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

Abstract

The present application belongs to the technical field of stem cell exosome preparation. More specifically, it relates to adipose-derived stem cells (ADSCs), conditioned medium (CM) thereof, and preparation method and use thereof in cosmetics and drugs. The present application provides a mesenchymal stem cell exosome preparation for delaying aging, wherein the exosome preparation is a conditioned medium obtained by culturing mesenchymal stem cells in a low-osmotic induction combined with epi-pen stimulation environment. Tests show that the conditioned medium can significantly enhance the proliferation and migration ability of senescent HSFs cells, reduce the expression of cell beta-galactosidase, reduce the expression of cell-related aging genes p16 and p53, and reduce the generation of intracellular ROS, and has a significant anti-aging effect.

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), methods for their preparation and use in cosmetics and pharmaceuticals. BACKGROUND

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

[0003] Adipose-derived mesenchymal stem cells are derived from adipose tissue and are a type of adult stem cell that has been extensively studied in the field of stem cells after bone marrow mesenchymal stem cells. They are abundant and easy to extract, making them ideal seed cells. Currently, Ding et al. [4] research has shown that human adipose-derived stem cells secrete hepatocyte growth factor and basic fibroblast growth factor, which can improve ovarian function during natural aging by activating the SIRT1 / FOXO1 signaling pathway. Cooper et al. [5]Human adipose-derived mesenchymal stem cell-conditioned medium was found to promote human skin fibroblast migration and ischemic wound healing. Exosomes regulate the biological functions of recipient cells by non-selectively releasing their bioactive components, such as microRNAs, mRNAs, and proteins, into recipient cells through binding with receptors. Therefore, the secretion amount and active components carried by exosomes will directly affect their functions. Recent studies have shown that, in addition to differences in MSC sources, different stimuli, such as drugs, cytokines, and hypoxia, have a significant impact on the secretion and function of exosomes. Appropriate interventions can not only increase the secretion of exosomes but also enrich the secretion of exosomes with certain specific components, such as drugs or cytokines, which can increase the yield and biological activity of exosomes. Shi et al. [6] It was found that 3,3'-biindolylmethane could increase the expression of Wnt11 protein in UCMSCs-Exos by activating the Wnt / β-catenin signaling pathway. Sung et al. [7] The effects of thrombin, hydrogen peroxide, and lipopolysaccharide treatment on MSCs-Exos secretion and wound healing treatment were compared. It was found that thrombin stimulation could significantly promote MSCs-Exos secretion and enrich it with VEGF and angiogenin and other growth factors; hypoxic treatment could change the active components carried by exosomes and enhance their efficacy. Wang et al. [8] It was found that hypoxia-induced ADSCs-Exos could 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] It was found that hypoxia-induced MSCs-Exos were rich in microRNA-210 and other microRNAs related to promoting angiogenesis, which could enhance their angiogenic effect. There is also evidence that hypoxia-induced ADSCs-Exos can improve the efficiency of angiogenesis by activating the PKA pathway

[10] In addition, Liu et al.

[11] found that hypoxia (1% O2) stimulated exosomes were rich in miR-216a-5p, which promoted 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. Normal physiological osmotic pressure is about 290-310 mosm / kg (isotonic). Low osmotic pressure (usually <250 mosm / kg) will directly affect the survival, proliferation, differentiation, and function of stem cells. Currently, there is no research on the effects of different osmotic pressures on the secretion and function of mesenchymal stem cell exosomes.

[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 Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing. Adv Wound Care (New Rochelle). 2018;7(9):299-308.

[0010] [6] Shi H, Xu X, Zhang B, et al. 3,3'-Diindolylmethane stimulates exosomal Wnt11 autocrine signaling in human umbilical cord mesenchymal stem cells to enhance wound healing [J]. Theranostics. 2017, 7(6): 1674-1688

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

[0012] [8] Wang J, Wu H, Peng Y, et al. Hypoxia adipose stem cell-derived exosomes promote high quality healing of diabetic wound involves activation of PI3K / 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 PKA Signaling 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 traumatic spinal cord injury by shifting microglial M1 / M2 polarization [J]. J Neuroinflammation. 2020, 17(1): 1-22. SUMMARY

[0016] The present inventors have found, through long-term and large-scale research, that the conditioned medium (CM) obtained by culturing adipose-derived stem cells (ADSCs) under the stimulation of epigallocatechin gallate combined with hypoosmotic pressure is rich in unique bioactive factors due to cell stress response and adaptive regulation, and through further research on the functional characteristics of the bioactive factors, it is found that the above-mentioned conditioned medium can significantly enhance the proliferation and migration ability of senescent HSFs cells, reduce the expression of cell beta-galactosidase, reduce the expression of cell-related senescence genes p16 and p53, and reduce the generation of intracellular ROS, thereby having a significant anti-aging effect.

[0017] The above-mentioned object of the present application is achieved by the following technical solutions.

[0018] The object of the present application is to provide a mesenchymal stem cell exosome preparation for delaying aging, wherein the exosome preparation is a conditioned medium obtained by culturing mesenchymal stem cells in a low-osmotic-pressure environment.

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

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

[0021] Osmotic pressure is an important physical parameter of cell microenvironment, normal physiological osmotic pressure is about 290-310mosm / kg (isotonic), and low osmotic pressure usually refers to < 250mosm / kg. The low osmotic pressure described in this paper refers to the osmotic pressure of 200-220mosm / kg; it is observed in the test that the osmotic pressure < 200mosm / kg causes many harms to the culture of mesenchymal stem cells, such as cell morphology and structure damage, proliferation and survival inhibition, functional property degradation, etc. And the conditioned medium obtained in the low osmotic environment of 220-250mosm / kg is not very satisfactory in various indicators in the aging HSFs cell model.

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

[0023] ①Primary dilution of DMEM medium

[0024] C1V1=C2V2, wherein 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] 310xV1=200xV2→V2=1.5V1, that is, 100mL of original DMEM is added with 50mL of ultrapure water.

[0026] ②Actual operation: take 100mL of standard DMEM, add 50mL of ultrapure water, mix well, and then measure the osmotic pressure with an osmometer, if it is too high, continue to add water, if it is too low, add a small amount of DMEM concentrate or NaCl; because the concentration of HCO3 - is reduced after dilution, the pH is easy to rise, so the pH can be adjusted to 7.2-7.4 with 1M HCl / NaOH, the diluted medium needs to be filtered with 0.22μm filter membrane to remove bacteria (avoid high pressure sterilization, which may change the osmotic pressure), and stored at 4℃ after packaging, and warmed to 37℃ before use.

[0027] In the present application, the mesenchymal stem cells are cultured in a low-osmotic pressure environment for 8-24 hours. The inventors have found that short-term low-osmotic culture is beneficial to the proliferation of adipose mesenchymal stem cells, and it is speculated that it may promote proliferation through the Hippo-YAP pathway (increased YAP nuclear translocation), but low-osmotic culture for >24 hours reduces the proliferation rate of adipose mesenchymal stem cells, increases apoptosis, and reduces the functionality of exosomes. More preferably, the mesenchymal stem cells are cultured in a low-osmotic pressure environment for 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours or 24 hours, and more preferably, the mesenchymal stem cells are cultured in a low-osmotic pressure environment for 10 hours or 15 hours.

[0028] Another innovative finding is that, under the stimulation of certain factors combined with low-osmotic induction, the most additive condition medium for anti-aging effect is obtained. Among these factors, the most prominent additive effect is epigallocatechin gallate, followed by melatonin, but it is worth mentioning that under the stimulation of certain factors, no or weak effect is observed. These factors currently identified include ascorbic acid, resveratrol, etc. Although epigallocatechin gallate, melatonin, ascorbic acid, resveratrol, etc. all have anti-aging effects, the stimulation effect on adipose stem cells is significantly different when combined with low-osmotic stimulation.

[0029] In the present application, the concentration of epigallocatechin gallate is 5-20 μmol / L, and more preferably, the concentration of epigallocatechin gallate 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 epigallocatechin gallate is 8 μmol / L, 12 μmol / L or 15 μmol / L.

[0030] In the present application, the mesenchymal stem cells can 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 stimulation effect of epigallocatechin gallate combined with low-osmotic stimulation on adipose mesenchymal stem cells is the strongest.

[0031] Another object of the present application is to provide the use of the mesenchymal stem cell exosome preparation in the preparation of a cosmetic or pharmaceutical product for delaying aging.

[0032] In the present application, the potential functional properties and mechanism analysis of the CM obtained by adipose stem cells induced by epigallocatechin gallate combined with low-osmotic stimulation to achieve additive anti-aging effect are as follows:

[0033] ①EpiPeptide combined with hypotonic stimulation can lead to more antioxidant enzymes, such as superoxide dismutase SOD2, glutathione peroxidase GSH-Px, etc. being released;

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

[0035] ③EpiPeptide combined with hypotonic stimulation delivers telomerase active ingredients (such as hTERT mRNA) through sEVs (exosomes);

[0036] ④EpiPeptide combined with hypotonic stimulation activates the Hippo-YAP pathway under stress, and promotes the secretion of VEGF, HGF, FGF-2 and other pro-angiogenic factors.

[0037] ⑤EpiPeptide combined with hypotonic stimulation promotes the secretion of anti-inflammatory factors such as IL-10 and IL-1RA, and inhibits TNF-α and IL-6; and regulates the polarization of macrophages to M2 type through the PGE2 / COX-2 pathway. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 A is the comparison of cell proliferation activity of each group;

[0039] Figure 1 B is the comparison of SA-β-gal staining positive rate of cells in each group;

[0040] Figure 2 A is the comparison of intracellular reactive oxygen species level of each group;

[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 is the comparison of relative expression amount of P16 and P53 genes of cells in each group;

[0043] Among them, compared with the P16 expression amount of the Control group cells, * P<0.05, ** P<0.01; compared with the P53 expression amount of the Control group cells, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0044] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

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

[0046] Example 1: Preparation of ADSCs-CM

[0047] ① Preparation of DMEM culture medium with an osmotic pressure of 200 mosm / kg: Take Gibco TM DMEM low-glucose medium (catalog number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure of 305 mosm / kg); take 100 ml of DMEM low-glucose medium (catalog number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure of 305 mosm / kg); TM In DMEM low-glucose medium, 52.5 mL of ultrapure water was added and mixed thoroughly. The osmotic pressure at this point was approximately 200 mosm / kg. HEPES (25 mM) was added to stabilize the pH to 7.2–7.4. 8 μmol / L epipeptide was added, and the mixture was filtered through a 0.22 μm filter for sterilization. The final product osmotic pressure was confirmed to be 200 ± 5 mosm / kg using a calibrated osmoremeter. After short-term culture of adipose-derived mesenchymal stem cells for 24 hours, the cell viability was measured to be >90%, and the cells showed normal morphology. The cells were then aliquoted and stored at 4°C as hypotonic medium for later use.

[0048] ② Take third-generation human adipose-derived mesenchymal stem cells in good growth condition, at a dose of 1×10⁻⁶. 6 One cell was seeded in a 100 mm culture dish (containing complete culture medium for human adipose-derived mesenchymal stem cells). When the cell confluence reached about 80%, the dish was discarded and replaced with hypotonic culture medium. After culturing in a 37°C, 5% CO2 incubator for 10 h, the supernatant was collected and centrifuged at 3000 r / min for 15 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain ADSCs-CM, which was stored at 4°C for later use.

[0049] Example 2: Preparation of ADSCs-CM

[0050] ① Preparation of DMEM culture medium with an osmotic pressure of 210 mosm / kg: Take Gibco TM DMEM low-glucose medium (catalog number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure of 305 mosm / kg); take 100 ml of DMEM low-glucose medium (catalog number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure of 305 mosm / kg); TMDMEM low glucose medium, add 45 mL ultrapure water, mix well, the osmotic pressure is about 210 mosm / kg at this time, add HEPES (25 mM) to stabilize pH to 7.2-7.4, add 12 μmol / L epirubicin, mix well, filter sterilization with 0.22 μm filter membrane, use calibrated osmometer to confirm that the final product osmotic pressure is 210±5 mosm / kg. After short-term culture of adipose-derived mesenchymal stem cells for 24 h, the survival rate is >90%, and the cell morphology is normal, then store at 4℃ after sub-packaging, and label as low osmotic medium for standby.

[0051] ②Take the well-grown third generation human adipose-derived mesenchymal stem cells, inoculate 1×10 6 cells in a 100 mm culture dish (containing human adipose-derived mesenchymal stem cell complete culture medium), when the cell confluence reaches about 80%, discard and replace with low osmotic medium, culture in a 37℃, 5% CO2 incubator for 12 h, then collect the culture supernatant, centrifuge at 3000 r / min for 15 min, filter the centrifuged supernatant with 0.22 μm filter membrane to obtain ADSCs-CM, store at 4℃ for standby.

[0052] Example 3, preparation of ADSCs-CM

[0053] ①DMEM medium with osmotic pressure of 220 mosm / kg: take Gibco DMEM low glucose medium (item number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure is 305 mosm / kg); take 100 mL Gibco DMEM low glucose medium, add 38.6 mL ultrapure water, mix well, the osmotic pressure is about 220 mosm / kg at this time, add HEPES (25 mM) to stabilize pH to 7.2-7.4, add 15 μmol / L epirubicin, mix well, filter sterilization with 0.22 μm filter membrane, use calibrated osmometer to confirm that the final product is 220±5 mosm / kg. After short-term culture of adipose-derived mesenchymal stem cells for 24 h, the survival rate is >90%, and the cell morphology is normal, then store at 4℃ after sub-packaging, and label as low osmotic medium for standby. TM DMEM low glucose medium (item number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure is 305 mosm / kg); take 100 mL Gibco DMEM low glucose medium, add 38.6 mL ultrapure water, mix well, the osmotic pressure is about 220 mosm / kg at this time, add HEPES (25 mM) to stabilize pH to 7.2-7.4, add 15 μmol / L epirubicin, mix well, filter sterilization with 0.22 μm filter membrane, use calibrated osmometer to confirm that the final product is 220±5 mosm / kg. After short-term culture of adipose-derived mesenchymal stem cells for 24 h, the survival rate is >90%, and the cell morphology is normal, then store at 4℃ after sub-packaging, and label as low osmotic medium for standby. TM DMEM low glucose medium (item number: 11885084, containing 1 g / L glucose, initial standard osmotic pressure is 305 mosm / kg); take 100 mL Gibco DMEM low glucose medium, add 38.6 mL ultrapure water, mix well, the osmotic pressure is about 220 mosm / kg at this time, add HEPES (25 mM) to stabilize pH to 7.2-7.4, add 15 μmol / L epirubicin, mix well, filter sterilization with 0.22 μm filter membrane, use calibrated osmometer to confirm that the final product is 220±5 mosm / kg. After short-term culture of adipose-derived mesenchymal stem cells for 24 h, the survival rate is >90%, and the cell morphology is normal, then store at 4℃ after sub-packaging, and label as low osmotic medium for standby.

[0054] ②Take the well-grown third generation human adipose-derived mesenchymal stem cells, inoculate 1×10 6 cells in a 100 mm culture dish (containing human adipose-derived mesenchymal stem cell complete culture medium), when the cell confluence reaches about 80%, discard and replace with low osmotic medium, culture in a 37℃, 5% CO2 incubator for 15 h, then collect the culture supernatant, centrifuge at 3000 r / min for 15 min, filter the centrifuged supernatant with 0.22 μm filter membrane to obtain ADSCs-CM, store at 4℃ for standby.

[0055] Comparative Example 1

[0056] The difference from Example 1 is that normal osmotic pressure culture is adopted, i.e. the culture medium is Gibco DMEM / F12 (1:1) medium containing 1 g / L glucose, and the initial standard osmotic pressure is 305 mosm / kg. TM DMEM low-sugar culture medium (item number: 11885084, containing 1 g / L glucose, and the initial standard osmotic pressure is 305 mosm / kg).

[0057] Comparative Example 2

[0058] The difference from Example 1 is that epigallocatechin 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 instead of epigallocatechin, and the other parameters refer to Example 1.

[0061] Comparative Example 4

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

[0063] Comparative Example 5

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

[0065] Test Example 1, Comparison of Effects of Conditioned Media from Different Sources on Aging Human Skin Fibroblasts

[0066] 1.1 Aging human skin fibroblast culture: Human skin fibroblasts were purchased from Shanghai Cell Bank, and the culture medium was composed of 1% double antibody, 10% fetal bovine serum by volume, and 89% DMEM / F12. The cells were subcultured to naturally aged cells for experiments (subcultured to more than 45 generations).

[0067] 1.2 CCK-8 method for detecting cell proliferation activity: 1×10 5

[0068] ​1.3 β-galactosidase staining: P45 generation human skin fibroblasts in good growth state were inoculated in 96-well plates at 1x10 4 cells per well, and cultured at 37°C in a 5% CO2 atmosphere for 24 h. The culture medium was discarded, and the cells were washed with PBS. The conditioned medium prepared in Examples 1 to 3 (T1 to T3) and Comparative Examples 1 to 5 (C1 to C5) was 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 stained with a β-galactosidase staining kit. The cell culture medium was removed, and the cells were washed once with PBS. An appropriate amount of SA-β-gal staining and fixing solution was added, and the cells were fixed at room temperature for 15 min. The fixing solution was removed, and the cells were washed with PBS for 3 min each time. The PBS was removed, and an appropriate amount of cell staining working solution was added. The cells were incubated at 37°C overnight. The senescent cells were observed under an optical microscope to be light blue to dark blue. The positive rate of the sample was expressed as a percentage of the number of positive samples to the total number of samples.

[0069] 1.4 Intracellular reactive oxygen species level: P45 generation human skin fibroblasts in good growth state were inoculated in 96-well plates at 1x10 5 cells per well, and cultured at 37°C. After the cells adhered to the wall, the culture medium was discarded, and the cells were washed with PBS. The conditioned medium prepared in Examples 1 to 3 (T1 to T3) and Comparative Examples 1 to 5 (C1 to C5) was added, respectively. The blank control group (Control) was added with DMEM medium containing 10% fetal bovine serum. After 72 h of culture, the intracellular reactive oxygen species level was detected with a reactive oxygen species detection kit. The cells were trypsinized, centrifuged, and the supernatant was discarded. The cells were washed with PBS, centrifuged, and the supernatant was discarded. 500 μL of 5 μmol / L DCFH-DA solution was added to each tube, and the cells were incubated at 37°C for 20 min. After centrifugation at 1200 r / min for 5 min, the supernatant was removed, and the cells were washed with serum-free DMEM for 2 to 3 times. The cells were resuspended with PBS and detected on a machine. The results were expressed as fluorescence intensity values.

[0070] 1.5 Real-time fluorescent quantitative PCR detection of cell-related senescence gene expression: P45 generation human skin fibroblasts in good growth state were inoculated in 96-well plates at 1x10 6Cells were inoculated in 100mm culture dish, cultured at 37℃, 5% CO2, after cell adhered, discarded the culture medium, washed with PBS, added the conditioned medium prepared by example 1~3 (T1~T3) and comparative example 1~5 (C1~C5), the blank control group (Control) added DMEM medium containing 10% fetal bovine serum, after 72h, centrifuged after digestion, added RNAiso plus reagent to extract total RNA, obtained cDNA by reverse transcription, and carried out RT-PCR reaction. According to the primer sequence and PCR kit instruction, completed the PCR reaction system, with β-actin as internal reference, through 2 -ΔΔCt Method, the gene expression of each group was statistically analyzed.

[0071] Table 1 primer sequence information

[0072]

[0073] 2. Statistical analysis: SPSS software was used for analysis, two independent sample comparison used independent sample t test, different group comparison used single factor analysis of variance, measurement data was expressed by x±s, P<0.05 was significantly different.

[0074] 3. Results

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

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

[0077] 2.3 Comparison of intracellular reactive oxygen species level of each group of cells: the intracellular reactive oxygen species level of each group of cells was shown in Figure 2A, after 72h culture, compared with the control group, the eight kinds of conditioned medium can significantly reduce the level of ROS in the aging HSF cells; among them, the ROS level of HSF cells cultured by the conditioned medium obtained by low osmotic pressure + epipelet combined stimulation (T1~T3) is the lowest, followed by low osmotic pressure + melatonin treatment group (C4).

[0078] 2.4 The expression of related aging genes in each group of cells: see Figure 2 B, compared with the control group, only the P16 and P53 expression levels in the HSF cells of the low osmotic pressure + epipelet treatment group (T1~T3) were significantly reduced (P<0.01); the P16 and P53 expression levels in the cells of the low osmotic pressure treatment group (C2) were significantly higher than those in the control group (P<0.01); the P16 expression level in the cells of the epipelet treatment group (C1) and the low osmotic pressure + ascorbic acid treatment group (C3) was significantly lower than that in the control group (P<0.05), but the P53 expression level was significantly higher than that in the control group (P<0.01); the P16 expression level in the cells of the low osmotic pressure + melatonin treatment group was not significantly different from that in the control group, but the P53 expression level was significantly lower than that in the control group; the P16 expression level in the cells of the low osmotic pressure + resveratrol treatment group (C4) was significantly higher than that in the control group (P<0.01), but the P53 expression level was not significantly different from that in the control group.

[0079] In summary, the decrease in cell viability is an important manifestation of skin aging, and each of the above treatment groups can improve the viability of aging cells, showing that they can alleviate some characteristics of endogenous aging of skin fibroblasts. β-galactosidase is a marker enzyme for aging, which increases with age. In the experiment, the low osmotic pressure + epipelet treatment group, the epipelet treatment group, the low osmotic pressure + melatonin treatment group and the low osmotic pressure + resveratrol treatment group can significantly reduce the percentage of SA-β-gal positive cells, indicating that the conditioned medium obtained by the above treatment groups can significantly delay the aging of fibroblasts; the conditioned medium obtained by each treatment group can significantly reduce the content of intracellular reactive oxygen species; among the expression of related aging genes, only the P16 and P53 expression levels in the HSF cells of the low osmotic pressure + epipelet treatment group (T1~T3) were significantly lower than those in the control group (P<0.01); the anti-apoptotic effect of the epipelet treatment group is stronger, but the other indicators are higher. Comprehensive analysis of the experimental results, epipelet may be able to enhance the viability of fibroblasts, but it cannot really rejuvenate aging cells, and low osmotic pressure can stimulate cell proliferation to a certain extent, but the P16 and P53 expression levels in the cells are significantly higher than those in the control group, which may be the response of the cells to low osmotic pressure stimulation.

[0080] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A mesenchymal stem cell exosome preparation for delaying aging, characterized in that, The exosome preparation is a conditioned medium obtained by culturing adipose-derived mesenchymal stem cells in a medium containing 5-20 μmol / L epipeptide with an osmotic pressure of 200-220 mosm / kg for 8-24 hours; the conditioned medium refers to a medium containing bioactive substances secreted or released by the mesenchymal stem cells after culturing them in a medium for a period of time.

2. The use of the mesenchymal stem cell exosome preparation according to claim 1 in the preparation of cosmetics or pharmaceuticals for delaying aging.

3. A cosmetic product for delaying skin aging, characterized in that, The cosmetic product comprises the mesenchymal stem cell exosome preparation according to claim 1.

4. A drug for delaying skin aging, characterized in that, The drug comprises the mesenchymal stem cell exosome preparation according to claim 1.

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