A method for cell anti-aging

Abstract

The present invention provides a method for cell anti-aging, in which a constructed cell aging model is first treated with mesenchymal stem cells for 12-48 hours, and then metformin is added for 12-48 hours. This combined therapy can significantly enhance the expression level of glutathione peroxidase 7 (GPx7) in aging IMR-90 cells, thereby greatly reversing the aging state of IMR-90 cells.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, in particular to a method for anti-aging of cells. Background Art

[0002] Aging refers to a series of physiological changes that occur in organisms as they age, including organ function decline, decreased cell regeneration ability, and slowed metabolism. These changes are usually accompanied by symptoms such as sagging skin, graying hair, memory loss, and slow movements, and may affect the functions of multiple systems such as the reproductive system and the immune system. The manifestations of human aging are diverse and involve multiple systems and organs, especially cell aging: over time, cells continue to divide and proliferate, which can cause DNA damage, causing the cell's repair ability to gradually decrease. At the same time, oxidative stress can also damage cell structure and DNA, leading to aging.

[0003] Stem cells are a type of cell with the potential for self-renewal and differentiation, and they play a key role in tissue repair and regeneration. With age, the number and function of stem cells will gradually decline, resulting in a weakening of tissue regeneration ability, which in turn causes various aging-related diseases. Therefore, stem cell therapy is regarded as a potential anti-aging strategy. In recent years, more and more clinical studies have begun to explore the application of mesenchymal stem cells in the field of anti-aging. These studies mainly focus on the therapeutic effects of mesenchymal stem cells on aging-related diseases such as frailty and chronic diseases. For example: Clinical trials of Lomecel-B: Lomecel-B is an allogeneic bone marrow mesenchymal stem cell therapy that has been used in many clinical trials for frailty and aging-related diseases. These trials have shown that intravenous injection of Lomecel-B can significantly improve functional indicators of aging, such as physical performance and inflammatory biomarkers.

[0004] Umbilical cord-derived mesenchymal stem cells (MSCs) have shown their potential in the field of anti-aging. However, in actual clinical applications and practices, there is still room for further improvement in their anti-aging effects. Although MSCs have multiple advantages such as self-renewal, multidirectional differentiation and immune regulation, they still face many challenges in precisely regulating their differentiation pathways, optimizing cell survival and function after transplantation, and ensuring long-term efficacy and safety. In order to fully explore the potential of MSCs in the field of anti-aging, researchers are committed to overcoming a series of technical difficulties, such as optimizing cell culture conditions, improving cell transplantation efficiency, and exploring more precise molecular regulatory methods. At the same time, in-depth research on the interaction mechanism between MSCs and the body's microenvironment, as well as their specific action pathways in the anti-aging process, will also provide strong support for improving the anti-aging effect of MSCs.

[0005] Metformin is a widely used hypoglycemic drug that has been found to have potential anti-aging effects in recent years. Its anti-aging mechanisms mainly include: activation of the AMPK pathway, antioxidant effects, anti-inflammatory effects, etc. However, metformin and mesenchymal stem cells (MSCs) each have certain potential in the field of anti-aging. Although metformin and mesenchymal stem cells each show unique advantages in the field of anti-aging, there are relatively few studies on the anti-aging effects of their combined use, and research in this field is still in its infancy. Summary of the invention

[0006] In view of this, the present invention aims to propose a cell anti-aging method, which achieves anti-aging effects by combining mesenchymal stem cells MSCs with metformin. Experimental results show that this combined therapy can significantly enhance the expression level of glutathione peroxidase 7 (GPx7) in aged IMR-90 cells, thereby greatly reversing the aging state of IMR-90 cells. More importantly, this combined therapy effectively reduces the potential inhibitory effect of metformin on IMR-90 cells when used alone, showing more superior safety and effectiveness.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] A method for cell anti-aging, the method comprising the following steps:

[0009] S1. Establishment of cell senescence model

[0010] The cells to be tested are inoculated in a culture medium and then cultured for 24-48 hours by adding D-galactose to the culture medium;

[0011] S2. Establishment of anti-aging model

[0012] The constructed cell aging model was first treated with mesenchymal stem cells for 12-48 hours, and then metformin was added for 12-48 hours; then the cells were tested for aging.

[0013] Furthermore, the seeding density of mesenchymal stem cells was 1×10 4 / cm 2 -1×10 5 / cm 2 , the concentration of metformin is 4-6mM / L.

[0014] Furthermore, the concentration of D-galactose in step S1 is 40 g / L-80 g / L.

[0015] Furthermore, the cells to be tested in step S1 are human embryonic lung fibroblasts IMR-90, and the cell seeding density is 1×10 4 / cm 2 .

[0016] Furthermore, in step S1, when the cell confluence reaches 80%, D-galactose is added.

[0017] Furthermore, in step S2, the source of the mesenchymal stem cells is umbilical cord-derived mesenchymal stem cells or embryo-derived mesenchymal stem cells.

[0018] Furthermore, the source of the mesenchymal stem cells is embryonic-derived mesenchymal stem cells.

[0019] Furthermore, aging detection includes detection of aging cells by β-galactosidase method and detection of GPx7 gene.

[0020] Compared with the prior art, the cell anti-aging method of the present invention has the following advantages:

[0021] The cell anti-aging method of the present invention achieves anti-aging effects by combining MSCs with metformin. Experimental results show that this combined therapy can significantly enhance the expression level of glutathione peroxidase 7 (GPx7) in aged IMR-90 cells, thereby greatly reversing the aging state of IMR-90 cells. More importantly, this combined therapy effectively reduces the potential inhibitory effect of metformin on IMR-90 cells when used alone, showing more superior safety and effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 The cell senescence at different D-gal concentrations in Example 3; (a) is 0 g / L, (b) is 10 g / L, (c) is 20 g / L, (d) is 40 g / L, and (e) is 80 g / L;

[0024] Figure 2 The different anti-aging treatment conditions in Example 4; (a) is group 1, (b) is group 2, (c) is group 3, (d) is group 4, (e) is group 5; (f) is group 6; (g) is group 7; (h) is group 8; (i) is group 9;

[0025] Figure 3 The test results of GPx7;

[0026] Figure 4 This is a comparison chart of the effects of MSCs and metformin on cells;

[0027] Figure 5 This is a comparison chart of the effects of seeding density and concentration on cells;

[0028] Figure 6 This is a comparison chart of the effects of MSCs sources on cells. DETAILED DESCRIPTION

[0029] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 IMR-90 cell recovery culture

[0032] Shake the cryotube containing 1 mL of IMR-90 cell suspension quickly in a 37°C water bath to thaw, add the thawed cell suspension to a 15 ml centrifuge tube containing 6 mL of complete culture medium (DMEM-H culture medium supplemented with 10% FBS, 1% NEAA) and mix well. Centrifuge at 1000 RPM for 5 minutes, discard the supernatant, add 6-8 mL of culture medium and blow evenly. Then add all the cell suspension to a T25 culture flask for culture (or add the cell suspension to a 6 cm dish).

[0033] If the cell density reaches 80%-90%, you can proceed to subculture. The method is briefly as follows: discard the culture supernatant, rinse the cells 1-2 times with PBS without calcium and magnesium ions, add 1-2ml digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture bottle, place it in a 37℃ incubator for digestion for 2-5 minutes, and then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take it back to the operating table, tap the culture bottle a few times, and add more than 5ml of complete culture medium containing 10% serum to terminate digestion. Centrifuge at 1000RPM for 5 minutes, discard the supernatant, add 1-2mL of culture solution and blow evenly. Add culture solution at 6-8ml / bottle, and divide the cell suspension into new dishes or bottles containing 6-8ml culture solution at a ratio of 1:2 to 1:5.

[0034] Example 2 Umbilical cord-derived mesenchymal stem cell resuscitation culture

[0035] Remove the frozen P4 MSCs from liquid nitrogen and quickly thaw them in a 37°C water bath. During the thawing process, gently shake the cryovial to ensure that the cells are evenly heated. After the contents of the cryovial melt, disinfect the mouth and outer wall of the cryovial with 75% alcohol, open the cryovial in a sterile workbench, gently blow the melted cell suspension with a pipette, and then inoculate the cells in a T75 culture flask and culture them in a constant temperature and humidity incubator at 37°C, DMEM / F12 medium, and 5% CO2.

[0036] Example 3 Establishment of Cell Aging Model

[0037] 1×10 4 / cm 2 When the confluence of IMR-90 cells reached 80%, 0 g / L, 10 g / L, 20 g / L, 40 g / L, 80 g / L, and 100 g / L of D-galactose D-gal were added to the culture medium, respectively, and cultured for 24 hours, after which the cells were tested for senescence.

[0038] Cell senescence was detected according to the β-galactosidase staining kit (Zhuocai, ZG-G10054), and the main method was as follows:

[0039] 1) Remove the cell culture medium, wash twice with PBS, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 min.

[0040] 2) Discard the fixative solution and wash the cells three times with PBS, 2 min each time.

[0041] 3) Remove the PBS with a pipette, add 1 mL of β-galactosidase staining solution to each well, and incubate at 37°C for 2 hours to overnight. Note: Do not incubate in a 37°C CO2 incubator.

[0042] During the staining process, the color development needs to be observed in time. If the expression level of β-galactosidase in the sample is high, the staining can be completed within a few hours. If the expression level of β-galactosidase is low, the incubation time needs to be appropriately extended. During this period, the plate needs to be sealed with plastic wrap or parafilm to prevent the evaporation of the liquid from affecting the staining results.

[0043] 4) Observe under an ordinary optical microscope. After the positive cells are colored, remove the staining solution.

[0044] 5) If the cells are covered with PBS, the staining is complete and the sample can be stored at 4°C for 1 week. Alternatively, the cells can be covered with 70% glycerol and stored at 4°C for a longer period of time.

[0045] The β-galactosidase staining kit uses X-Gal as a substrate. Under the catalysis of senescence-specific β-galactosidase, a dark blue product is generated. Under an optical microscope, cells or tissues expressing β-galactosidase that have turned blue can be observed. The kit only stains senescent cells and will not stain normally cultured pre-senescent cells, quiescent cells, immortalized cells, or tumor cells.

[0046] The staining results are as follows Figure 1 As shown, Figure 1 (a) There are almost no stained cells at 0 g / L D-gal. Figure 1 (b)- Figure 1 (e) The senescence staining of cells becomes more obvious with the increase of D-gal concentration, indicating that the senescence of IMR-90 cells becomes more obvious with the increase of D-gal concentration, and 80g / L D-gal concentration is the best for inducing senescence of IMR-90 cells. When the concentration of D-gal is 100g / L (not shown), the cells die, indicating that the concentration is too high and not suitable for modeling.

[0047] Therefore, the following cell senescence model was constructed using D-gal at a concentration of 80 g / L.

[0048] Example 4 Anti-aging model screening

[0049] 1. Model construction

[0050] Group 1: normal control group, treated with 0g / L D-gal for 72h, i.e., no D-gal treatment;

[0051] Group 2: Aging model group, treated with 80 g / L D-gal for 24 h to establish the model, and then cultured in a medium without D-gal for another 48 h;

[0052] Group 3: Experimental group A, treated with 80 g / L D-gal for 24 h to establish the model, then treated with MSCs for 24 h, and then cultured in a medium without D-gal for another 24 h;

[0053] Group 4: Experimental group B, treated with 80 g / L D-gal for 24 h to establish the model, then treated with 5 mM / L metformin for 24 h, and then cultured in a medium without D-gal for another 24 h;

[0054] Group 5: Experimental group C, treated with 80 g / L D-gal for 24 hours to establish the model, then treated with MSCs for 24 hours, and then continued to add 5 mM / L metformin for 24 hours.

[0055] Group 6: Experimental group D, treated with 80 g / L D-gal for 24 hours to establish the model, then treated with MSCs for 24 hours, and then changed to 5 mM / L metformin for 24 hours.

[0056] Group 7: Experimental group E, treated with 80 g / L D-gal for 24 hours to establish the model, then treated with 5 mM / L metformin for 24 hours, and then continued to add MSCs for 24 hours.

[0057] Group 8: Experimental group F, treated with 80 g / L D-gal for 24 hours to establish the model, then treated with 5 mM / L metformin for 24 hours, and then changed to MSCs for 24 hours.

[0058] Group 9: Experimental group G, treated with 80 g / L D-gal for 24 h to establish the model, and then treated with MSCs and metformin for 48 h.

[0059] MSCs are mesenchymal stem cells derived from embryos. The seeding density of MSCs is 2×10 4 / cm 2 ; This experiment used 6-well plates for inoculation.

[0060] 2. Aging detection

[0061] The β-galactosidase staining kit (Zhuocai, ZG-G10054) was also used to detect cell senescence. The results are as follows Figure 2 shown.

[0062] Figure 2 They are different anti-aging models. As can be seen from the figure, 80g / L concentration of D-gal in group 2 significantly induced the senescence of IMR-90 cells; group 3 showed that the addition of MSCs could significantly reduce the senescence of IMR-90 cells; group 4 showed that the addition of metformin could reduce the senescence of IMR-90 cells, but significantly inhibited the proliferation of cells; group 5 showed that the combination of metformin and MSCs reduced the senescence of IMR-90 cells and reversed the inhibition of metformin on the growth of IMR-90 cells; groups 6-9 changed the use and order of MSCs and metformin, and MSCs could reverse the inhibition of metformin on cells, and they all had a certain combined anti-aging effect, but the effects were weaker than those of group 5. From this experiment, group 5 was the best anti-aging solution.

[0063] From the above description, it can be seen that the combination of mesenchymal stem cells and metformin inhibits D-gal-induced IMR-90 cell senescence, and the order of using mesenchymal stem cells and metformin will also affect the senescence of IMR-90 cells.

[0064] 3. Detection of GPx7 gene

[0065] Studies have found that when cells age, the expression level of glutathione peroxidase 7 (GPx7) decreases, and the degree of cell aging can be observed through the expression of GPx7.

[0066] The GPx7 test consists of the following steps:

[0067] Step 1: Total RNA extraction

[0068] Before total RNA extraction, soak the consumables involved, such as large gun tips, medium gun tips, small gun tips and all utensils in DEPC water overnight, dry them, sterilize them at 121℃ for 30min, and dry them in a 56℃ oven for later use, or purchase RNase-free consumables. Precool the anhydrous ethanol, chloroform and isopropanol used at -20℃ for later use.

[0069] The specific method is as follows:

[0070] 1) Aspirate the culture medium in a biosafety cabinet, wash three times with PBS, add 1 mL of Trizol to each well of a 6-well plate and place on ice for 5 min.

[0071] 2) Centrifuge at 12000r / min and 4℃ for 10min, and transfer the supernatant to a new EP tube.

[0072] 3) Add 200 μL of chloroform, shake for 15 seconds, and place on ice for 5 minutes.

[0073] 4) Centrifuge at 12000r / min and 4℃ for 10min. Transfer the supernatant water phase to a new EP tube.

[0074] 5) Add 0.5 mL of isopropanol to 1 mL of Trizol, shake to mix, and place in a -20°C refrigerator to settle for 1 hour.

[0075] 6) Centrifuge at 12000 r / min and 4°C for 10 min, discard the supernatant and retain the precipitate at the bottom of the tube.

[0076] 7) Add 75% ethanol to rinse (add 250 μL of DEPC water to 750 μL of pre-cooled anhydrous ethanol to prepare 1 mL of mixed solution), and repeat the rinse once.

[0077] 8) Centrifuge at 12000 r / min and 4°C for 10 min. Discard the supernatant and place in a biosafety cabinet at room temperature for 15 min.

[0078] 9) Add 20 μL of DEPC water to each tube and place at 55°C to dissolve the RNA sample.

[0079] 10) Determine the RNA OD value for quantification.

[0080] Step 2: cDNA reverse transcription

[0081] The specific method is as follows:

[0082] 1) In a biosafety cabinet, add 2 μg RNA template, 5 μL random primers (0.1 μg / μL), 1 μL dNTPs (10 mM / L), and DEPC water to make up to 20 μL system.

[0083] 2) Place in a 65°C water bath for 5 minutes and then quickly place in an ice bath for 2 minutes.

[0084] 3) Then add 4 μL 5×Bffer, 2 μL 0.1M DTT, and 1 μL RNase inhibitor and mix well.

[0085] 4) Incubate in a 37°C water bath for 2 min.

[0086] 5) Add 1 μL M-MLV reverse transcriptase, incubate in a 25°C water bath for 10 min, and then continue incubation in a 37°C water bath for 1-2 h.

[0087] 6) Quickly place the sample in a 70°C water bath and incubate for 15 minutes to terminate the reaction.

[0088] 7) After quantification, the samples were stored in a -20°C refrigerator for future use.

[0089] Step 3: qPCR detection of target gene expression

[0090] The primer sequence GPx7 used in real-time quantitative PCR detection is as follows:

[0091] Table 1 Primer sequence list

[0092] Primer sequences Serial Number Upstream AGCAGGACTTCTACGACTT SEQ ID NO.1 Downstream TTCACCACCAGGGACA SEQ ID NO.2

[0093] The reaction conditions used for real-time fluorescence quantitative PCR were: 95℃ pre-denaturation for 2min, 95℃ denaturation for 30s, 60℃ annealing for 30s, 40 cycles. Then the melting curve stage was performed, 95℃ for 1min, 55℃ for 1min, 95℃ for 15S. The experimental method was as follows: before the experiment, all reagents (sterile water, 50×ROX Reference Dye, SYBR Green qPCR mastermix) were placed on ice. The sample addition operation was carried out strictly according to the SybrGreen qPCR mastermix instructions, and the data were processed using the ΔΔCT method.

[0094] GPx7 was detected in cells from Group 1 to Group 9. The results are as follows Figure 3As shown, group 1 was the negative control; group 2 80g / L concentration of D-gal significantly inhibited the expression of GPx7 gene in IMR-90 cells; group 3 mesenchymal stem cells promoted the expression of GPx7 gene in IMR-90 cells after modeling; group 4 metformin promoted the expression of GPx7 gene in IMR-90 cells after modeling; group 5 mesenchymal stem cells combined with metformin greatly promoted the expression of GPx7 gene in IMR-90 cells after modeling; group 6-group 9 mesenchymal stem cells combined with metformin could also promote the expression of GPx7 gene, but they were all weaker than group 5, indicating that the use of mesenchymal stem cells first and the continuous use of mesenchymal stem cells can weaken the inhibitory effect of metformin on cells. This result shows that the anti-aging scheme of group 5 is the best.

[0095] From the above description, the combination of mesenchymal stem cells and metformin significantly enhanced the expression level of GPx7 gene, and changing the order of using mesenchymal stem cells and metformin will also affect the expression level of GPx7 gene.

[0096] Example 5 Effects of MSCs and Metformin on Cells

[0097] In order to clarify the effects of MSCs and metformin on cells, the following experiment was designed:

[0098] Table 2 Comparison scheme

[0099]

[0100] CCK-8 is a rapid and highly sensitive detection kit widely used in cell proliferation and cytotoxicity detection. Its working principle is: in the presence of electron coupling reagent, it can be reduced by dehydrogenase in the cell mitochondria to generate water-soluble formazan, which is orange-yellow and can be used to judge cell proliferation based on the depth of the color. Generally, the color of formazan is proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450nm using an enzyme reader, and this OD value can indirectly reflect the number of living cells.

[0101] The specific experimental operation method is: aspirate the culture medium, add 100 μL of CCK-8 diluent to each well (the ratio of cell culture medium to CCK-8 is 10:1), place it in a 37°C CO2 incubator for incubation for 1 hour, and then measure the absorbance value at a wavelength of 450 nm.

[0102] The results are as follows Figure 4 As shown, it can be found that 5mM / L metformin can inhibit the proliferation of IMR-90 cells, and the addition of MSCs can significantly reduce the inhibitory effect of metformin on IMR-90 cells.

[0103] Example 6 MSCs seeding density and metformin concentration screening

[0104] In order to determine the seeding density of MSCs and the concentration of metformin, the following experiment was conducted, that is, compared with Group 5 in Example 4, other conditions remained unchanged, and only the appropriate seeding density of MSCs or the concentration of metformin was changed.

[0105] Table 3 Inoculation density and concentration comparison scheme

[0106]

[0107] GPx7 was detected in cells from Group 1 to Group 6. The results are as follows Figure 5 As shown, Group 1 is a negative control; the seeding density of MSCs and the concentration of metformin used in Group 2 have the best effect. If the seeding density of MSCs is too low or too high, and the concentration of metformin does not meet 4-6mM / L, the anti-aging effect of cells will be affected. Only by following the scheme of this application can better effects be achieved.

[0108] Example 7 Source of Mesenchymal Stem Cells

[0109] In order to determine the effect of the source of MSCs on cell anti-aging, the following experiment was conducted. Compared with Group 5 in Example 4, other conditions remained unchanged, and only the source of MSCs was changed.

[0110] Table 4 Comparison of MSCs sources

[0111]

[0112]

[0113] GPx7 was detected in cells from Group 1 to Group 4. The results are as follows Figure 6 As shown, Group 1 is the negative control group (normally cultured IMR-90 cells); through comparison, it was found that embryonic-derived mesenchymal stem cells have a better effect in promoting the expression of endogenous GPx7 in IMR-90 cells and have a better anti-aging effect.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for anti-aging of cells, characterized in that: The method comprises the following steps: S1. Establishment of cell senescence model The cells to be tested are inoculated in a culture medium and then cultured for 24-48 hours by adding D-galactose to the culture medium; S2. Establishment of anti-aging model The constructed cell aging model was first treated with mesenchymal stem cells for 12-48 hours, and then metformin was added for 12-48 hours; then the cells were tested for aging.

2. The method for anti-aging of cells according to claim 1, characterized in that: The seeding density of mesenchymal stem cells was 1×10 4 / cm 2 -1×10 5 / cm 2 , the concentration of metformin is 4-6mM / L.

3. The cell anti-aging method according to claim 1, characterized in that: The concentration of D-galactose in step S1 is 40 g / L-80 g / L.

4. The method for anti-aging of cells according to claim 1, characterized in that: The cells to be tested in step S1 are human embryonic lung fibroblasts IMR-90, and the cell seeding density is 1×10 4 / cm 2 .

5. The cell anti-aging method according to claim 1, characterized in that: In step S1, when the cell confluence reaches 80%, D-galactose is added.

6. The cell anti-aging method according to claim 1, characterized in that: In step S2, the source of the mesenchymal stem cells is umbilical cord-derived mesenchymal stem cells or embryo-derived mesenchymal stem cells.

7. The cell anti-aging method according to claim 6, characterized in that: The source of mesenchymal stem cells is embryonic mesenchymal stem cells.

8. The cell anti-aging method according to claim 1, characterized in that: Aging detection includes the detection of senescent cells using the β-galactosidase method and the detection of the GPx7 gene.

Images