Culture medium for improving Klotho protein secreted by mesenchymal stem cells and application of culture medium

Abstract

The invention provides a culture medium for improving Klotho protein secreted by mesenchymal stem cells and application of the culture medium, and particularly, the culture medium comprises a basic culture medium, statins and a ROCK inhibitor. The culture medium can effectively improve the Klotho protein secretion level of the mesenchymal stem cells, does not affect the biological activity of the mesenchymal stem cells, and ensures the biological function integrity of the mesenchymal stem cells; meanwhile, operation is easy, cost is low, and good clinical application value is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell engineering and biomedical technology, in particular, the present application relates to a culture medium for improving the secretion of Klotho protein by mesenchymal stem cells and its application, more particularly, the present application relates to a culture medium and its use, a method for improving the secretion of Klotho protein by mesenchymal stem cells in vitro, and a mesenchymal stem cell product and a preparation method thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSC) are a kind of adult stem cells with self-renewal, multi-directional differentiation potential and immune regulation function, which have various sources and can be widely obtained from bone marrow, adipose tissue, umbilical cord blood, umbilical cord and other tissues, and have broad application prospects in regenerative medicine, anti-aging and cell therapy for various diseases.

[0003] Klotho protein is a key anti-aging signal molecule, which is mainly secreted by the kidney, and studies have confirmed that MSC can also secrete a small amount of Klotho protein. This protein has multiple and important physiological functions, not only can be used as a co-receptor of fibroblast growth factor 23 (FGF23) to regulate calcium and phosphorus homeostasis, but also can reduce inflammation by inhibiting the NF-κB signaling pathway, regulate mitochondrial function, reduce the generation of active oxygen to inhibit oxidative stress, and play a role in neuroprotection, delaying cell aging and promoting tissue regeneration. A large number of studies have shown that the secretion level of Klotho protein is closely related to the occurrence and development of the body's aging process and various aging-related diseases such as osteoporosis, cardiovascular disease, and neurodegenerative disease. In MSC-mediated cell therapy, the Klotho protein secreted by MSC is one of the core paracrine molecules that play a therapeutic effect, which participates in the treatment process by strengthening anti-inflammatory, antioxidant and tissue repair functions, so improving the level of Klotho protein secreted by MSC can significantly enhance its therapeutic efficacy for related diseases.

[0004] At present, there are few methods for improving the secretion of Klotho protein by MSC, and there are problems such as poor effect, complex operation, high cost, or possible adverse effects on the biological activity of MSC. Therefore, developing a simple, efficient, low-cost and non-damaging MSC biological activity method for improving the secretion of Klotho protein by MSC is of great significance for promoting the application of MSC in regenerative medicine and disease treatment. SUMMARY

[0005] The present application is based on the discovery and understanding of the inventors of the following facts and problems:

[0006] Klotho protein is an important anti-aging protein, mainly secreted by the kidney and brain, and has multiple biological functions. When mesenchymal stem cells secrete Klotho, synergistic effects are produced, enhancing their repair and tissue protection capabilities. However, the level of Klotho protein secreted by MSCs themselves is low, which is a key factor limiting their therapeutic effect. Currently, the main technical methods for increasing the secretion of Klotho protein by MSCs are lentiviral transfection and vesicle delivery, etc. These methods are complex and costly to operate. Through a large amount of creative labor, the present application screens a culture medium, which can effectively increase the level of Klotho protein secreted by MSCs without damaging the biological activity of MSCs by adjusting the components and proportions of the culture medium.

[0007] Therefore, in the first aspect of the present application, the present application provides a culture medium. According to an embodiment of the present application, the culture medium comprises a basic culture medium, a statin and a ROCK inhibitor. The culture medium proposed by the present application can effectively increase the level of Klotho protein secreted by MSCs, and has simple operation, low cost and does not affect the integrity of the biological function of MSCs, and has good clinical application safety.

[0008] According to an embodiment of the present application, the statin is selected from at least one of atorvastatin, rosuvastatin, simvastatin or pravastatin.

[0009] According to an embodiment of the present application, the ROCK inhibitor is selected from at least one of Y27632, Fasudil, SR3677, GSK429286, TQ05105, AS1892802 or CAY10622.

[0010] According to an embodiment of the present application, the final concentration of the statin is 0.1 μmol / L~20 μmol / L; and / or the final concentration of the ROCK inhibitor is 5 μmol / L ~200 μmol / L.

[0011] According to an embodiment of the present application, the basic culture medium comprises at least one of DMEM culture medium DMEM / F12, MEM, alpha-MEM culture medium, RPMI-1640 culture medium or other commercial culture media for MSC culture.

[0012] According to an embodiment of the present application, the basic culture medium further comprises at least one of serum or serum substitute, and / or the final volume fraction of the serum or serum substitute is 2%~25%.

[0013] In a second aspect of the present application, the present application provides the use of the culture medium as described above in at least one of the following: (1) increasing the secretion of Klotho protein by mesenchymal stem cells; and (2) preparing a mesenchymal stem cell product containing Klotho protein.

[0014] In a third aspect of the present application, the present application provides a method for increasing the secretion of Klotho protein by mesenchymal stem cells in vitro. According to an embodiment of the present application, the method comprises: subjecting the mesenchymal stem cells to a first culture treatment in the culture medium as described above. The method according to the present application can efficiently increase the level of Klotho protein secreted by MSCs, and has the advantages of simple operation, low cost and no influence on the integrity of the biological function of MSCs, and good safety in clinical application.

[0015] In a fourth aspect of the present application, the present application provides a method for preparing a mesenchymal stem cell product. According to an embodiment of the present application, the method comprises: subjecting the mesenchymal stem cells to a first culture treatment in the culture medium as described above to obtain the mesenchymal stem cell product containing Klotho protein. The method according to the present application can significantly increase the level of Klotho protein secreted by MSCs by adding statins and ROCK inhibitors in the culture system of MSCs, and has the advantages of simple operation and low cost in clinical application.

[0016] According to an embodiment of the present application, the conditions of the first culture treatment are 32-40℃, 2-8% volume fraction of CO2 and saturated humidity; and / or the time of the first culture treatment is 10-28h.

[0017] In a fifth aspect of the present application, the present application provides a mesenchymal stem cell product. According to an embodiment of the present application, the mesenchymal stem cell product is prepared by the method as described above. The mesenchymal stem cell product according to the present application can secrete high levels of Klotho protein while maintaining good biological function integrity.

[0018] The present application has at least the following advantages: The culture medium according to the present application can effectively increase the level of Klotho protein secreted by mesenchymal stem cells while maintaining the biological activity, ensuring the integrity of the biological function of mesenchymal stem cells; and has the advantages of simple operation, low cost and good clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a statistical chart of the effect of different drugs on the level of Klotho protein secreted by MSC cells according to an embodiment of the present application.

[0020] Figure 2is a statistical graph of the level of Klotho protein secreted from MSC cells induced by atorvastatin according to an embodiment of the present application.

[0021] Figure 3 is a statistical graph of the level of Klotho protein secreted from MSC cells induced by Y27632 according to an embodiment of the present application.

[0022] Figure 4 is a statistical graph of the level of Klotho protein secreted from MSC cells induced by atorvastatin and Y27632 according to an embodiment of the present application.

[0023] Figure 5 is a statistical graph of the level of Klotho protein secreted from MSC cells induced by atorvastatin and Y27632 alone and in combination according to an embodiment of the present application.

[0024] Figure 6 is a statistical graph of the number of MSC cells expanded after atorvastatin and Y27632 alone and in combination according to an embodiment of the present application.

[0025] Figure 7 is a statistical graph of the level of Klotho protein secreted from a single MSC cell after atorvastatin and Y27632 alone and in combination according to an embodiment of the present application. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below with reference to the attached drawings. The embodiments described below are examples of the present application, and are not intended to limit the present application.

[0027] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximate descriptions of the ranges and are understood to be open-ended. Each range disclosed herein is to be understood to include individual values, and sub-ranges, falling within the stated range. In other words, any numerical range disclosed herein is intended to include all sub-ranges falling within the broader range, and every numerical value within that range, even if not explicitly stated.

[0028] In addition, the terms "first", "second", etc. are used herein only to describe the order of the features, and are not used to denote or imply relative importance or a number of the features. Thus, the features with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise explicitly specified.

[0029] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present document have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present application. Throughout this application, other terms of art are to be understood according to their general accepted meanings in the field of the present application, unless otherwise specifically defined herein. Throughout this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, to the extent that any definition or usage provided by the Dictionary of Cell and Molecular Biology is inconsistent with the definitions provided herein, the definitions provided above are controlling.

[0030] As used herein, the term "mesenchymal stem cell" or "MSC" is an adult stem cell present in various tissues, with the ability of self-renewal and multi-directional differentiation (differentiating into osteoblasts, chondrocytes, adipocytes or, under certain conditions, also trans-tissue differentiation into neural cells, hepatocytes, etc.). The mesenchymal stem cell has low immunogenicity and immunoregulatory function, and its therapeutic effect is not mainly dependent on direct differentiation replacement, but on improving the microenvironment of the injury site through secretion of factors (paracrine) and immunoregulation to activate endogenous repair. Compared with embryonic stem cells and induced pluripotent stem cells (iPSCs), it has low immunogenicity and strong immunoregulatory capacity, and low tumorigenic risk; compared with ordinary somatic cells, it also has strong repair and regulation capacity.

[0031] The technical solutions of the present application are described in detail as follows: Culture medium and use thereof In some embodiments of the present application, a culture medium is provided. According to an embodiment of the present application, the culture medium comprises a basal medium, a statin and a ROCK inhibitor. The culture medium provided by the present application can effectively improve the level of Klotho protein secreted by MSCs. By adding only two reagents, a statin and a ROCK inhibitor, to the conventional culture system of MSCs, the level of Klotho protein secreted by MSCs can be increased by 2-10 times, without the need for complex equipment and operation procedures. The statin and the ROCK inhibitor used are both common biological reagents, which are relatively inexpensive, thereby reducing the cost of experiments and clinical applications. Moreover, they do not affect the integrity of the biological functions of MSCs, and have good safety for clinical applications.

[0032] According to an embodiment of the present application, the statin is selected from at least one of atorvastatin, rosuvastatin, simvastatin or pravastatin. The statin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, which blocks the mevalonate pathway by inhibiting HMG-CoA reductase, thereby inhibiting the isoprenylation activation of small G protein RhoA, causing its inactivation. RhoA and its downstream signaling molecules have a negative regulatory effect on Klotho gene transcription, and its inactivation can relieve the inhibition of Klotho, leading to up-regulation of Klotho mRNA and protein expression.

[0033] According to embodiments of the present invention, the ROCK inhibitor is selected from at least one of Y27632, Fasudil, SR3677, GSK429286, TQ05105, AS1892802, or CAY10622. ROCK is a downstream effector molecule of RhoA; increased RhoA activity inhibits Klotho gene transcription. The ROCK inhibitor can inhibit ROCK activity, thereby inhibiting the RhoA / ROCK pathway, relieving epigenetic repression of the Klotho gene, and upregulating Klotho transcription and expression. The ROCK inhibitor and the statin drug work synergistically to inhibit the RhoA / ROCK signaling pathway, thereby significantly increasing the level of Klotho protein secreted by MSCs.

[0034] According to embodiments of the present invention, the final concentration of the statin drug is 0.1 μmol / L to 20 μmol / L. Exemplarily, the final concentration of the statin drug is 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, 10 μmol / L, 12 μmol / L, 14 μmol / L, 15 μmol / L, 16 μmol / L, 18 μmol / L, or 20 μmol / L, or a range between any two of the above values, such as 0.5 μmol / L to 18 μmol / L, 1 μmol / L to 15 μmol / L, or 1 μmol / L to 12 μmol / L. According to some preferred embodiments of the present invention, the final concentration of the statin drug is 0.5 μmol / L to 10 μmol / L.

[0035] According to embodiments of the present invention, the final concentration of the ROCK inhibitor is 5 μmol / L to 200 μmol / L. Exemplarily, the final concentration of the ROCK inhibitor is 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 30 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, 140 μmol / L, 150 μmol / L, 160 μmol / L, 180 μmol / L, or 200 μmol / L, or a range between any two of the above values, such as 5 μmol / L to 180 μmol / L, 10 μmol / L to 160 μmol / L, or 10 μmol / L to 120 μmol / L. According to some preferred embodiments of the present invention, the final concentration of the ROCK inhibitor is 10 μmol / L to 100 μmol / L.

[0036] According to embodiments of the present invention, the basal culture medium includes at least one of DMEM medium (DMEM / F12), MEM, α-MEM medium, RPMI-1640 medium, or other commercially available culture media for MSC culture. The basal culture medium provides the essential nutrients and physicochemical environment for the survival and proliferation of mesenchymal stem cells: maintaining osmotic pressure and pH balance, providing basic energy and carbon sources, and various vitamins and amino acids, etc.

[0037] According to embodiments of the present invention, the basal culture medium further comprises at least one of serum or serum substitutes. The serum or serum substitutes can provide abundant growth factors (such as PDGF, FGF, IGF, TGF-β, etc.), hormones, and lipids, promoting the survival and proliferation of mesenchymal stem cells, nutrient transport, and maintaining the multi-lineage differentiation potential and immunomodulatory function of mesenchymal stem cells.

[0038] According to embodiments of the present invention, the serum comprises at least one of fetal bovine serum (FBS) or human serum.

[0039] According to embodiments of the present invention, the serum substitute includes at least one of platelet lysis buffer or currently commercially available serum substitutes (e.g., Ultraser™ G, Ultraser' G Serum Substitute, or Helios' Ultra GRO™).

[0040] According to embodiments of the present invention, the final volume fraction of the serum or serum substitute is 2% to 25%. Exemplarily, the final volume fraction of the serum or serum substitute is 2%, 5%, 8%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, or 25%, or a range between any two of the above values. According to some preferred embodiments of the present invention, the final volume fraction of the serum or serum substitute is 5% to 20%.

[0041] It should be noted that the culture medium system proposed in this invention is also applicable to serum-free chemical culture media. By adding the statin drug and ROCK inhibitor described in this invention to the serum-free chemical culture medium system, the level of Klotho protein secreted by mesenchymal stem cells can also be increased.

[0042] In some embodiments of the present invention, the present invention proposes the use of the aforementioned culture medium in at least one of the following: (1) Increase the secretion of Klotho protein by mesenchymal stem cells; (2) Prepare mesenchymal stem cell products containing Klotho protein.

[0043] Methods and mesenchymal stem cell products In some embodiments of the present invention, a method for increasing the secretion of Klotho protein by mesenchymal stem cells (MSCs) in vitro is proposed. According to embodiments of the present invention, the method includes: performing a first culture treatment on the MSCs in the aforementioned culture medium. The method proposed in this invention can effectively increase the level of Klotho protein secretion by MSCs simply by adding two reagents—statins and ROCK inhibitors—to the conventional MSC culture system, without requiring complex equipment or procedures; the statins and ROCK inhibitors used are commonly used biological reagents, relatively inexpensive, which can reduce the cost of experimental and clinical applications; and it does not affect the integrity of the biological function of MSCs, possessing good safety for clinical application.

[0044] According to embodiments of the present invention, the mesenchymal stem cells are not limited in origin, including those isolated from at least one of the following tissues: bone marrow, adipose tissue, umbilical cord blood, and umbilical cord.

[0045] According to an embodiment of the present invention, before performing the first culture treatment, the mesenchymal stem cells are subjected to a second culture treatment in a basal culture medium; the cell fusion degree of the product of the second culture treatment is 50% to 70%, and exemplarily, the cell fusion degree of the product of the second culture treatment is 50%, 55%, 60%, 65% or 70%.

[0046] It should be noted that the method proposed in this invention for increasing the secretion of Klotho protein by mesenchymal stem cells in vitro is for non-disease treatment purposes.

[0047] In some embodiments of the present invention, a method for preparing mesenchymal stem cell products is provided. According to embodiments of the present invention, the method includes: performing a first culture treatment on the mesenchymal stem cells in the aforementioned culture medium to obtain the mesenchymal stem cell product, wherein the mesenchymal stem cell product contains Klotho protein. This method significantly increases the level of Klotho protein secreted by MSCs by adding statins and ROCK inhibitors to the MSC culture system; it is simple to operate and can reduce the cost of clinical application.

[0048] According to embodiments of the present invention, the mesenchymal stem cells are not limited in origin, including those isolated from at least one of the following tissues: bone marrow, adipose tissue, umbilical cord blood, and umbilical cord.

[0049] According to an embodiment of the present invention, before performing the first culture treatment, the mesenchymal stem cells are subjected to a second culture treatment in a basal culture medium; the cell fusion degree of the product of the second culture treatment is 50% to 70%, and exemplarily, the cell fusion degree of the product of the second culture treatment is 50%, 55%, 60%, 65% or 70%.

[0050] According to an embodiment of the present invention, the conditions for the first culture treatment are 32°C to 40°C, 2 to 8% CO2 by volume and saturated humidity. According to some preferred embodiments of the present invention, the conditions for the first culture treatment are 37°C, 5% CO2 by volume and saturated humidity.

[0051] According to an embodiment of the present invention, the first culture treatment time is 10 h to 28 h. For example, the first culture treatment time is 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h or 28 h, or a range between any two of the above values. According to some preferred embodiments of the present invention, the first culture treatment time is 12 h to 24 h.

[0052] In some embodiments of the present invention, a mesenchymal stem cell product is proposed. According to embodiments of the present invention, the mesenchymal stem cell product is prepared using the methods described above. The mesenchymal stem cell product proposed by the present invention can secrete high levels of Klotho protein while maintaining good biological functional integrity.

[0053] According to embodiments of the present invention, the mesenchymal stem cell product includes at least one of mesenchymal stem cell drugs, medical devices / tissue engineering products, immunomodulators, or cosmetic medical aesthetic products.

[0054] According to embodiments of the present invention, the mesenchymal stem cell drug is used to treat at least one of neurodegenerative diseases, chronic kidney disease, cardiovascular disease, osteoarthritis, aging-related syndromes and metabolic diseases, and fibrotic diseases.

[0055] According to embodiments of the present invention, the mesenchymal stem cell product may be in liquid, semi-solid, or solid form.

[0056] According to embodiments of the present invention, the dosage form of the product includes at least one of an injectable formulation, an oral formulation, or an inhaled formulation.

[0057] According to an embodiment of the present invention, the injectable formulation is an injection solution or a lyophilized powder for injection.

[0058] Embodiments of the present invention will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0059] Example 1 Screening of drug additives MSC Cell Resuscitation and Culture: MSC cells were isolated from previously preserved human umbilical cord tissue. The culture medium used was MSC-specific basal medium (purchased from Dakota Biotechnology), supplemented with 5% EliteGro-Adv (GMP) serum substitute (purchased from Dakota Biotechnology). First, the cryovial cap was clamped with hemostatic forceps and immersed in a 37°C water bath. The cap should remain above the water surface during immersion. The cryovial was gently agitated during immersion. When only rice-grain-sized ice crystals remained in the cryovial, it was immediately sprayed with 75% alcohol and wiped clean before being placed in a biosafety cabinet. The revived cells were aspirated using a 5 mL pipette and added to a 15 mL centrifuge tube containing 9 mL of complete culture medium. The tube was labeled and centrifuged at 300×g at room temperature for 5 min, discarding the supernatant. The cells were resuspended in an appropriate amount of complete culture medium, counted, and the cell concentration was adjusted to 3.8×10⁻⁶ cells / mL. 4 Cells / mL were seeded into 12-well culture plates, 1 mL per well, and incubated in an incubator at 37°C, 5% CO2, and saturated humidity.

[0060] Induction culture: First, dissolve the drugs atorvastatin (purchased from MCE), Y27632 (purchased from MCE), troglitazone (purchased from MCE), calcitriol (purchased from MCE), and berberine (TargetMol) in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5), and then dilute with culture medium to the appropriate concentration. When the cell confluence reached 50%–70%, the cells were divided into groups, with three replicate wells in each group. Based on the cells' response to each drug, different concentration gradients of various drugs were added (Atorvastatin, 0.01µM–1µM; Y27632, 5µM–20µM; Troglitazone, 5µM–50µM; Calcitriol, 5nM–200nM; Berberine, 1µM–20µM). The control group consisted of cells with an equal volume of culture medium replacing the drug additives. The cells were then placed in an incubator at 37°C, 5% CO2, and saturated humidity for further culture.

[0061] Klotho protein assay: When the cell confluence reached approximately 85-95%, the culture supernatant of each group of cells was collected, and the concentration of Klotho protein was detected using an ELISA kit (purchased from Delos Biotechnology Co., Ltd.). Refer to the ELISA kit instructions for specific methods.

[0062] This embodiment screened several drugs that can affect Klotho protein, including statins (atorvastatin), ROCK inhibitors (Y27632), thiazolidinediones (troglitazone), vitamins (calcitriol), and berberine (Coptis chinensis extract). Specific experimental results are attached. Figure 1 As shown, atorvastatin (Ator) and Y27632 significantly increased the secretion of Klotho protein in MSC cells, while troglitazone had no effect on Klotho protein secretion. Calcitriol and Berberine inhibited Klotho protein secretion in MSC cells. Therefore, the experimental results indicate that statins (atorvastatin) and the ROCK inhibitor (Y27632) are significantly more effective than other drug additives in increasing Klotho protein secretion in MSC cells.

[0063] Example 2: Effects of different concentrations of atorvastatin on Klotho protein secretion by MSC cells The MSC cell resuscitation and culture method is shown in Example 1.

[0064] Induction culture: Atorvastatin (purchased from MCE) was dissolved in DMSO and then diluted to different concentrations (0.5–10 μmol / L) with culture medium (the serum-added basal medium described in Example 1). When the cell confluence reached 50%–70%, the cells were divided into groups, with three replicate wells in each group. Different concentrations (0.5–10 μmol / L) of atorvastatin were added to each group. The control group consisted of cells with an equal volume of culture medium replacing the drug additive. The cells were then cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0065] Klotho protein assay: When the cell confluence reached approximately 85-95%, the culture supernatant of each group of cells was collected, and the concentration of Klotho protein was detected using an ELISA kit (purchased from Delos Biotechnology Co., Ltd.). Refer to the ELISA kit instructions for specific methods.

[0066] Specific experimental results are attached. Figure 2 As shown, atorvastatin can significantly increase the secretion level of Klotho protein in MSC cells. When atorvastatin is used alone, a final concentration of not less than 1 μM can significantly increase the level of Klotho protein secreted by MSC cells; the effect is best at a final concentration of 5 μM, which can increase the level of Klotho protein secreted by MSC cells by 4 times.

[0067] Example 3: Effects of different concentrations of Y27632 on the secretion of Klotho protein by MSC cells The MSC cell resuscitation and culture method is shown in Example 1.

[0068] Induction culture: Y27632 (purchased from MCE) was dissolved in DMSO and then diluted to a suitable concentration (10-80 μmol / L) with the medium described in Example 1. When the cell confluence reached approximately 50%-70%, the cells were divided into groups with three replicate wells in each group. Different concentrations (10-80 μmol / L) of Y27632 were added to each group. The control group consisted of cells with an equal volume of medium replacing the drug additive. The cells were then cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0069] Klotho protein assay: When the cell confluence reached approximately 85-95%, the culture supernatant of each group of cells was collected, and the concentration of Klotho protein was detected using an ELISA kit (purchased from Delos Biotechnology Co., Ltd.). Refer to the ELISA kit instructions for specific methods.

[0070] Specific experimental results are attached. Figure 3As shown, Y27632 (Y27) can significantly increase the secretion of Klotho protein in MSC cells. When Y27 is used alone, its increase in the secretion level of Klotho protein in MSC cells shows a certain dose-dependent effect. When the final concentration of Y27 is 80 μmol / L, it can significantly increase the secretion level of Klotho protein in MSC cells by 4 to 5 times.

[0071] Example 4: Effect of atorvastatin combined with Y27632 on Klotho protein secretion in MSC cells The MSC cell resuscitation and culture method is shown in Example 1.

[0072] Induction culture: Atorvastatin (purchased from MCE) and Y27632 (purchased from MCE) were dissolved in DMSO and diluted to suitable concentrations. The two drugs were then combined at different concentrations. When cell confluence reached approximately 50%–70%, the cells were grouped and the drugs were added according to the concentration: low concentration group L (Atorvastatin, 0.5 µM; Y27632, 10 µM), medium concentration group M (Atorvastatin, 1 µM; Y27632, 20 µM), high concentration group H (Atorvastatin, 5 µM; Y27632, 40 µM), and a control group (the control group received an equal volume of culture medium instead of the drug additive). Each group had three replicate wells. After drug addition, the cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0073] Klotho protein assay: When cell confluence reached approximately 85-95%, cells were counted to determine the average Klotho protein secretion per cell. The culture supernatant from each group of cells was collected, and the Klotho protein concentration was detected using an ELISA kit (purchased from Delos Biotechnology Co., Ltd.). Refer to the ELISA kit instructions for specific methods.

[0074] Specific experimental results are attached. Figure 4 As shown, atorvastatin combined with Y27632 can significantly increase the secretion of Klotho protein in MSC cells in a dose-dependent manner; in the high-dose group of the combined treatment, the secretion level of Klotho protein in MSC cells can be increased by about 6.5 times. (See attached image.) Figure 5 As shown, compared with the single-drug groups, the combined-drug group showed a significant increase in the secretion of Klotho protein in MSC cells. (See attached image.) Figure 6 As shown, compared to the control group, the total number of MSC cells expanded significantly in the single-drug group, while the total number of cells expanded in the combined-drug group did not change significantly, indicating that the two drug additives do not have an adverse effect on the growth and expansion of MSC cells and are safe. (See attached image)Figure 7 As shown, the average Klotho protein secretion per MSC cell in the drug-treated group was significantly increased compared to the control group, and this was even more pronounced in the combined drug-treated group. Moreover, the average Klotho protein secretion per MSC cell in the combined drug-treated group was significantly higher than that in the drug-treated group alone.

[0075] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0076] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a culture medium for increasing secretion of Klotho protein from mesenchymal stem cells, characterized in that, The culture medium is composed of a basal medium, atorvastatin and a ROCK inhibitor Y27632; The final concentration of the atorvastatin is 0.1 μmol / L~20 μmol / L; The final concentration of the ROCK inhibitor Y27632 is 5 μmol / L ~200 μmol / L.

2. Use according to claim 1, characterized in that, The basal medium comprises at least one of DMEM medium DMEM / F12, MEM, α-MEM medium or RPMI-1640 medium.

3. Use according to claim 1, characterized in that, The basal medium further comprises at least one of serum or serum substitute, and / or The final volume fraction of the serum or serum substitute is 2%~25%.

4. A method for increasing Klotho protein secretion from mesenchymal stem cells in vitro, characterized by, Comprising: The mesenchymal stem cells are subjected to a first culture treatment in the culture medium according to any one of claims 1~3.

5. A method of preparing a mesenchymal stem cell product, characterized in that, Comprising: The mesenchymal stem cells are subjected to a first culture treatment in the culture medium according to any one of claims 1~3 to obtain the mesenchymal stem cell product containing Klotho protein.

6. The method for increasing Klotho protein secretion from mesenchymal stem cells in vitro according to claim 4 or the method for preparing a mesenchymal stem cell product according to claim 5, wherein, The condition of the first culture treatment is 32℃~40℃, 2~8% volume fraction of CO2 and saturated humidity; and / or The time of the first culture treatment is 10 h~28 h.

7. A mesenchymal stem cell product, characterized in that, The mesenchymal stem cell product is obtained by the method according to claim 5 or 6.

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