1 signaling pathway. Cooper et al. [5]It has been found that the conditioned medium of human adipose mesenchymal stem cells can promote the migration of human skin fibroblasts and ischemic wound healing. Exosomes regulate the biological functions of recipient cells by binding to recipient cells and non-selectively releasing bioactive components such as microRNA, mRNA, and proteins contained therein. Therefore, the secretion amount of exosomes and the active components carried by them will directly affect their functions. Recent studies have shown that in addition to differences in the source of MSCs, different stimuli such as drugs, cytokines, and hypoxia also have a great impact on the secretion and function of exosomes. By using appropriate intervention measures, not only can the secretion amount of exosomes be increased, but the secreted exosomes can also be enriched with certain specific components. For example, drug or cytokine stimulation can increase the yield and biological activity of exosomes. Shi et al. [6] suggested that 3,3′-diindolylmethane can increase the expression of Wnt11 protein in UCMSCs-Exos by activating the Wnt / β-catenin signaling pathway. Sung et al. [7] compared the effects of thrombin, hydrogen peroxide, and lipopolysaccharide treatments on the secretion of MSCs-Exos and the therapeutic effect of skin wound healing, and found that thrombin stimulation can significantly promote the secretion of MSCs-Exos and enrich them with various growth factors such as VEGF and angiopoietin; hypoxia treatment can change the active components carried by exosomes and enhance their efficacy. Wang et al. [8] found that ADSCs-Exos induced by hypoxia can significantly increase the gene expression of TGF-β, EGF, and bFGF in fibroblasts, promote diabetic wound healing, and inhibit inflammation through the PI3K / Akt pathway. Zhu et al. [9] showed that MSCs-Exos produced after hypoxia treatment are rich in microRNA related to promoting angiogenesis such as microRNA-210, which can enhance their angiogenesis-promoting effect. There is also evidence that ADSCs-Exos secreted after hypoxia stimulation can improve the efficiency of angiogenesis by activating the PKA pathway

[10] . In addition, Liu et al.

[11] found that exosomes produced by hypoxia (1% O2) stimulation are rich in miR-216a-5p, which promotes the repair of spinal cord injury by stimulating the TLR4 / NF-κB / PI3K / Akt signaling pathway.

[0004] Osmotic pressure is an important physical parameter of the cell microenvironment, and the normal physiological osmotic pressure is about 290-310 mosm / kg (isotonic). Hypotonicity (usually referring to <250 mosm / kg) will directly affect the survival, proliferation, differentiation, and function of stem cells. At present, there is no study on the secretion and function of mesenchymal stem cell exosomes under different osmotic pressure stimulations.

[0005] [1] Wang T, Jian Z, Baskys A, et al. MSC-derived exosomes protect against oxidative stress-induced skin injury via adaptive regulation of the NRF2 defense system[J]. Biomaterials. 2020, 257: 1-18.

[0006] [2] Zhang W, Bai X, Zhao B, et al. Cell-free therapy based on adipose tissue stem cell-derived exosomes promotes wound healing via the PI3K / Akt signaling pathway[J]. Exp Cell Res. 2018, 370(2): 333-342.

[0007] [3] Deng M, Yu T Z, Li D, et al. Human umbilical cord mesenchymal stem cell-derived and dermal fibroblast-derived extracellular vesicles protect dermal fibroblasts from ultraviolet radiation-induced photoaging in vitro[J]. Photochem Photobiol Sci. 2020, 19(3): 406-414.

[0008] [4] Ding C, Zou Q, Wang F, et al. HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1 / FOXO1 Signaling Pathway. Cell Physiol Biochem. 2018;45(4): 1316-1332.

[0009] [5]Cooper DR,Wang C,Patel R,et al.Human Adipose-Derived Stem CellConditioned Media and Exosomes Containing MALAT1Promote Human DermalFibroblast Migration and Ischemic WoundHealing.Adv Wound Care(New Rochelle).2018;7(9):299-308.

[0010] [6]Shi H,Xu X,Zhang B,et al.3,3'-Diindolylmethane stimulates exosomalWnt11 autocrine signaling in human umbilical cord mesenchymal stem cells toenhance wound healing[J].Theranostics.2017,7(6):1674-1688

[0011] [7]Sung D K,Chang Y S,Sung S I,et al.Thrombin Preconditioning ofExtracellular Vesicles Derived from Mesenchymal Stem Cells AcceleratesCutaneous Wound Healing by Boosting Their Biogenesis and Enriching CargoContent[J].J Clin Med.2019,8(4):1-16.

[0012] [8]Wang J,Wu H,Peng Y,et al.Hypoxia adipose stem cell-derivedexosomes promote highquality healing of diabetic wound involves activation ofPI3K / Akt pathways[J].J Nanobiotechnology.2021,19(1):1-13.

[0013] [9] Zhu J, Lu K, Zhang N, et al. Myocardial reparative functions of exosomes from mesenchymal stem cells are enhanced by hypoxia treatment of the cells via transferring microRNA-210 in an nSMase2-dependent way[J]. Artif Cells Nanomed Biotechnol. 2018, 46(8):1659-1670.

[0014]

[10] Xue C, Shen Y, Li X, et al. Exosomes Derived from Hypo xia-Treated Human Adipose Mesenchymal Stem Cells Enhance Angi ogenesis Through the PKASignaling Pathway[J]. Stem Cells Dev. 2018, 27(7):456-465.

[0015]

[11] Liu W, Rong Y, Wang J, et al. Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair trau matic spinal cord injury by shifting microglial M1 / M2 polarization[J]. J Neuroinflammation. 2020, 17(1):1-22. Summary of the Invention

[0016] After long-term and extensive research, the present inventors found that the conditioned medium (CM) obtained by culturing adipose-derived stem cells (ADSCs) under the stimulation of epithalon combined with hypoosmotic conditions is rich in unique bioactive factors due to cellular stress responses and adaptive regulation. Further studies on its functional characteristics revealed that the above-mentioned conditioned medium can significantly enhance the proliferation and migration abilities of senescent HSFs cells; reduce the expression of cellular β-galactosidase; reduce the expression of the cellular senescence-related genes p16 and p53, and reduce the generation of intracellular ROS, showing a significant anti-aging effect.

[0017] The above object of the present invention is achieved by the following technical solutions:

[0018] The object of the present invention is to provide a mesenchymal stem cell exosome preparation for delaying aging, and the exosome preparation is a conditioned medium obtained by culturing mesenchymal stem cells in a hypoosmotic environment.

[0019] In this article, the conditioned medium (CM) refers to the medium collected after culturing mesenchymal stem cells in a conventional medium for a period of time, which contains bioactive substances secreted or released by the mesenchymal stem cells. These substances include: soluble proteins (such as growth factors, cytokines, chemokines), extracellular vesicles (such as exosomes, microvesicles), metabolites (such as lactic acid, amino acids, nucleotides), and nucleic acids (such as miRNAs, mRNAs, circRNAs). The CM components produced by different cells (such as mesenchymal stem cells, tumor cells, immune cells) under different culture conditions vary significantly, and products with different physiological activities can be obtained by regulating these factors.

[0020] In this article, the collection of the conditioned medium can be carried out through the following steps: collecting the supernatant, centrifuging to remove cell debris, and filtering and sterilizing. Centrifugation is to remove cell debris, and its parameters can be 2000 - 4000 r / min for 10 - 20 minutes; the filtering and sterilization can be carried out using a 0.22 μm ultrafiltration membrane.

[0021] Osmotic pressure is an important physical parameter of the cell microenvironment. The normal physiological osmotic pressure is about 290 - 310 mosm / kg (isotonic), while low osmotic pressure usually refers to < 250 mosm / kg. In this article, the specific low osmotic pressure mentioned refers to an osmotic pressure of 200 - 220 mosm / kg; during the experiment, it was observed that an osmotic pressure < 200 mosm / kg caused various harms to the culture of mesenchymal stem cells, such as damage to cell morphology and structure, inhibition of proliferation and survival, and degradation of functional characteristics. However, the conditioned medium obtained in a low osmotic environment with an osmotic pressure of 220 - 250 mosm / kg did not yield very satisfactory results in the senescent HSFs cell model.

[0022] The control of osmotic pressure can be achieved by diluting the DMEM medium. The osmotic pressure of standard DMEM and RPMI - 1640 media is usually 290 - 310 mosm / kg (close to the physiological level), and the osmotic pressure can be reduced by diluting the medium with ultrapure water. Conversely, adding NaCl, sucrose, or mannitol, etc. can increase the osmotic pressure. The specific control method can be carried out using conventional methods. Specifically in this invention, taking the preparation of DMEM medium with an osmotic pressure of 200 mosm / kg as an example:

[0023] ① Preliminary dilution of DMEM medium

[0024] C1V1 = C2V2, where C1 is the osmotic pressure of the original DMEM medium, C2 is the target osmotic pressure, V1 is the volume of the original DMEM medium; V2 is the total volume after dilution.

[0025] 310×V1 = 200×V2 → V2 = 1.5V1, that is, for every 100 mL of the original DMEM, add 50 mL of ultrapure water.

[0026] ② Actual operation: Take 100 mL of standard DMEM, add 50 mL of ultrapure water, mix well and measure the osmotic pressure using an osmometer. If it is too high, continue to add water; if it is too low, supplement a small amount of DMEM concentrate or NaCl; since the concentration of HCO3 - decreases after dilution and the pH is likely to increase, the pH can be finely adjusted to 7.2 - 7.4 with 1M HCl / NaOH. The diluted medium needs to be filtered and sterilized with a 0.22 μm filter membrane (to avoid autoclaving, which may change the osmotic pressure), aliquoted and stored at 4°C, and warmed to 37°C before use.

[0027] In this article, the mesenchymal stem cells are cultured in a hypoosmotic environment for 8 to 24 hours. The inventors found that short-term hypoosmotic culture is beneficial to the proliferation of adipose mesenchymal stem cells. It is speculated that it may promote proliferation through the Hippo-YAP pathway (increased YAP nuclear translocation). However, when the hypoosmotic culture exceeds 24 hours, the proliferation rate of adipose mesenchymal stem cells decreases, apoptosis increases, and the functionality of exosomes decreases. More preferably, the mesenchymal stem cells are cultured in a hypoosmotic environment for 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, or 24 hours. More preferably, the mesenchymal stem cells are cultured in a hypoosmotic environment for 10 hours or 15 hours.

[0028] Another innovative finding is that under the stimulation of certain factors combined with hypoosmotic induction, conditioned media with the greatest anti-aging effect enhancement can be obtained. Among these factors, Epithalon shows the most prominent enhancement effect, followed by melatonin. It is worth mentioning that under the stimulation of certain factors, there is no effect or a weak effect. Currently identified factors include ascorbic acid, resveratrol, etc. Although Epithalon, melatonin, ascorbic acid, resveratrol, etc. all have anti-aging effects, the stimulating effects on adipose stem cells are significantly different under combined hypoosmotic stimulation.

[0029] In this article, the concentration of Epithalon is 5 to 20 μmol / L. More preferably, the concentration of Epithalon can be 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 11 μmol / L, 12 μmol / L, 13 μmol / L, 14 μmol / L, 15 μmol / L, 16 μmol / L, 17 μmol / L, 18 μmol / L, 19 μmol / L, or 20 μmol / L. More preferably, the concentration of Epithalon is 8 μmol / L, 12 μmol / L, or 15 μmol / L.

[0030] In this article, the mesenchymal stem cells can in principle include but are not limited to: adipose mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose stem cells. Among these mesenchymal stem cells, the stimulating effect of Epithalon combined with hypoosmotic stimulation on adipose mesenchymal stem cells is the strongest.

[0031] Another object of the present invention is to provide the use of the mesenchymal stem cell exosome preparation in the preparation of anti-aging cosmetics or drugs.

[0032] In this article, the potential functional characteristics and mechanism analysis of the CM obtained by inducing adipose stem cells with Epithalon combined with hypoosmotic stimulation to achieve anti-aging effect enhancement:

[0033] ①The combination of epipeptide and hypotonic stimulation may lead to the release of more antioxidant enzymes, such as superoxide dismutase SOD2 and glutathione peroxidase GSH-Px;

[0034] ②The combination of epipeptide and hypotonic stimulation inhibits the p16INK4a / p21 pathway and reduces the activity of senescence-associated β-galactosidase (SA-β-gal), and this mechanism was also confirmed in subsequent experiments;

[0035] ③The combination of epipeptide and hypotonic stimulation transfers telomerase active components (such as hTERT mRNA) through sEVs (exosomes);

[0036] ④The combination of epipeptide and hypotonic stimulation stress-activates the Hippo-YAP pathway and promotes the secretion of angiogenesis-promoting factors such as VEGF, HGF, and FGF-2.

[0037] ⑤The combination of epipeptide and hypotonic stimulation promotes the secretion of anti-inflammatory factors such as IL-10 and IL-1RA and inhibits TNF-α and IL-6; regulates the polarization of macrophages to the M2 type through the PGE2 / COX-2 pathway. Description of the Drawings

[0038] Figure 1 A shows the comparison of cell proliferation viability among groups;

[0039] Figure 1 B shows the comparison of the positive rate of SA-β-gal staining of cells among groups;

[0040] Figure 2 A shows the comparison of the intracellular reactive oxygen species levels among groups;

[0041] Figure 1 and Figure 2 In A, compared with the Control group, * P < 0.05, ** P < 0.01; compared with the TI group, # P < 0.05, ## P < 0.01.

[0042] Figure 2 B shows the comparison of the relative expression levels of P16 and P53 genes of cells among groups;

[0043] Among them, compared with the P16 expression level of cells in the Control group, * P < 0.05, ** P < 0.01; compared with the P53 expression level of cells in the Control group, # P < 0.05, ## P < 0.01. Detailed Implementation Modes

[0044] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0046] Example 1: Preparation of ADSCs-CM

[0047] ① Preparation of DMEM medium with an osmotic pressure of 200 mosm / kg: Take Gibco TM DMEM low-glucose medium (product number: 11885084, containing 1 g / L Glucose, initial standard osmotic pressure of 305 mosm / kg); Take 100 mL of Gibco TM DMEM low-glucose medium, add 52.5 mL of ultrapure water and mix well. At this time, measure the osmotic pressure to be approximately 200 mosm / kg. Add HEPES (25 mM) to stabilize the pH to 7.2 - 7.4, add 8 μmol / L epithalon, mix well and filter through a 0.22 μm filter membrane to sterilize. Use a calibrated osmometer to confirm that the osmotic pressure of the final product is 200 ± 5 mosm / kg. After culturing with adipose-derived mesenchymal stem cells for 24 h, measure the cell viability > 90%, and after the cell morphology is normal, aliquot and store at 4°C, labeled as hypotonic medium, for later use.

[0048] ② Take the third-generation human adipose-derived mesenchymal stem cells in good growth state and inoculate 1×10 6 cells into a 100 mm culture dish (containing complete medium for human adipose-derived mesenchymal stem cells). When the cell confluence reaches about 80%, discard the medium and replace it with hypotonic medium. Culture in a 37°C, 5% CO2 incubator for 10 h, then collect the culture medium supernatant, centrifuge at 3000 r / min for 15 min, and filter the centrifuged supernatant through a 0.22 μm filter membrane to obtain ADSCs-CM, which is stored at 4°C for later use.

[0049] Example 2: Preparation of ADSCs-CM

[0050] ① Preparation of DMEM medium with an osmotic pressure of 210 mosm / kg: Take Gibco TM DMEM low-glucose medium (product number: 11885084, containing 1 g / L Glucose, initial standard osmotic pressure of 305 mosm / kg); Take 100 mL of Gibco TMDMEM low-glucose medium was mixed with 45 mL of ultrapure water. At this time, the measured osmotic pressure was about 210 mosm / kg. HEPES (25 mM) was added to stabilize the pH to 7.2 - 7.4. 12 μmol / L Epithalon was added and after mixing, it was filtered through a 0.22 μm filter membrane to sterilize. A calibrated osmometer was used to confirm that the osmotic pressure of the final product was 210 ± 5 mosm / kg. After short-term culturing with adipose-derived mesenchymal stem cells for 24 h, the survival rate was measured to be >90%, and after the cell morphology was normal, it was aliquoted and stored at 4°C, labeled as hypotonic medium for standby.

[0051] ② Take the 3rd passage of human adipose-derived mesenchymal stem cells in good growth state and inoculate 6 1×10 cells into a 100 mm culture dish (containing complete medium for human adipose-derived mesenchymal stem cells). When the cell confluence reached about 80%, discard the medium and replace it with hypotonic medium. Culture in an incubator at 37°C and 5% CO2 for 12 h, then collect the culture medium supernatant, centrifuge at 3000 r / min for 15 min, filter the centrifuged supernatant through a 0.22 μm filter membrane to obtain ADSCs-CM, and store it at 4°C for standby.

[0052] Example 3: Preparation of ADSCs-CM

[0053] ① Preparation of DMEM medium with an osmotic pressure of 220 mosm / kg: Take Gibco TM DMEM low-glucose medium (product number: 11885084, containing 1 g / L Glucose, initial standard osmotic pressure of 305 mosm / kg); Take 100 mL of Gibco TM DMEM low-glucose medium, add 38.6 mL of ultrapure water and mix well. At this time, the measured osmotic pressure was about 220 mosm / kg. Add HEPES (25 mM) to stabilize the pH to 7.2 - 7.4. Add 15 μmol / L Epithalon, mix well and filter through a 0.22 μm filter membrane to sterilize. Use a calibrated osmometer to confirm that the final product was 220 ± 5 mosm / kg. After short-term culturing with adipose-derived mesenchymal stem cells for 24 h, the survival rate was measured to be >90%, and after the cell morphology was normal, it was aliquoted and stored at 4°C, labeled as hypotonic medium for standby.

[0054] ② Take the 3rd passage of human adipose-derived mesenchymal stem cells in good growth state and inoculate 6 1×10 cells into a 100 mm culture dish (containing complete medium for human adipose-derived mesenchymal stem cells). When the cell confluence reached about 80%, discard the medium and replace it with hypotonic medium. Culture in an incubator at 37°C and 5% CO2 for 15 h, then collect the culture medium supernatant, centrifuge at 3000 r / min for 15 min, filter the centrifuged supernatant through a 0.22 μm filter membrane to obtain ADSCs-CM, and store it at 4°C for standby.

[0055] Comparative Example 1

[0056] The difference from Example 1 is that normal osmotic pressure culture is adopted, that is, the culture medium uses Gib co TM DMEM low-sugar culture medium (product number: 11885084, containing 1 g / L Glucose, initial standard osmotic pressure is 305 mosm / kg).

[0057] Comparative Example 2

[0058] The difference from Example 1 is that epithalon is not added, and the other parameters refer to Example 1.

[0059] Comparative Example 3

[0060] The difference from Example 1 is that ascorbic acid is used to replace epithalon, and the other parameters refer to Example 1.

[0061] Comparative Example 4

[0062] The difference from Example 1 is that melatonin is used to replace epithalon, and the other parameters refer to Example 1.

[0063] Comparative Example 5

[0064] The difference from Example 1 is that resveratrol is used to replace epithalon, and the other parameters refer to Example 1.

[0065] Test Example 1. Comparison of the effects of conditioned media from different sources on senescent human skin fibroblasts

[0066] 1.1 Culture of senescent human skin fibroblasts: Human skin fibroblasts were purchased from Shanghai Cell Bank. The culture medium consisted of 1% double antibody, 10% fetal bovine serum by volume, and 89% DMEM / F12. After subculturing them to naturally senescent cells, experiments were carried out (subcultured to more than 45 generations).

[0067] 1.2 Detection of cell proliferation activity by CCK-8 method: Take P45 human fibroblasts with good growth status and inoculate 1×10 5 cells per well in a 96-well plate. Add 200 μL of culture medium to each well and culture at 37 °C and 5% CO2 for 24 h. Discard the original culture medium, wash 3 times with PBS, and add the conditioned media prepared in Examples 1-3 (T1-T3) and Comparative Examples 1-5 (C1-C5) respectively. The blank control group (Control) was added with DMEM medium containing 10% fetal bovine serum by volume. After culturing for 24 h, discard the culture medium in the wells, add a mixture of 10 μL of CCK-8 and 10 μL of serum-free DMEM to each well, incubate in a cell culture incubator for 2.5 h, and measure the absorbance value at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0068] 1. 3β - galactosidase staining: Take P45 human skin fibroblasts in good growth state, inoculate 1×10 4 cells per well into a 96 - well plate, culture at 37°C and 5% CO₂ for 24 h, discard the medium, wash with PBS, and add the conditioned media prepared in Examples 1 - 3 (T1 - T3) and Comparative Examples 1 - 5 (C1 - C5) respectively. The blank control group (Control) adds DMEM medium containing 10% fetal bovine serum by volume. After culturing for 72 h, operate according to the β - galactosidase detection kit. Aspirate the cell medium, wash once with PBS, add an appropriate amount of SA - β - gal staining fixative, fix at room temperature for 15 min, aspirate the cell fixative, add PBS and wash 3 times, 3 min each time. Aspirate the PBS, add an appropriate amount of cell staining working solution, incubate overnight at 37°C. Observe under an optical microscope that senescent cells are light blue to dark blue. The positive rate of the specimens is expressed as the percentage of positive specimens in the total number of specimens.

[0069] 1.4 Intracellular reactive oxygen species (ROS) level: Take P45 human skin fibroblasts in good growth state, inoculate 1×10 5 cells per well into a 96 - well plate, culture at 37°C and 5% CO₂. After the cells adhere, discard the medium, wash with PBS, and add the conditioned media prepared in Examples 1 - 3 (T1 - T3) and Comparative Examples 1 - 5 (C1 - C5) respectively. The blank control group (Control) adds DMEM medium containing 10% fetal bovine serum by volume. Detect the intracellular ROS level with a ROS detection kit at 72 h of culture. Digest the cells with trypsin and centrifuge, discard the supernatant, add PBS to wash, centrifuge and discard the supernatant. Add 500 μL of 5 μmol / L DCFH - DA solution to each tube, incubate at 37°C for 20 min, centrifuge at 1200 r / min for 5 min and then remove the supernatant. Wash the cells two or three times with serum - free DMEM, resuspend with PBS and detect on the machine. The results are expressed as fluorescence intensity values.

[0070] 1.5 Real - time fluorescence quantitative PCR to detect the expression of cell - related senescence genes: Take P45 human skin fibroblasts in good growth state, inoculate 1×10 6Cells were seeded in 100 mm culture dishes and cultured at 37 °C under 5% CO2. After the cells adhered, the medium was discarded, and the cells were washed with PBS. Conditioned media prepared in Examples 1-3 (T1-T3) and Comparative Examples 1-5 (C1-C5) were added respectively. The blank control group (Control) was added with DMEM medium containing 10% fetal bovine serum. After 72 h of culture, the cells were digested and centrifuged. RNAiso plus reagent was added to each well to extract total cellular RNA, and cDNA was obtained by reverse transcription for RT-PCR reaction. The PCR reaction system was completed according to the primer sequences and the PCR kit instructions. Using β-actin as an internal reference, the gene expression levels of each group were statistically analyzed by the 2 -ΔΔCt method.

[0071] Table 1 Primer sequence information

[0072]

[0073] 2. Statistical analysis: SPSS software was used for analysis. Independent-samples t-test was used for comparison between two independent samples, and one-way ANOVA was used for comparison between different groups. Measurement data were expressed as x±s, and P<0.05 was considered statistically significant.

[0074] 3. Results

[0075] 2.1 Comparison of cell proliferation ability in each group: The effects of different conditioned media on the proliferation activity of senescent HSF cells are shown in Figure 1 Figure A. From the results, we found that compared with the control group (100%), all conditioned media groups significantly stimulated the growth of HSF cells (P<0.05); among them, the epithalamin treatment group (C1) had the strongest stimulating effect on senescent HSF cells, followed by the hypotonic + epithalamin treatment groups (T1-T3), then the hypotonic + melatonin treatment group (C4), and the worst was the hypotonic treatment group (C2).

[0076] 2.2 Positive rate of SA-β-gal staining in each group of cells: The results of SA-β-gal staining are shown in Figure 1 Figure B. The number of blue-green senescent cells in the hypotonic + epithalamin treatment groups (T1-T3) was the least, and the positive rate of SA-β-gal staining was about 50%; followed by the epithalamin treatment group (C1) and the hypotonic + melatonin treatment group (C4), then the hypotonic + resveratrol treatment group (C5); there was no significant difference in the positive rate of SA-β-gal staining between the hypotonic treatment group (C2) and the hypotonic + ascorbic acid treatment group (C3) and the control group (P>0.05), and there was a significant difference compared with the T1 group.

[0077] 2.3 Comparison of intracellular reactive oxygen species levels in each group of cells: The results of intracellular reactive oxygen species levels in each group of cells are shown in Figure 2After culturing for 72 h, compared with the control group, the levels of intracellular reactive oxygen species (ROS) in senescent HSF cells were significantly decreased by all eight conditioned media. Among them, the lowest ROS levels were observed in HSF cells cultured with the conditioned media obtained from hypotonic + epithalon co-stimulation (T1-T3), followed by the hypotonic + melatonin treatment group (C4).

[0078] 2.4 Expression levels of cell-related senescence genes in each group: See Figure 2 B, compared with the control group, only in the hypotonic + epithalon treatment group (T1-T3) were the expression levels of P16 and P53 in HSF cells significantly decreased (P < 0.01). The expression levels of P16 and P53 in the hypotonic treatment group (C2) were significantly higher than those in the control group (P < 0.01). The expression level of P16 in the epithalon treatment group (C1) and the hypotonic + ascorbic acid treatment group (C3) was significantly lower than that in the control group (P < 0.05), but the expression level of P53 was significantly higher than that in the control group (P < 0.01). There was no significant difference in the expression level of P16 between the hypotonic + melatonin treatment group and the control group, but the expression level of P53 was significantly lower than that in the control group. The expression level of P16 in the hypotonic + resveratrol treatment group (C4) was significantly higher than that in the control group (P < 0.01), but there was no significant difference in the expression level of P53 compared with the control group.

[0079] In summary, the decrease in cell viability is an important manifestation of skin aging. All the above treatment groups can improve the viability of senescent cells, indicating that they can alleviate some characteristics of endogenous aging of skin fibroblasts. β-galactosidase is a marker enzyme of senescence, and its level increases with age. In the experiment, the hypotonic + epithalon treatment group, epithalon treatment group, hypotonic + melatonin treatment group, and hypotonic + resveratrol treatment group could all significantly reduce the percentage of SA-β-gal positive cells, indicating that the conditioned media obtained from the above treatment groups could significantly delay the senescence of fibroblasts. The conditioned media obtained from each treatment group could significantly reduce the intracellular content of reactive oxygen species. Among the expressions of related senescence genes, only in the hypotonic + epithalon treatment group (T1-T3) were the expression levels of P16 and P53 in HSF cells significantly lower than those in the control group (P < 0.01). The anti-apoptotic effect of the epithalon treatment group was stronger, but the other indicators were higher. Based on the comprehensive analysis of the experimental results, epithalon may enhance the viability of fibroblasts, but it cannot truly rejuvenate senescent cells. Hypotonic treatment can stimulate cell proliferation to a certain extent, but the expression levels of P16 and P53 in its cells are significantly higher than those in the control group, which may be the reaction of cells to hypotonic stimulation.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included within the protection scope of the present invention.

Claims

1. A mesenchymal stem cell exosome preparation for delaying aging, characterized in that, The exosome preparation is a conditioned medium obtained by culturing mesenchymal stem cells in a hypoosmotic environment.

2. The mesenchymal stem cell exosome preparation according to claim 1, wherein The hypoosmotic pressure refers to an osmotic pressure of 200 - 220 mosm / kg.

3. The mesenchymal stem cell exosome preparation according to claim 1 or 2, characterized in that, The mesenchymal stem cells are cultured in a hypoosmotic environment for 8 - 24 hours.

4. The mesenchymal stem cell exosome preparation according to claim 3, characterized in that, Epithalon is added for stimulation during the culturing of the mesenchymal stem cells in a hypoosmotic environment.

5. The mesenchymal stem cell exosome preparation according to claim 4, characterized in that, The concentration of Epithalon is 5 - 20 μmol / L.

6. The mesenchymal stem cell exosome preparation according to any one of claims 1 to 5, characterized in that, The mesenchymal stem cells are adipose stem cells.

7. Use of the mesenchymal stem cell exosome preparation according to any one of claims 1 - 6 in the preparation of an anti-aging cosmetic or drug.

8. A cosmetic for delaying skin aging, characterized in that, The cosmetic contains the mesenchymal stem cell exosome preparation according to any one of claims 1 - 6.

9. A drug for delaying skin aging, characterized in that, The drug contains the mesenchymal stem cell exosome preparation according to any one of claims 1 - 6.

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