Culture medium for improving secretion of klotho protein by mesenchymal stem cells and application thereof

Abstract

The application provides a culture medium for improving secretion of Klotho protein by mesenchymal stem cells and an application thereof, and particularly relates to the culture medium which comprises a basic culture medium, a statin and a ROCK inhibitor. The culture medium can effectively improve the level of the secretion of Klotho protein by the mesenchymal stem cells while not affecting the biological activity of the mesenchymal stem cells, and guarantees the biological function integrity of the mesenchymal stem cells. Meanwhile, the culture medium has simple operation, low cost and good clinical application value.

Description

Technical Field

[0001] This invention relates to the fields of cell engineering and biomedical technology. Specifically, this invention relates to a culture medium for enhancing the secretion of Klotho protein by mesenchymal stem cells and its application. More specifically, this invention relates to a culture medium and its uses, a method for enhancing the secretion of Klotho protein by mesenchymal stem cells in vitro, and mesenchymal stem cell products and their preparation methods. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal, multi-lineage differentiation potential and immunomodulatory functions. They have multiple sources and can be widely obtained from various tissues such as bone marrow, adipose tissue, umbilical cord blood, and umbilical cord. They have broad application prospects in regenerative medicine, anti-aging, and cell therapy for various diseases.

[0003] Klotho protein is a key anti-aging signaling molecule, primarily secreted by the kidneys, and studies have confirmed that MSCs also secrete small amounts of Klotho protein. This protein possesses diverse and important physiological functions. It not only acts as a co-receptor for fibroblast growth factor 23 (FGF23) to regulate calcium and phosphorus homeostasis, but also reduces inflammation by inhibiting the NF-κB signaling pathway, regulates mitochondrial function, reduces reactive oxygen species production to inhibit oxidative stress, and exerts neuroprotective effects, delays cellular senescence, and promotes tissue regeneration. Numerous studies have shown that the secretion level of Klotho protein is closely related to the aging process and the development of various age-related diseases such as osteoporosis, cardiovascular disease, and neurodegenerative diseases. In MSC-mediated cell therapy, the secreted Klotho protein is one of the core paracrine molecules that exert therapeutic effects, participating in the treatment process by enhancing anti-inflammatory, antioxidant, and tissue repair functions. Therefore, increasing the level of Klotho protein secreted by MSCs can significantly enhance its therapeutic efficacy against related diseases.

[0004] Currently, there are few techniques for enhancing Klotho protein secretion by MSCs, and those that exist suffer from problems such as poor efficacy, complex operation, high cost, or potential adverse effects on MSC bioactivity. Therefore, developing a simple, efficient, low-cost method to enhance Klotho protein secretion by MSCs without compromising their bioactivity is of great significance for promoting the application of MSCs in regenerative medicine and disease treatment. Summary of the Invention

[0005] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0006] Klotho protein is an important anti-aging protein, mainly secreted by the kidneys and brain, and has multiple biological functions. When mesenchymal stem cells (MSCs) secrete Klotho, a synergistic effect occurs, 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 efficacy. Currently, the main techniques for increasing Klotho protein secretion by MSCs are lentiviral transfection and vesicle delivery, which are complex and costly. This invention, through extensive creative work, has screened a culture medium that, by adjusting its components and ratios, can effectively increase the level of Klotho protein secreted by MSCs without impairing their biological activity.

[0007] Therefore, in a first aspect, the present invention provides a culture medium. According to embodiments of the present invention, the culture medium comprises: a basal culture medium, a statin drug, and a ROCK inhibitor. The culture medium proposed in this invention can effectively increase the level of Klotho protein secreted by MSCs, and is simple to operate, low in cost, and does not affect the integrity of the biological function of MSCs, thus possessing good safety for clinical application.

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

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

[0010] According to embodiments of the present invention, the final concentration of the statin drug is 0.1 μmol / L to 20 μmol / L; and / or the final concentration of the ROCK inhibitor is 5 μmol / L to 200 μmol / L.

[0011] 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.

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

[0013] In a second aspect of the invention, the invention proposes the use of the aforementioned culture medium in at least one of the following: (1) enhancing the secretion of Klotho protein by mesenchymal stem cells; (2) preparing mesenchymal stem cell products containing Klotho protein.

[0014] In a third aspect, the present invention provides a method for in vitro enhancement of Klotho protein secretion by mesenchymal stem cells (MSCs). According to an embodiment 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 efficiently enhance the level of Klotho protein secretion by MSCs, is simple to operate, low in cost, and does not affect the integrity of the biological function of MSCs, thus possessing good safety for clinical application.

[0015] In a fourth aspect, the present invention provides a method for preparing a mesenchymal stem cell product. According to an embodiment of the 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, the mesenchymal stem cell product containing 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.

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

[0017] In a fifth aspect, the present invention provides a mesenchymal stem cell product. According to an embodiment of the present invention, the mesenchymal stem cell product is prepared using the method 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.

[0018] The beneficial effects of this invention are at least as follows:

[0019] The culture medium proposed in this invention can effectively increase the level of Klotho protein secreted by mesenchymal stem cells without affecting their biological activity, thus ensuring the integrity of the biological function of mesenchymal stem cells. At the same time, it is simple to operate and low in cost, and has good clinical application value. Attached Figure Description

[0020] Figure 1 This is a statistical graph showing the effect of different drugs on the level of Klotho protein secreted by MSC cells according to embodiments of the present invention.

[0021] Figure 2 This is a statistical graph showing the level of Klotho protein secreted by MSC cells induced by atorvastatin according to an embodiment of the present invention.

[0022] Figure 3 This is a statistical graph showing the level of Klotho protein secreted by Y27632-induced MSC cells according to an embodiment of the present invention.

[0023] Figure 4 This is a statistical graph showing the level of Klotho protein secreted by MSC cells induced by the combined use of atorvastatin and Y27632 according to an embodiment of the present invention.

[0024] Figure 5 This is a statistical graph showing the levels of Klotho protein secreted by MSC cells induced by atorvastatin and Y27632 alone and in combination, according to embodiments of the present invention.

[0025] Figure 6 This is a statistical chart showing the number of MSC cells expanded after atorvastatin and Y27632 were used alone and in combination, according to embodiments of the present invention.

[0026] Figure 7 This is a statistical graph showing the level of Klotho protein secreted by a single MSC cell after atorvastatin and Y27632 were used alone and in combination, according to embodiments of the present invention. Detailed Implementation

[0027] Embodiments of the present invention are described in detail below, 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 present invention, and should not be construed as limiting the present invention.

[0028] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0030] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. In the description of this invention, the terms used herein have been explained and described; these explanations and descriptions are merely for the purpose of facilitating understanding and should not be construed as limiting the scope of protection of this invention.

[0031] In this article, the term "mesenchymal stem cells" or "MSCs" refers to adult stem cells found in various tissues, possessing self-renewal and multi-lineage differentiation capabilities (they can differentiate into osteoblasts, chondrocytes, adipocytes, or, under certain conditions, differentiate across germ layers into nerve cells, hepatocytes, etc.). These mesenchymal stem cells exhibit low immunogenicity and immunomodulatory functions. Their therapeutic efficacy does not primarily rely on direct differentiation and replacement, but rather on improving the microenvironment of the damaged site and activating endogenous repair through the secretion of factors (paracrine signaling) and immunomodulation. Compared to embryonic stem cells and induced pluripotent stem cells (iPSCs), they possess low immunogenicity and strong immunomodulatory capabilities, resulting in a low risk of tumorigenesis; compared to ordinary somatic cells, they exhibit powerful repair and regulatory capabilities.

[0032] The technical solution of this application is described in detail below:

[0033] Culture media and their uses

[0034] In some embodiments of the present invention, a culture medium is proposed. According to embodiments of the present invention, the culture medium comprises: a basal culture medium, statins, and a ROCK inhibitor. The culture medium proposed in this invention can effectively increase the level of Klotho protein secreted by MSCs. By simply adding statins and ROCK inhibitors to the conventional MSC culture system, the level of Klotho protein secreted by MSCs can be increased by 2 to 10 times, without the need for complex equipment and procedures. The statins and ROCK inhibitors used are commonly used biological reagents, relatively inexpensive, which can reduce the cost of experimental and clinical applications. Furthermore, it does not affect the integrity of the biological functions of MSCs and has good safety for clinical application.

[0035] According to embodiments of the present invention, the statin is selected from at least one of atorvastatin, rosuvastatin, simvastatin, or pravastatin. The statin is a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, which inhibits HMG-CoA reductase, blocks the mevalonate pathway, and thereby inhibits the isopreneization activation of the small G protein RhoA, thus inactivating it. RhoA and its downstream signaling molecules have a negative regulatory effect on Klotho gene transcription; its inactivation can relieve the inhibition of Klotho, leading to upregulation of Klotho mRNA and protein expression.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] According to embodiments of the present invention, the serum comprises at least one of fetal bovine serum (FBS) or human serum.

[0042] 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™).

[0043] 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%.

[0044] 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.

[0045] 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:

[0046] (1) Increase the secretion of Klotho protein by mesenchymal stem cells; (2) Prepare mesenchymal stem cell products containing Klotho protein.

[0047] Methods and mesenchymal stem cell products

[0048] 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.

[0049] 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.

[0050] 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%.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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%.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] According to embodiments of the present invention, the mesenchymal stem cell product may be in liquid, semi-solid, or solid form.

[0061] 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.

[0062] According to an embodiment of the present invention, the injectable formulation is an injection solution or a lyophilized powder for injection.

[0063] 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 present invention, and should not be construed as limiting the present invention.

[0064] Example 1 Screening of drug additives

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Example 2: Effects of different concentrations of atorvastatin on Klotho protein secretion by MSC cells

[0070] The MSC cell resuscitation and culture method is shown in Example 1.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] Example 3: Effects of different concentrations of Y27632 on the secretion of Klotho protein by MSC cells

[0075] The MSC cell resuscitation and culture method is shown in Example 1.

[0076] 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.

[0077] 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.

[0078] Specific experimental results are attached. Figure 3 As 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.

[0079] Example 4: Effect of atorvastatin combined with Y27632 on Klotho protein secretion in MSC cells

[0080] The MSC cell resuscitation and culture method is shown in Example 1.

[0081] 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.

[0082] 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.

[0083] 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 6As 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.

[0084] 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.

[0085] 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. The use of a culture medium in enhancing the secretion of Klotho protein by mesenchymal stem cells, characterized in that, The culture medium consists of the following substances: basal culture medium, atorvastatin, and ROCK inhibitor Y27632; The final concentration of atorvastatin is 0.5 μmol / L to 20 μmol / L; The final concentration of the ROCK inhibitor Y27632 is 10 μmol / L ~ 200 μmol / L.

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

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

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

5. A method for preparing mesenchymal stem cell products, characterized in that, include: The mesenchymal stem cells are subjected to a first culture treatment in the culture medium described in any one of claims 1 to 3 to obtain the mesenchymal stem cell product, wherein the mesenchymal stem cell product contains Klotho protein.

6. The method for increasing the secretion of Klotho protein by mesenchymal stem cells in vitro according to claim 4, or the method for preparing mesenchymal stem cell products according to claim 5, characterized in that, The conditions for the first culture treatment were 32℃~40℃, 2~8% CO2 (volume fraction), and saturated humidity; and / or The first culture treatment lasted for 10 h to 28 h.

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