CN115261311A - Application of ginsenoside Rg2 in promoting mesenchymal stem cell proliferation in vitro and inhibiting replicative senescence - Google Patents

Application of ginsenoside Rg2 in promoting mesenchymal stem cell proliferation in vitro and inhibiting replicative senescence Download PDF

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CN115261311A
CN115261311A CN202210739739.9A CN202210739739A CN115261311A CN 115261311 A CN115261311 A CN 115261311A CN 202210739739 A CN202210739739 A CN 202210739739A CN 115261311 A CN115261311 A CN 115261311A
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王楠
张同存
车丽娜
黄磊
赵崇杰
何红鹏
罗学刚
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Tianjin University of Science and Technology
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Abstract

The invention discloses an application of ginsenoside Rg2 in promoting mesenchymal stem cell proliferation and inhibiting replicative senescence in vitro, wherein the chemical structural formula of the ginsenoside Rg2 is as follows:
Figure DDA0003717362220000011
the invention discovers that the ginsenoside Rg2 can be used for promoting the in-vitro proliferation of the mesenchymal stem cells so as to improve the efficiency of in-vitro amplification of the mesenchymal stem cells. The invention provides important guarantee for the in vitro amplification, dryness maintenance and subsequent application of the mesenchymal stem cells, provides enough stem cells for clinical treatment and scientific research, and has important economic value and social significance.

Description

Application of ginsenoside Rg2 in promoting mesenchymal stem cell proliferation in vitro and inhibiting replicative senescence
Technical Field
The invention belongs to the technical field of medicines, relates to stem cells, and particularly relates to application of ginsenoside Rg2 in promoting mesenchymal stem cell in-vitro proliferation and inhibiting replicative senescence.
Background
Stem cells (stem cells) refer to cells with high self-renewal replication capacity and multidirectional differentiation potential, are important seed cells in tissue engineering and regenerative medicine, and have great application potential. Stem cells can differentiate into various functional cells of the body under certain conditions. Stem cells can be classified into totipotent, unipotent and pluripotent stem cells according to their developmental potential. The stem cells can be further divided into embryonic stem cells and adult stem cells according to the development stage of the stem cells.
Mesenchymal Stem Cells (MSCs) belong to adult stem cells, are widely distributed in various tissues such as bone marrow, placenta, fat, peripheral blood, umbilical cord, muscle and the like, are cultured in vitro to grow in an attached manner, express various surface markers, can be directionally differentiated into various cells such as bone, cartilage, fat, myoblasts, nerve and the like, and have wide application prospects in the aspect of tissue injury repair. However, MSCs require long-term subculture expansion to sufficient numbers to achieve clinically administered doses, and thus, aging of mesenchymal stem cells may limit their therapeutic applications.
Like any normal somatic cell, mesenchymal stem cells have a limited lifespan in vitro. After a certain number of cell divisions, MSCs enter the senescence stage, morphologically characterized by an increase in cell morphology, irregularity, and eventually stop proliferating. Meanwhile, the mesenchymal stem cells have reduced differentiation potential after long-term subculture in vitro. Therefore, promotion of mesenchymal stem cell proliferation in vitro and inhibition of replicative senescence are important for applications such as gene engineering, tissue engineering, cell regeneration therapy, and cell culture meat.
Ginsenoside is a steroid compound, and is considered to be a main active ingredient in ginseng. Part of ginsenoside has been proved to have various pharmacological activities such as anti-inflammatory, anticancer, anti-aging, etc. These ginsenosides exert their effects through mechanisms such as autophagy, apoptosis, cell cycle arrest, anti-metastasis and epigenetic changes. Ginsenosides all have a similar basic structure and all contain a steroid core of 30 carbons arranged in four rings. They are divided into two groups according to the glycosidic architecture: dammarane type and oleanane type. Dammarane types include two classes: panaxadiol type-A, aglycone is 20 (S) -protopanaxadiol, and contains most ginsenosides such as ginsenoside Rb1, rb2, rb3, rc, rd, rg3, rh2 and glycoside PD; panaxatriol-B type, 20 (S) -protopanaxatriol, contains ginsenoside Re, rg1, rg2, rh1 and glycosyl PT.
Ginsenoside Rg2, whose chemical name is (3 beta, 6 alpha, 12 beta) -3, 12, 20-trihydroxy dammara-24-en-6-yl-2-O- (6-deoxy-alpha-L-mannopyranosyl) -beta-D-glucopyranoside, has been proved to have various effects of resisting hypertension, diabetes, depression, neuroprotection and the like. However, the influence of ginsenoside Rg2 on the proliferation and replicative senescence of mesenchymal stem cells has not been reported so far.
The chemical structural formula of the ginsenoside Rg2 is as follows:
Figure BDA0003717362200000021
through searching, the following patent publications relevant to the patent application of the invention are found:
application of ginsenoside 20 (R) -25-OH-Rg3 in promoting in-vitro proliferation of umbilical cord mesenchymal stem cells (CN 110055214A) discloses application of ginsenoside 20 (R) -25-OH-Rg3 in promoting in-vitro proliferation of umbilical cord mesenchymal stem cells. The invention discovers that 20 (R) -25-OH-Rg3 has a less proliferation promoting effect on umbilical cord mesenchymal stem cells than 20 (R) -Rg3, but 20 (R) -25-OH-Rg3 can promote proliferation of umbilical cord mesenchymal stem cells without influencing phenotypes of the umbilical cord mesenchymal stem cells, can maintain cell purity and stem cell characteristics of the umbilical cord mesenchymal stem cells, and can provide more seed cells for research of umbilical cord mesenchymal stem cells.
By contrast, the present patent application is substantially different from the above patent publications. Firstly, the chemical structural formula of the ginsenoside Rg2 is different from that of the ginsenoside 20 (R) -25-OH-Rg 3. In addition, the cells used by the invention also comprise the pig bone marrow mesenchymal stem cells besides the human umbilical cord mesenchymal stem cells, and have important significance for the applications of poultry cell regeneration treatment, cell culture meat and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the application of ginsenoside Rg2 in promoting the in vitro proliferation of mesenchymal stem cells and inhibiting replicative senescence.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the application of the ginsenoside Rg2 in promoting mesenchymal stem cell proliferation and inhibiting replicative senescence in vitro is disclosed, wherein the chemical structural formula of the ginsenoside Rg2 is as follows:
Figure BDA0003717362200000031
further, the application is the application of the ginsenoside Rg2 in serving as and/or preparing a medicament for promoting mesenchymal stem cell proliferation and inhibiting replicative senescence in vitro.
Further, the application refers to the application of the ginsenoside Rg2 in the preparation of and/or application of a medicament for promoting the proliferation of human mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 50-400 mu M.
Further, the application is the application of the ginsenoside Rg2 in serving as and/or preparing a medicament for inhibiting replicative senescence of human-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 50-200 mu M.
Further, the application refers to the application of the ginsenoside Rg2 in the preparation of and/or application of a medicament for promoting the proliferation of porcine-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 25-300 mu M.
Further, the application is the application of the ginsenoside Rg2 in serving as and/or preparing a medicament for inhibiting replicative senescence of porcine-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 25-100 mu M.
Further, the ginsenoside Rg2 is high-purity ginsenoside Rg2, and the purity is more than or equal to 98%.
The invention has the advantages and positive effects that:
1. the invention discovers that the ginsenoside Rg2 can be used for promoting the in-vitro proliferation of the mesenchymal stem cells so as to improve the efficiency of in-vitro amplification of the mesenchymal stem cells. The invention provides important guarantee for the in vitro amplification, dryness maintenance and subsequent application of the mesenchymal stem cells, provides enough stem cells for clinical treatment and scientific research, and has important economic value and social significance. As shown in fig. 2 and fig. 5EdU experimental results, the cell proliferation rate is significantly increased after adding ginsenoside Rg 2.
2. The ginsenoside Rg2 can promote the proliferation of the mesenchymal stem cells, can inhibit the replicative senescence of the mesenchymal stem cells, has the prospect of being developed into a culture medium for promoting the in-vitro proliferation of the mesenchymal stem cells, and is very important for the applications of gene engineering, tissue engineering, cell regeneration treatment, cell culture meat and the like. Can also provide enough cells for tissue engineering and stem cell therapy for treating bone defect, cartilage defect, liver fibrosis, myocardial infarction, pulmonary fibrosis, diabetes and the like, and simultaneously reduce the immunological rejection of organisms. As shown in fig. 3 and fig. 6, the results of beta-galactosidase staining experiments show that the senescent cell rate is significantly reduced after the ginsenoside Rg2 is added.
3. The invention discovers that the ginsenoside Rg2 has the function of promoting the proliferation of human-derived and pig-derived mesenchymal stem cells in vitro and also has the function of inhibiting the replicative senescence of the mesenchymal stem cells. Therefore, the ginsenoside Rg2 has the prospect of being developed into a culture medium for promoting the in-vitro proliferation of the mesenchymal stem cells, and is very important for the applications of gene engineering, tissue engineering, cell regeneration treatment, cell culture meat and the like.
4. The invention finds that the preferable concentration of the ginsenoside Rg2 is 50-400 mu M, and the ginsenoside Rg2 is obviously applied to promoting the proliferation of the human mesenchymal stem cells in vitro. The preferable concentration of the ginsenoside Rg2 is 50-200 mu M, and the ginsenoside Rg2 is obviously applied to the in vitro inhibition of replicative senescence of the human mesenchymal stem cells. The preferable concentration of the ginsenoside Rg2 is 25-300 mu M, and the ginsenoside Rg2 is obviously applied to promoting the proliferation of the pig-derived mesenchymal stem cells in vitro. The preferable concentration of the ginsenoside Rg2 is 25-100 mu M, and the ginsenoside Rg2 is obviously applied to inhibiting the replicative senescence of the porcine mesenchymal stem cells in vitro. As shown in the MTT experimental result of figure 1, after the ginsenoside Rg2 (50-400 mu M) with different concentrations is added, the proliferation rate of the human mesenchymal stem cells is obviously increased. As shown in the MTT experimental result of FIG. 4, after the ginsenoside Rg2 (25-300 μ M) with different concentrations is added, the proliferation rate of the pig-derived mesenchymal stem cells is obviously increased.
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FIG. 1 shows the MTT experimental results of in vitro culture of human mesenchymal stem cells by ginsenoside Rg 2;
FIG. 2 shows the result of an EdU experiment for in vitro culture of human mesenchymal stem cells by ginsenoside Rg 2; wherein, the left graph is the EdU staining result of the control group and the administration group, and the right graph is the quantitative analysis;
FIG. 3 shows the result of staining with cell senescence beta-galactosidase of in vitro cultured human mesenchymal stem cells of ginsenoside Rg2 in the present invention; wherein, the upper graph is the beta-galactosidase staining result of the control group and the administration group, and the lower graph is the quantitative analysis;
FIG. 4 shows the MTT experimental results of in vitro culture of porcine-derived mesenchymal stem cells by ginsenoside Rg 2;
FIG. 5 shows the result of an EdU experiment for in vitro culture of pig-derived mesenchymal stem cells by ginsenoside Rg 2; wherein, the left graph is the EdU staining result of the control group and the administration group, and the right graph is the quantitative analysis;
FIG. 6 shows the result of staining with cell senescence beta-galactosidase of in vitro cultured porcine mesenchymal stem cells with ginsenoside Rg 2; wherein, the upper graph is the beta-galactosidase staining result of the control group and the administration group, and the lower graph is the quantitative analysis.
Detailed Description
The present invention is described in detail below with reference to the following examples, which are intended to be illustrative and not limiting, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The application of the ginsenoside Rg2 in promoting the proliferation of mesenchymal stem cells and inhibiting replicative senescence in vitro, wherein the chemical structural formula of the ginsenoside Rg2 is as follows:
Figure BDA0003717362200000051
preferably, the application refers to the application of the ginsenoside Rg2 in the preparation of the in vitro mesenchymal stem cell proliferation promoting and replicative senescence inhibiting medicines.
Preferably, the application refers to the application of the ginsenoside Rg2 in the preparation of and/or application of a medicament for promoting the proliferation of human mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 50-400 mu M.
Preferably, the application is the application of the ginsenoside Rg2 in serving as and/or preparing a medicament for inhibiting replicative senescence of human-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 50-200 mu M.
Preferably, the application refers to the application of the ginsenoside Rg2 in the preparation of and/or application of a medicament for promoting the proliferation of porcine-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 25-300 mu M.
Preferably, the application refers to the application of the ginsenoside Rg2 in the preparation of and/or application of a medicament for inhibiting the replicative senescence of porcine-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 25-100 mu M.
Further, the ginsenoside Rg2 is high-purity ginsenoside Rg2, and the purity is more than or equal to 98%.
Specifically, the preparation and detection examples are as follows:
example 1
Culture of human umbilical cord-derived mesenchymal stem cells (HMSCs)
Taking P0 generation HMSCs cells (purchased from Tianjin Kaesibo Biotech Co., ltd.), using complete medium (DMEM/F12 medium + penicillin 100IU/mL + streptomycin 100. Mu.g/mL +10% fetal bovine serum) at 37 ℃ and 5% CO2Culturing in the environment, and changing the culture solution every two days. When the cell isAnd (3) digesting and passaging when the growth density reaches 80-90%, using the HMSCs cells from generation 2 to generation 5 for proliferation experiments, and using the human umbilical cord mesenchymal stem cells from generation 10 for cell senescence beta-galactosidase staining experiments.
Example 2
P3-generation HMSCs cells were used for MTT experiments.
The experiment is divided into a blank group, a control group and an administration group. Ginsenoside Rg2 in the administration group was dissolved, filtered, and mixed at 6 concentrations (25. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 300. Mu.M, and 400. Mu.M).
HMSCs of generation P3 at 5X 103Inoculating the cells/well into a 96-well plate, culturing for 24 hours, adding 6 culture media with different concentrations of ginsenoside Rg2 into the 96-well plate after the cells adhere to the wall, wherein 6 wells are formed in each component. After dosing for 48h, 10uL of MTT solution was added to each well, incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated. Add 100uLDMSO to each well, shake for 10 minutes, make the crystal fully melt. The wavelength was chosen to be 490mm and the absorbance of each well was measured on an enzyme linked immunosorbent instrument.
Proliferation rate = ODAdministration set-ODBlank group/ODControl group-ODBlank group。
The experimental results are as follows: OD values of the control group and the administration groups with different concentrations are shown in figure 1, the ginsenoside Rg2 with different concentrations can effectively promote the in-vitro proliferation of human umbilical cord mesenchymal stem cells, and compared with the proliferation rate of 100% of the control group, the proliferation rates of 200 mu M and 300 mu M of the ginsenoside Rg2 administration groups are as high as 142.67% +/-1.02% and 135.32% +/-2.14%.
Example 3
P3 passage HMSCs cells were used for EdU experiments.
Wherein, the stationary liquid is: containing 4% by volume of paraformaldehyde solution in 10mM phosphate buffered saline;
the washing liquid is: a 3% by volume Bovine Serum Albumin (BSA) solution in 10mM phosphate buffered saline;
the liquid permeability is: contains 0.3-3.5% by volume of polyethylene glycol octyl phenyl ether (TritonX-100) solution and 10mM phosphate buffer solution.
Divided into a control group and a dosing group. The concentration of ginsenoside Rg2 in the administration group is 200 μ M. The concentration range selected is between 50 and 400. Mu.M. HMSCs of generation P3 at 5X 106The density of individual/well was seeded in 6-well plates. The control group was cultured in a conventional medium, and the administration group was cultured in a medium containing 200. Mu.M, after 48 hours, 2 XEdU working solution was preheated at 37 ℃ and added in an equal volume to 6-well plates so that the final EdU concentration in the 6-well plates became 1X. After incubation for 2h, the medium was removed, 1ml of fixative was added and fixed at room temperature for 15-30min. The fixative was removed and the cells were washed 3 times with 1ml of wash solution per well, 3-5min each time. The wash solution was removed, 1ml of the permeate was added and incubated at room temperature for 10-15min. The permeate was removed and the cells were washed 1-2 times for 3-5min with 1ml of wash solution per well. And removing the washing liquid in the previous step. Add 500. Mu.l of Click reaction per well and shake the plate gently to ensure that the Click reaction can cover the sample evenly. Incubate for 30min at room temperature in the dark. The Click reaction solution was aspirated off, and the cells were washed 3 times for 3-5min with 1ml of washing solution per well. The wash solution was aspirated off, 1ml of 1XHoechst33342 solution was added per well and incubated for 10min at room temperature in the absence of light. The 1XHoechst33342 solution was aspirated and the cells were washed 3 times for 3-5min with 1ml of wash solution per well. Fluorescence detection and photography can then be performed. Proliferating cells were labeled with bright red fluorescence and nuclei were blue fluorescence, and the proportion of red fluorescent cells was calculated.
The experimental results are as follows: the EdU experiment results of the control group and the administration group are shown in figure 2, the ginsenoside Rg2 can effectively promote the in-vitro proliferation of the human umbilical cord mesenchymal stem cells, and compared with the control group, the red fluorescent cell ratio of the administration group is increased by 18.46% +/-1.42%.
Example 4
HMSCs cells from P10 generation were used for staining with beta-galactosidase.
Divided into control group and administration group. The concentration of the ginsenoside Rg2 in the administration group is 100 MuM, and the selected concentration range is 50-200 MuM. HMSCs of generation P10 at 5X 106The density of cells/well was seeded in 6-well plates. The control group was cultured in a conventional medium, and the administration group was cultured in a medium containing 100. Mu.M for a long period of timeAnd (4) sucking out cell culture solution, washing the cell culture solution once by using PBS (phosphate buffer solution), adding 1mL of beta-galactosidase staining fixing solution, and fixing the cell culture solution at room temperature for 15min. The fixative was aspirated and the cells were washed 3 times with pre-cooled PBS. PBS was aspirated and 1mL of staining solution was added to each well. Incubate overnight in 37 ℃ incubator. Observed under a common optical microscope and photographed. Different visual fields are randomly selected, and the proportion of aged cells is calculated.
The experimental results are as follows: the results of the beta-galactosidase staining experiments of the control group and the administration group are shown in fig. 3, the ginsenoside Rg2 can effectively inhibit the replicative senescence of the human umbilical cord mesenchymal stem cells, and compared with the control group, the senescence proportion of the human umbilical cord mesenchymal stem cells of the administration group is reduced by 32.76% +/-3.10%.
Example 5
Isolation and culture of porcine bone marrow mesenchymal stem cells (BMSCs)
A newborn piglet of one week old is taken, anesthetized and killed by 3% sodium pentobarbital, ankle joints are cut off at ankles, soles are removed, and connective tissues at the tail ends of bones are scraped along the backbone. The tibia and humerus were preserved in cold PBS supplemented with 5% penicillin/streptomycin. The ends of the femur and tibia were cut under sterile conditions, and the bone marrow filler was flushed from the incision with complete medium (DMEM/F12 medium + penicillin 100IU/mL + streptomycin 100. Mu.g/mL +10% fetal bovine serum) to collect BMSCs. The 200 mesh screen filters the cell suspension to remove bone marrow needles, muscle and cell mass. Dilution of cells to 1X 10 by addition of complete Medium5one/mL at 100mm2In a dish. Cultured under the culture conditions of 5% CO2 at 37 ℃. The porcine bone marrow mesenchymal stem cells from generation 2 to generation 5 are used for proliferation experiments, and the porcine bone marrow mesenchymal stem cells from generation 10 are used for cell senescence beta-galactosidase staining experiments.
Example 6
P3-generation BMSCs cells were used for MTT experiments.
The experiment was divided into a blank group, a control group and a dosing group. Ginsenoside Rg2 in the administration group was dissolved, filtered, and mixed at 6 concentrations (25. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 300. Mu.M, and 400. Mu.M). BMSCs of generation P3 at 5X 103The density of each well is inoculated in a 96-well plate and cultured for 24 hoursWhen the cells adhere to the wall, 6 culture mediums added with ginsenoside Rg2 with different concentrations are respectively added into a 96-well plate, and 6 wells are formed in each component. After 48h of dosing, 10uL of MTT solution was added to each well, incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated away. Add 100u LDMSO to each hole, shake for 10 minutes, make the crystal fully melt. The wavelength was chosen to be 490mm and the absorbance of each well was measured on an enzyme linked immunosorbent instrument.
Proliferation rate = ODAdministration set-ODBlank group/ODControl group-ODBlank group。
The experimental results are as follows: the OD values of the control group and the administration groups with different concentrations are shown in figure 4, the ginsenoside Rg2 with different concentrations can effectively promote the in-vitro proliferation of the mesenchymal stem cells of the pig bone marrow, and compared with the proliferation rate of 100% of the control group, the proliferation rates of the administration groups with 100 mu M and 200 mu M of the ginsenoside Rg2 are as high as 145.17% +/-1.72% and 137.81% +/-0.19%.
Example 7
P3-generation BMSCs cells were used for EdU experiments.
Wherein, the stationary liquid is: containing 4% by volume of paraformaldehyde solution in 10mM phosphate buffer solution;
the washing liquid is: a solution containing 3% Bovine Serum Albumin (BSA) by volume in a 10mM phosphate buffered saline;
the liquid permeability is: contains 0.3-3.5% by volume of polyethylene glycol octyl phenyl ether (TritonX-100) solution and 10mM phosphate buffer solution.
Divided into control group and administration group. The concentration of ginsenoside Rg2 in the administration group is 100 μ M. The concentration range selected is between 25 and 300. Mu.M. BMSCs of generation P3 at 5X 106The density of individual/well was seeded in 6-well plates. The control group was cultured in a conventional medium, and the administration group was cultured in a medium containing 100. Mu.M, and after 48 hours, a 2 XEdU working solution was preheated at 37 ℃ and added in an equal volume to 6-well plates so that the final EdU concentration in the 6-well plates became 1X. After incubation for 2h, the medium was removed, 1ml of fixative was added and fixed at room temperature for 15-30min. The fixative was removed and the cells were washed 3 times with 1ml of wash solution per well, 3-5min each time. The washing solution was removed and 1ml of a permeation solution was addedAnd incubating for 10-15min at room temperature. The permeate was removed and the cells were washed 1-2 times for 3-5min with 1ml of wash solution per well. And removing the washing liquid in the previous step. Add 500. Mu.l of Click reaction per well and shake the plate gently to ensure that the Click reaction can cover the sample evenly. Incubate for 30min at room temperature in the dark. The Click reaction solution was aspirated off, and the cells were washed 3 times for 3-5min with 1ml of washing solution per well. The wash solution was aspirated off, 1ml of 1XHoechst33342 solution was added per well and incubated for 10min at room temperature in the absence of light. The 1XHoechst33342 solution was aspirated and the cells were washed 3 times for 3-5min with 1ml of wash solution per well. Fluorescence detection can then be performed and photographed. Proliferating cells were labeled with bright red fluorescence and nuclei were blue fluorescence, and the proportion of red fluorescent cells was calculated.
The experimental results are as follows: the EdU experiment results of the control group and the administration group are shown in fig. 5, the ginsenoside Rg2 can effectively promote the in vitro proliferation of the pig bone marrow mesenchymal stem cells, and compared with the control group, the red fluorescent cell ratio of the administration group is increased by 12.65% ± 2.19%.
Example 8
BMSCs cells of P10 generation are used for beta-galactosidase staining experiment and are divided into a control group and a dosing group. The concentration of the ginsenoside Rg2 in the administration group is 50 MuM, and the selected concentration ranges are all between 25 and 100 MuM.
BMSCs of generation P10 are divided into 5 × 106The density of cells/well was seeded in 6-well plates. The control group was cultured in a conventional medium, the administration group was cultured for a long period to P10 passage with a medium added with 50. Mu.M, the cell culture solution was aspirated, washed once with PBS, and fixed for 15min at room temperature by adding 1mL of a beta-galactosidase staining fixative. The fixative was aspirated and the cells were washed 3 times with pre-cooled PBS. PBS was aspirated and 1mL of staining solution was added to each well. Incubate overnight in 37 ℃ incubator. Observed and photographed under a normal optical microscope. Different visual fields are randomly selected, and the proportion of aged cells is calculated.
The experimental results are as follows: the results of the beta-galactosidase staining experiments of the control group and the administration group are shown in fig. 6, the ginsenoside Rg2 can effectively inhibit the replicative senescence of the porcine bone marrow mesenchymal stem cells, and compared with the control group, the senescence proportion of the porcine bone marrow mesenchymal stem cells of the administration group is reduced by 32.19% +/-3.10%.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, alterations and modifications are possible without departing from the spirit and scope of this disclosure and appended claims, and accordingly, the scope of this disclosure is not limited to the embodiments disclosed.

Claims (7)

1. The application of the ginsenoside Rg2 in promoting the proliferation of mesenchymal stem cells and inhibiting replicative senescence in vitro, wherein the chemical structural formula of the ginsenoside Rg2 is as follows:
Figure FDA0003717362190000011
2. use according to claim 1, characterized in that: the application is the application of the ginsenoside Rg2 in the preparation of and/or the application in the preparation of medicaments for promoting the proliferation of mesenchymal stem cells and inhibiting replicative senescence in vitro.
3. Use according to claim 1 or 2, characterized in that: the application refers to the application of the ginsenoside Rg2 in the preparation and/or application of a medicament for promoting the proliferation of human mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 50-400 mu M.
4. Use according to claim 1 or 2, characterized in that: the application refers to the application of the ginsenoside Rg2 in serving as and/or preparing a medicament for inhibiting replicative senescence of human-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 50-200 mu M.
5. Use according to claim 1 or 2, characterized in that: the application refers to the application of the ginsenoside Rg2 in the preparation and/or the application of medicines for promoting the proliferation of the pig-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 25-300 mu M.
6. Use according to claim 1 or 2, characterized in that: the application refers to the application of the ginsenoside Rg2 in serving as and/or preparing a medicament for inhibiting replicative senescence of porcine-derived mesenchymal stem cells in vitro, wherein the concentration of the ginsenoside Rg2 is 25-100 mu M.
7. Use according to claim 1 or 2, characterized in that: the ginsenoside Rg2 is high-purity ginsenoside Rg2, and the purity is more than or equal to 98%.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116832047A (en) * 2023-07-04 2023-10-03 中国医学科学院基础医学研究所 Application of ginsenoside Rg1 and derivatives thereof in vitro antagonism of replicative senescence of sub-totipotent stem cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244253A (en) * 2021-06-11 2021-08-13 浙江大学 Application of ginsenoside Rg2 in preparation of medicine for treating bone marrow depression and medicine containing ginsenoside Rg2

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244253A (en) * 2021-06-11 2021-08-13 浙江大学 Application of ginsenoside Rg2 in preparation of medicine for treating bone marrow depression and medicine containing ginsenoside Rg2

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
何方;余丽梅;: "人参皂苷Rg1对成体干细胞特性与功能的影响", 中国药理学通报, no. 03 *
郭少三;黄畅;韩大良;周希瑜;汪保和;: "人参茎叶皂苷对体外条件下小鼠骨髓间充质干细胞和粒-巨噬系祖细胞增殖的影响", 中医药导报, no. 06 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116832047A (en) * 2023-07-04 2023-10-03 中国医学科学院基础医学研究所 Application of ginsenoside Rg1 and derivatives thereof in vitro antagonism of replicative senescence of sub-totipotent stem cells
CN116832047B (en) * 2023-07-04 2024-04-12 中国医学科学院基础医学研究所 Application of ginsenoside Rg1 and derivatives thereof in vitro antagonism of replicative senescence of sub-totipotent stem cells

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