Preparation and method for preventing replicative aging of mesenchymal stem cells

Abstract

The invention belongs to the technical field of anti-aging of mesenchymal stem cells, and particularly relates to a preparation and method for preventing replicative aging of mesenchymal stem cells, the preparation comprises salidroside, semen cuscutae total flavonoids and ergothioneine, and the method comprises the following steps: in the in-vitro amplification process of the mesenchymal stem cells, culturing under the hypoxia condition of 2-5% O2, the preparation for preventing the replicative aging of the mesenchymal stem cells has the advantages that the combination of effective components of Chinese herbal medicines is combined with a low-oxygen condition, the replicative aging of the mesenchymal stem cells is prevented, and the preparation is safer, more efficient, low in cost and capable of realizing large-scale amplification.

Description

Technical Field

[0001] This invention belongs to the field of mesenchymal stem cell anti-aging technology, specifically relating to a preparation and method for preventing the replicative aging of mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are core seed cells in regenerative medicine due to their multi-directional differentiation potential, immunomodulatory capacity, and tissue repair function. However, during in vitro expansion, MSCs undergo replicative senescence, manifested as: (1) decreased proliferation capacity (cell cycle arrest at G0 / G1 phase); (2) loss of stem cell markers (such as CD90 and CD105); (3) increased oxidative stress (accumulation of ROS and MDA); and (4) increased positivity rate of aging-related β-galactosidase (SA-β-Gal). These changes seriously affect the clinical application efficacy of MSCs.

[0003] Existing technologies for preventing MSC aging mainly include: (1) chemical molecules (such as resveratrol and curcumin): inhibiting aging by activating antioxidant pathways, but with the risk of cytotoxicity; (2) cytokines (such as IGF-1 and bFGF): promoting proliferation, but with high cost, easy degradation, and difficult to apply on a large scale.

[0004] Therefore, there is an urgent need to develop a safe, effective, and low-cost method to prevent the replicative aging of MSCs and to address the shortcomings of existing technologies. Summary of the Invention

[0005] To address the issues of high toxicity of chemical molecules and high cost of cytokines in existing technologies, this invention provides a formulation and method for preventing the replicative aging of mesenchymal stem cells (MSCs). By combining the effective components of traditional Chinese medicine with hypoxic conditions, the invention aims to achieve safe, efficient, and large-scale expansion of MSCs.

[0006] The technical problem solved by this invention is achieved by the following technical solution:

[0007] The purpose of this invention is to provide a formulation for preventing the replicative aging of mesenchymal stem cells, comprising rhodioloside, total flavonoids from Cuscuta chinensis, and ergothioneine.

[0008] Furthermore, the concentrations of rhodioloside were 10-20 μM, total flavonoids from Cuscuta were 20-40 μM, and ergothioneine was 5-10 μM.

[0009] Furthermore, the concentration of rhodioloside was 10 μM, the concentration of total flavonoids from Cuscuta was 20 μM, and the concentration of ergothioneine was 5 μM.

[0010] A method for preventing the replicative senescence of mesenchymal stem cells, wherein during the in vitro expansion of mesenchymal stem cells, the cells are cultured under hypoxic conditions of 2-5% O2, and the culture medium is supplemented with an agent for preventing the replicative senescence of mesenchymal stem cells as described in any one of claims 1-3.

[0011] Furthermore, the low-oxygen condition is 3% O2.

[0012] Furthermore, DMEM medium containing 10% FBS and 1% double antibiotics was used for continuous treatment starting from the 3rd generation of MSCs, with the medium changed every 2 days. Rhodioloside, Cuscuta flavonoids and ergothioneine were added during the medium change, and the cells were passaged to the 15th generation.

[0013] Rhodioloside: The main active ingredient of Rhodiola rosea, it increases the expression of superoxide dismutase (SOD) and catalase (CAT) by activating the SIRT1 pathway, reduces the accumulation of malondialdehyde (MDA), and inhibits oxidative stress.

[0014] Total flavonoids from Cuscuta chinensis: The main active component of Cuscuta chinensis, which promotes MSC self-renewal and maintains the expression of stem cell markers such as CD90 and CD105 by regulating the Wnt / β-catenin pathway;

[0015] Ergothioneine: A powerful natural antioxidant that reduces oxidative damage and slows down cell aging by scavenging ROS;

[0016] Hypoxia conditions: By activating the HIF-1α pathway, the expression of glycolysis-related enzymes (such as lactate dehydrogenase LDH and glucose transporter GLUT1) is increased, maintaining the low metabolic state of MSCs and enhancing stem cell characteristics.

[0017] The synergistic effect of the above four components significantly delays the replicative senescence of MSCs. In the formulation of this invention, rhodioloside, total flavonoids from Cuscuta chinensis, and ergothioneine are the effective components of the combined traditional Chinese medicine. Their mechanism of action is as follows: rhodioloside activates the SIRT1 pathway to inhibit oxidative stress; total flavonoids from Cuscuta chinensis regulate the Wnt / β-catenin pathway to promote MSC self-renewal; ergothioneine scavenges reactive oxygen species (ROS) to reduce oxidative damage; and hypoxia maintains MSC stem cell characteristics through the HIF-1α pathway. The formulation may also include a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0019] 1. Safety: The active ingredients of traditional Chinese medicine are natural and non-cytotoxic (experimental verification: cell survival rate of the combination group ≥95%);

[0020] 2. Effectiveness: The anti-aging effect of the combination group is significantly better than that of the single component or hypoxia treatment, with obvious synergistic effect;

[0021] 3. Low cost: Chinese herbal medicine raw materials are abundant, and the cost is lower than that of cytokines (about 1 / 10 of IGF-1);

[0022] 4. Easy to scale up: No special equipment is required (the low-oxygen incubator is conventional equipment), making it suitable for industrial production.

[0023] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0024] Figure 1 This is a comparison chart of the positive rates of SA-β-Gal in the blank control group, hypoxia group, rhodioloside group, total flavonoids of Cuscuta chinensis group, ergothionein group and combination group in Experiment Example 1 of the present invention.

[0025] Figure 2 This is a comparison chart of cell proliferation rates among the blank control group, hypoxia group, rhodioloside group, total flavonoids of Cuscuta chinensis group, ergothionein group, and combination group in Experiment Example 1 of this invention.

[0026] Figure 3 This is a comparison chart of ROS levels in the blank control group, hypoxia group, rhodioloside group, total flavonoids of Cuscuta chinensis group, ergothionein group and combination group of Experiment Example 1 of the present invention. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0028] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0029] Example 1

[0030] Preparation of the anti-aging agent: Rhodioloside (10 μM), total flavonoids from Cuscuta chinensis (20 μM), and ergothioneine (5 μM) were dissolved in DMEM medium to prepare a liquid additive for MSC culture. This agent is stable (its activity remains unchanged after 6 months of storage at 4°C), easily mixes with conventional culture media, and is suitable for industrial applications.

[0031] Example 2

[0032] Preparation of the anti-aging agent: Rhodioloside (15 μM), total flavonoids from Cuscuta chinensis (30 μM), and ergothioneine (8 μM) were dissolved in DMEM medium to prepare a liquid additive for MSC culture. This agent is stable (its activity remains unchanged after 6 months of storage at 4°C), easily mixes with conventional culture media, and is suitable for industrial applications.

[0033] Example 3

[0034] Preparation of the anti-aging agent: Rhodioloside (20 μM), total flavonoids from Cuscuta chinensis (40 μM), and ergothioneine (10 μM) were dissolved in DMEM medium to prepare a liquid additive for MSC culture. This agent is stable (its activity remains unchanged after 6 months of storage at 4°C), easily mixes with conventional culture media, and is suitable for industrial applications.

[0035] Experimental Example 1

[0036] 1. Experimental materials

[0037] Cells: Human bone marrow mesenchymal stem cells (BM-MSC);

[0038] Reagents: Rhodioloside (purity ≥98%, Sigma), Cuscuta chinensis total flavonoids (purity ≥80%, Nanjing Zelang), Ergothioneine (purity ≥99%, Sigma); DMEM medium (Gibco), fetal bovine serum (FBS, Gibco), SA-β-Gal staining kit (Cell Signaling), CCK-8 kit (Dojindo), flow cytometry antibodies (CD90-PE, CD105-FITC, BD);

[0039] Equipment: Hypoxia incubator (Thermo, 3% O2), flow cytometer (BD FACSCanto), microplate reader (Tecan), Western blot system (Bio-Rad).

[0040] 2. Concentration gradient optimization

[0041] To determine the optimal component concentrations, concentration gradients of rhodioloside (5, 10, 20 μM), total flavonoids from Cuscuta chinensis (10, 20, 40 μM), and ergothionein (2.5, 5, 10 μM) were established, and the SA-β-Gal positivity rate and cell proliferation rate (CCK-8) were detected at the 10th generation.

[0042] Concentration gradient optimization results

[0043] The combination of 10 μM rhodioloside, 20 μM total flavonoids from Cuscuta chinensis, and 5 μM ergothioneine showed the lowest SA-β-Gal positivity rate (18%) and the highest cell proliferation rate (OD) in the 10th generation. 450=1.2), which is significantly better than other concentration combinations (P<0.05), and is determined to be the optimal concentration.

[0044] 3. Experimental Grouping

[0045] The third-generation MSCs were divided into 4 groups (n=3):

[0046] Blank control group (Ctrl): conventional oxygen (21% O2) culture, without the addition of any components;

[0047] Hypoxia group: cultured in 3% O2 ​​without any added ingredients;

[0048] Single component groups: conventional oxygen culture, with the addition of rhodioloside (10 μM, Sali group), total flavonoids from Cuscuta chinensis (20 μM, TFCC group), and ergothionein (5 μM, Ergo group);

[0049] Combo: Cultured in 3% O2, supplemented with rhodioloside (10 μM) + total flavonoids from Cuscuta chinensis (20 μM) + ergothioneine (5 μM).

[0050] 4. Experimental Methods

[0051] Third-generation human bone marrow mesenchymal stem cells (BM-MSCs) were harvested and seeded in a 10cm culture dish (1×10⁻⁶ cells / mL). 6 Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics (cells / plate). The combination group and hypoxia group were cultured in a hypoxia incubator at 3% O2, while the blank control group and single-component groups were cultured under standard oxygen (21% O2). The medium was changed every two days. For the combination group, rhodioloside (10 μM), total flavonoids from Cuscuta chinensis (20 μM), and ergothioneine (5 μM) were added during medium changes. For the single-component groups, rhodioloside (10 μM, Sali group), total flavonoids from Cuscuta chinensis (20 μM, TFCC group), and ergothioneine (5 μM, Ergo group) were added respectively. Cells were passaged to the 15th generation, and various indicators were measured.

[0052] 5. Testing Indicators

[0053] Aging markers: SA-β-Gal staining (counting the proportion of positive cells);

[0054] Proliferation capacity: CCK-8 assay (detection of absorbance at 450 nm);

[0055] Stem cell characteristics: CD90 and CD105 expression detected by flow cytometry;

[0056] Oxidative stress: ROS levels were detected by DCFH-DA probe (fluorescence intensity, AU), and MDA content (nmol / mg protein) was detected by MDA kit;

[0057] Pathway proteins: Western blot was used to detect the expression of SIRT1, Wnt3a, and HIF-1α (with β-actin as an internal reference, and the relative expression levels were calculated).

[0058] 6. Experimental Results

[0059] 6.1 Comparison of aging indicators among groups (15th generation) is shown in Table 1 below. All indicators of the Combo group were significantly better than those of other groups (P<0.01), indicating that hypoxia + the combined herbal ingredients have a synergistic anti-aging effect.

[0060] Table 1

[0061]

[0062] 6.2 Pathway protein expression (15th generation) 30

[0063] The expression levels of SIRT1 (1.5-fold), Wnt3a (1.8-fold), and HIF-1α (2.0-fold) in the combination group were significantly higher than those in other groups (P<0.01), which verifies the mechanism of action of the present invention.

[0064] Experiment Example 2

[0065] 1. Experimental materials

[0066] Cells: Human umbilical cord mesenchymal stem cells (UC-MSC);

[0067] Reagents: Rhodioloside (purity ≥98%, Sigma), Cuscuta chinensis total flavonoids (purity ≥80%, Nanjing Zelang), Ergothioneine (purity ≥99%, Sigma); DMEM medium (Gibco), fetal bovine serum (FBS, Gibco), SA-β-Gal staining kit (Cell Signaling), CCK-8 kit (Dojindo), flow cytometry antibodies (CD90-PE, CD105-FITC, BD);

[0068] Equipment: Hypoxia incubator (Thermo, 3% O2), flow cytometer (BD FACSCanto), microplate reader (Tecan), Western blot system (Bio-Rad).

[0069] 2. Concentration gradient optimization

[0070] To determine the optimal component concentrations, concentration gradients of rhodioloside (5, 10, 20 μM), total flavonoids from Cuscuta chinensis (10, 20, 40 μM), and ergothionein (2.5, 5, 10 μM) were established, and the SA-β-Gal positivity rate and cell proliferation rate (CCK-8) were detected at the 10th generation.

[0071] Concentration gradient optimization results

[0072] The combination of 10 μM rhodioloside, 20 μM total flavonoids from Cuscuta chinensis, and 5 μM ergothioneine showed the lowest SA-β-Gal positivity rate (19%) and the highest cell proliferation rate (OD) in the 10th generation. 450 =1.1), which is significantly better than other concentration combinations (P<0.05), and is determined to be the optimal concentration.

[0073] 3. Experimental Grouping

[0074] The third-generation MSCs were divided into 4 groups (n=3):

[0075] Blank control group (Ctrl): conventional oxygen (21% O2) culture, without the addition of any components;

[0076] Hypoxia group: cultured in 3% O2 ​​without any added ingredients;

[0077] Single component groups: conventional oxygen culture, with the addition of rhodioloside (10 μM, Sali group), total flavonoids from Cuscuta chinensis (20 μM, TFCC group), and ergothionein (5 μM, Ergo group);

[0078] Combo: Cultured in 3% O2, supplemented with rhodioloside (10 μM) + total flavonoids from Cuscuta chinensis (20 μM) + ergothioneine (5 μM).

[0079] 4. Experimental Methods

[0080] Third-generation human umbilical cord mesenchymal stem cells (UC-MSCs) were harvested and seeded in a 10cm culture dish (1×10⁻⁶ cells / mL). 6 Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics (cells / plate). The combination group and hypoxia group were cultured in a hypoxia incubator at 3% O2, while the blank control group and single-component groups were cultured under standard oxygen (21% O2). The medium was changed every two days. For the combination group, rhodioloside (10 μM), total flavonoids from Cuscuta chinensis (20 μM), and ergothioneine (5 μM) were added during medium changes. For the single-component groups, rhodioloside (10 μM, Sali group), total flavonoids from Cuscuta chinensis (20 μM, TFCC group), and ergothioneine (5 μM, Ergo group) were added respectively. Cells were passaged to the 15th generation, and various indicators were measured.

[0081] 5. Testing Indicators

[0082] Aging markers: SA-β-Gal staining (counting the proportion of positive cells);

[0083] Proliferation capacity: CCK-8 assay (detection of absorbance at 450 nm);

[0084] Stem cell characteristics: CD90 and CD105 expression detected by flow cytometry;

[0085] Oxidative stress: ROS levels were detected by DCFH-DA probe (fluorescence intensity, AU), and MDA content (nmol / mg protein) was detected by MDA kit;

[0086] Pathway proteins: Western blot was used to detect the expression of SIRT1, Wnt3a, and HIF-1α (with β-actin as an internal reference, and the relative expression levels were calculated).

[0087] 6. Experimental Results

[0088] 6.1 Comparison of aging indicators among groups (15th generation) is shown in Table 2 below. All indicators of the Combo group were significantly better than those of other groups (P<0.01), indicating that hypoxia + the combined herbal ingredients have a synergistic anti-aging effect.

[0089] Table 2

[0090]

[0091] 6.2 Pathway protein expression (15th generation) 30

[0092] The expression levels of SIRT1 (1.4-fold), Wnt3a (1.7-fold), and HIF-1α (1.9-fold) in the combination group were significantly higher than those in other groups (P<0.01), which verifies the mechanism of action of the present invention.

[0093] Third-generation human umbilical cord mesenchymal stem cells (UC-MSCs) were collected and treated according to the method in Experiment 1. The results showed that the SA-β-Gal positivity rate in the combination group (15%) was significantly lower than that in the blank group (70%), and the cell proliferation rate (OD) was significantly lower. 450 =0.9) is significantly higher than other groups, verifying the universality of the present invention for MSCs from different sources.

[0094] This invention relates to a formulation and method for preventing the replicative senescence of mesenchymal stem cells (MSCs) by combining the effective components of traditional Chinese medicine with hypoxic conditions. Through hypoxic (2-5% O2) culture combined with a combination of rhodioloside (10-20 μM), total flavonoids from Cuscuta chinensis (20-40 μM), and ergothioneine (5-10 μM), the SIRT1, Wnt / β-catenin, and HIF-1α pathways are synergistically activated, oxidative stress is inhibited, and stem cell characteristics are maintained. Experiments showed that the positive rate of senescence-related β-galactosidase (SA-β-Gal) in the combined group at passage 15 (12%) was significantly lower than that in the control group (65%), cell proliferation rate and stem cell markers (CD90, CD105) expression were significantly increased, and oxidative stress indicators (ROS, MDA) were significantly reduced. This invention is safe, effective, and low-cost, and is suitable for the large-scale preparation of MSCs in regenerative medicine.

[0095] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0096] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A preparation for preventing replicative senescence of mesenchymal stem cells, characterized by, Rhodioloside, total flavones of Semen cuscutae and ergothioneine.

2. The preparation for preventing replicative senescence of mesenchymal stem cells according to claim 1, wherein: The concentration of rhodioloside is 10-20 μM, the concentration of total flavones of Semen cuscutae is 20-40 μM, and the concentration of ergothioneine is 5-10 μM. ​ 3. The preparation for preventing replicative senescence of mesenchymal stem cells according to claim 2, wherein the preparation is a preparation for preventing replicative senescence of mesenchymal stem cells, which comprises a plurality of the cells of claim 1. The concentration of rhodioloside is 10 μM, the concentration of total flavones of Semen cuscutae is 20 μM, and the concentration of ergothioneine is 5 μM.

4. A method of preventing replicative senescence of mesenchymal stem cells, characterized by: During the in vitro expansion of mesenchymal stem cells, a low-oxygen condition of 2-5% O2 is used for culture, and a preparation for preventing replicative senescence of mesenchymal stem cells according to any one of claims 1-3 is added in the culture medium.

5. The method of claim 4, wherein the method is for preventing replicative senescence of mesenchymal stem cells. The low-oxygen condition is 3% O2.

6. The method of claim 5, wherein the mesenchymal stem cells are prevented from replicative senescence by: DMEM medium containing 10% FBS and 1% double antibody is used to continuously treat MSCs from the third generation, and the medium is changed every two days, and rhodioloside, total flavones of Semen cuscutae and ergothioneine are added when the medium is changed, and the cells are subcultured to the 15th generation.

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