A method for preparing stem cells with high proliferation ability

Abstract

The present invention relates to a method for preparing stem cells with high proliferation ability, and belongs to the field of biological technology. The specific steps are to inoculate stem cells into a cell culture dish coated with an extracellular matrix, add a culture medium containing gentian glycoside, and continue to culture in a cell culture incubator for a certain period of time. The preferred stem cells are bone marrow mesenchymal stem cells, and the concentration of gentian glycoside in the culture medium is 25μM-50μM. The cell culture dish coated with an extracellular matrix is ​​obtained by culturing bone marrow mesenchymal stem cells in a specific culture medium and performing a series of treatments. This method can not only significantly improve the proliferation ability of stem cells, but also maintain their dryness, reduce dependence on extracellular matrix, and reduce culture costs, thereby providing an efficient stem cell preparation method for fields such as regenerative medicine and cell therapy.

Description

Technical Field

[0001] The invention belongs to the field of biological technology, and in particular relates to a method for preparing stem cells with high proliferation ability. Background Art

[0002] Stem cells, as a revolutionary discovery in the field of life sciences, have become a key element in the fields of regenerative medicine, cell therapy, and tissue engineering due to their excellent self-renewal ability and multidirectional differentiation potential. Stem cells have the ability to differentiate into a variety of mature cell types, providing new hope for the treatment of a variety of diseases such as Parkinson's disease, heart disease, diabetes, and osteoarthritis. Before realizing these therapeutic potentials, a crucial link is the in vitro expansion of stem cells, that is, increasing the number of stem cells under controlled laboratory conditions while maintaining their pluripotency and genetic stability.

[0003] The extracellular matrix (ECM) is a key component of the cell microenvironment and plays a vital role in the biological behavior of stem cells. ECM not only provides the necessary physical support for stem cells to maintain their structural integrity and stability, but also participates in the regulation of stem cell proliferation, differentiation and function by interacting with a variety of receptors on the cell surface. These interactions include but are not limited to cell adhesion, signal transduction and intercellular communication, which are essential for maintaining the pluripotency of stem cells and inducing their differentiation into specific cell lineages.

[0004] However, the process of obtaining ECM is often complicated and accompanied by high costs. This not only limits the extensiveness of stem cell research and application, but also increases the economic burden of related research and treatment. Therefore, how to effectively accelerate the proliferation of stem cells to reduce dependence on and use of ECM has become an urgent problem to be solved in the current field of stem cell research.

[0005] Amarogentin is a unique secoiridoid glycoside, mainly derived from the roots of Swertia and Gentiana plants. Its structural formula is shown in Formula I, and its CAS No.: 21018-84-8. Amarogentin can effectively scavenge free radicals and protect cells from oxidative damage, thereby helping to prevent a variety of diseases related to oxidative stress. At the same time, amarogentin has also shown significant effects in anti-tumor and anti-diabetic aspects. In addition, it also shows certain effects in liver protection, immunomodulation and vascular metabolism. In summary, as a pleiotropic natural compound, Amarogentin has a very broad application prospect in the field of pharmacology. Summary of the invention

[0006] The object of the present invention is to improve the method for preparing stem cells with high proliferation ability and high stem cell stemness maintenance ability.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing stem cells with high proliferation ability, the method comprising the following steps:

[0009] (1) Inoculating stem cells into a cell culture dish coated with an extracellular matrix;

[0010] (2) adding a culture medium containing gentian glycoside to the cell culture dish;

[0011] (3) placing the cell culture dish in a cell culture incubator and continuing to culture for a certain period of time to obtain stem cells with high proliferation ability.

[0012] Preferably, the stem cells are bone marrow mesenchymal stem cells.

[0013] Preferably, the method for preparing the extracellular matrix-coated cell culture dish comprises the following steps:

[0014] (1) Bone marrow mesenchymal stem cells were seeded into a cell culture dish, complete culture medium containing 10% FBS was added, and the cell culture plate was placed in a cell culture incubator for culture;

[0015] (2) When the cell density reaches 75-90% or more, the culture medium is replaced with an extracellular matrix induction medium containing 40-100 μg / mL ascorbic acid and cultured for 12-14 days;

[0016] (3) Remove the extracellular matrix induction medium and add PBS containing 20mmol / L NH4OH and 0.5-1% Triton X-100 for decellularization at room temperature for 30 minutes;

[0017] (4) After washing with PBS 2-3 times, add PBS solution containing 100 U / mL DNase and place in a cell culture incubator for 1-2 hours;

[0018] (5) Remove the PBS solution containing DNase, wash with PBS 2-3 times, and obtain a cell culture dish coated with extracellular matrix.

[0019] Preferably, the extracellular matrix induction medium is an α-MEM medium containing 5 μmol / L forskolin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 200 μg / L recombinant human neuregulin β1, and 10% fetal bovine serum.

[0020] Preferably, the concentration of gentian glycoside in the gentian glycoside-containing culture medium is 25 μM-50 μM;

[0021] The culture medium is DMEM culture medium containing 10% FBS;

[0022] The certain culturing time is culturing for more than or equal to 48 hours.

[0023] Secondly, the present invention provides a method for improving the ability of stem cells to maintain stemness, the method comprising the following steps:

[0024] (1) Inoculating stem cells into a cell culture dish coated with an extracellular matrix;

[0025] (2) adding a culture medium containing gentian glycoside to the cell culture dish;

[0026] (3) placing the cell culture dish in a cell culture incubator, and when the cell density reaches about 80-90%, inoculating the cells into a culture dish coated with an extracellular matrix at a ratio of 1:2, and adding a culture medium containing gentianin for cell passage.

[0027] Preferably, the stem cells are bone marrow mesenchymal stem cells;

[0028] The extracellular matrix-coated cell culture dish is prepared according to the method for preparing the extracellular matrix-coated cell culture dish as claimed in claim 3;

[0029] The concentration of gentian glycoside in the gentian glycoside-containing culture medium is 25 μM-50 μM;

[0030] The culture medium is DMEM medium containing 10% FBS.

[0031] Secondly, the present invention provides the application of gentian glycosides in enhancing the application effect of extracellular matrix in stem cell culture, wherein the application effect of extracellular matrix in stem cell culture is the effect of the extracellular matrix in enhancing stem cell proliferation and maintaining stem cell stemness.

[0032] Preferably, the extracellular matrix is ​​used to coat a cell culture dish to obtain an extracellular matrix-coated cell culture dish;

[0033] The extracellular matrix-coated cell culture dish is prepared according to the method for preparing the extracellular matrix-coated cell culture dish as claimed in claim 3;

[0034] The method of using the gentian glycoside is to prepare a culture medium containing the gentian glycoside;

[0035] The concentration of gentioside in the gentioside-containing culture medium is 25 μM-50 μM.

[0036] The culture medium is DMEM medium containing 10% FBS.

[0037] Secondly, the present invention provides an application of a composition for enhancing the proliferation ability and the maintenance ability of stem cells, wherein the composition is composed of a culture medium containing gentian glycoside and a cell culture dish coated with an extracellular matrix;

[0038] The stem cells are bone marrow mesenchymal stem cells;

[0039] The concentration of gentian glycoside in the gentian glycoside-containing culture medium is 25 μM-50 μM;

[0040] The culture medium is DMEM medium containing 10% FBS.

[0041] The extracellular matrix-coated cell culture dish is prepared according to the method for preparing the extracellular matrix-coated cell culture dish described in claim 3.

[0042] The beneficial effects of the present invention are:

[0043] 1. The present invention provides a method for improving the proliferation ability of stem cells. Compared with the use of extracellular matrix or gentian glycoside alone, the combined use of the two can significantly improve the proliferation ability of bone marrow mesenchymal stem cells. This provides a new and effective strategy for obtaining a large number of high-quality stem cell populations.

[0044] 2. The present invention provides a method for maintaining and enhancing the stemness of stem cells. The synergistic effect of gentian glycosides and extracellular matrix can significantly upregulate the expression levels of stemness-related genes Sox2 and Oct4 in stem cells. This is conducive to delaying the loss of stemness of stem cells during long-term in vitro culture, thereby ensuring that stem cells maintain their multidirectional differentiation potential.

[0045] 3. The present invention reduces the dependence on extracellular matrix and reduces the culture cost. Traditional stem cell culture requires a large amount of extracellular matrix, but the acquisition process of extracellular matrix is ​​complicated and costly. The present invention provides a new method, which can reduce the demand for extracellular matrix through the synergistic effect of gentian glycosides and extracellular matrix, thereby reducing the culture cost.

[0046] 4. The present invention provides a new approach for the clinical application of stem cells. The synergistic effect of gentian glycosides and extracellular matrix revealed in the present invention not only helps to expand high-quality stem cells in vitro, but also is expected to improve the efficacy of stem cells in clinical applications such as regenerative medicine, providing a new technical approach for promoting the clinical transformation of stem cells.

[0047] 5. The present invention provides a new application field for gentian glycoside, expands the new use of the natural product gentian glycoside in the field of stem cell research and application, and lays a foundation for the in-depth development of the compound in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The results of the analysis of the proliferation ability of cells in different experimental treatment groups in Example 4;

[0049] Figure 2 The results of the analysis of the expression levels of the cell cycle protein Cyclin-D1 in the cells of the different experimental treatment groups in Example 5;

[0050] Figure 3 The relative expression level analysis results of the stemness gene Sox2 in cells of different experimental treatment groups in Example 6;

[0051] Figure 4 The relative expression level analysis results of the stem cell gene Oct4 in cells of different experimental treatment groups in Example 6. DETAILED DESCRIPTION

[0052] Next, we will explain the present invention in more detail with reference to the accompanying drawings and embodiments.

[0053] Example 1

[0054] (1) Bone marrow mesenchymal stem cells were inoculated into a cell culture dish, DMEM complete medium containing 10% FBS was added, and the cell culture plate was placed in a cell culture incubator at 37° C. and 5% CO 2 for culture;

[0055] (2) When the cell density reached more than 80%, the cells were replaced with an extracellular matrix induction medium containing 50 μg / mL ascorbic acid (α-MEM medium containing 5 μmol / L forskolin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 200 μg / L recombinant human neuregulin β1, and 10% fetal bovine serum) and cultured for 12 days until a white film appeared on the bottom of the cell culture dish;

[0056] (3) The extracellular matrix induction medium was removed and PBS containing 20 mmol / L NH4OH and 0.5% Triton X-100 was added for decellularization at room temperature for 30 min;

[0057] (4) Wash with PBS three times to remove cell debris, add PBS solution containing 100 U / mL DNase, place in a cell culture incubator, and treat for 2 h;

[0058] (5) Remove the PBS solution containing DNase, wash with PBS three times, and obtain a cell culture dish containing extracellular matrix.

[0059] Example 2

[0060] (1) Bone marrow mesenchymal stem cells were inoculated into a cell culture dish, DMEM complete medium containing 10% FBS was added, and the cell culture plate was placed in a cell culture incubator at 37° C. and 5% CO 2 for culture;

[0061] (2) When the cell density reached more than 75%, the cells were replaced with an extracellular matrix induction medium containing 40 μg / mL ascorbic acid (α-MEM medium containing 5 μmol / L forskolin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 200 μg / L recombinant human neuregulin β1, and 10% fetal bovine serum) and cultured for 14 days until a white film appeared on the bottom of the cell culture dish;

[0062] (3) The extracellular matrix induction medium was removed and PBS containing 20 mmol / L NH4OH and 1% Triton X-100 was added for decellularization at room temperature for 15 min;

[0063] (4) After washing with PBS 2-3 times to remove cell debris, add PBS solution containing 100 U / mL DNase and place in a cell culture incubator for 1 hour;

[0064] (5) Remove the PBS solution containing DNase, wash twice with PBS, and obtain a cell culture dish containing extracellular matrix.

[0065] Example 3

[0066] (1) Bone marrow mesenchymal stem cells were inoculated into a cell culture dish, DMEM complete medium containing 10% FBS was added, and the cell culture plate was placed in a cell culture incubator at 37° C. and 5% CO 2 for culture;

[0067] (2) When the cell density reached more than 90%, the cells were replaced with an extracellular matrix induction medium (α-MEM medium containing 5 μmol / L forskolin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 200 μg / L recombinant human neuregulin β1, and 10% fetal bovine serum) containing 100 μg / mL ascorbic acid and cultured for 14 days until a white film appeared on the bottom of the cell culture dish;

[0068] (3) The extracellular matrix induction medium was removed and PBS containing 20 mmol / L NH4OH and 1% Triton X-100 was added for decellularization at room temperature for 20 min;

[0069] (4) After washing with PBS to remove cell debris, add PBS solution containing 100 U / mL DNase and place in a cell culture incubator for 1.5 h;

[0070] (5) Remove the PBS solution containing DNase, wash with PBS three times, and obtain a cell culture dish containing extracellular matrix.

[0071] Example 4

[0072] (1) Cultivating bone marrow mesenchymal stem cells according to the following cell treatment method:

[0073] Control group: Bone marrow mesenchymal stem cells were inoculated into commercially available ordinary cell culture dishes (Corning cell culture dishes), and after adding DMEM complete medium, the culture dishes were placed in a cell culture incubator and cultured for 48 hours;

[0074] Treatment group 1: Bone marrow mesenchymal stem cells were seeded into commercially available ordinary cell culture dishes, and DMEM complete medium containing 1 μM gentianin (purchased from Vicogen Biotechnology Co., Ltd.) was added, and the culture dishes were placed in a cell culture incubator for 48 h;

[0075] Treatment group 2: Bone marrow mesenchymal stem cells were seeded into commercially available ordinary cell culture dishes, and after adding DMEM complete medium containing 25 μM gentianin, the culture dishes were placed in a cell culture incubator and cultured for 48 h;

[0076] Treatment group 3: Bone marrow mesenchymal stem cells were seeded into commercially available ordinary cell culture dishes, and DMEM complete medium containing 50 μM gentianin was added, and the culture dishes were placed in a cell culture incubator for 48 h;

[0077] Treatment group 4: Bone marrow mesenchymal stem cells were inoculated into a cell culture dish coated with an extracellular matrix according to the method of Example 1, and after adding DMEM complete medium, the culture dish was placed in a cell culture incubator and cultured for 48 hours;

[0078] Treatment group 5: Bone marrow mesenchymal stem cells were inoculated into a cell culture dish coated with an extracellular matrix according to the method of Example 1, and DMEM complete medium containing 1 μM gentianin was added, and the culture dish was placed in a cell culture incubator for 48 hours;

[0079] Treatment group 6: Bone marrow mesenchymal stem cells were seeded into a cell culture dish coated with an extracellular matrix according to the method of Example 1, and DMEM complete medium containing 25 μM gentianin was added, and the culture dish was placed in a cell culture incubator for 48 hours;

[0080] Treatment group 7: Bone marrow mesenchymal stem cells were inoculated into a cell culture dish coated with an extracellular matrix according to the method of Example 1, and DMEM complete medium containing 50 μM gentianin was added, and the culture dish was placed in a cell culture incubator for 48 hours;

[0081] (2) After the culture is completed, the culture medium of each group of cells is removed, trypsin is added to digest the cells, and each group of cells is prepared into a cell suspension;

[0082] (3) Inoculate 100 μL per well, with 1×10 cells 4 The cell suspension of each group was inoculated into a 96-well plate, and each group was set up with 3 replicate wells;

[0083] (4) Place the cell culture plate in a cell culture incubator and continue to culture for 48 hours, then add 10 μL of CCK-8 reagent to each well, place the 96-well cell culture plate in a cell culture incubator, and continue to incubate for 3 hours;

[0084] (5) After the incubation, the 96-well cell culture plate was placed in an ELISA reader to detect the absorbance of various cells at 450 nm, and the absorbance differences of various cells were calculated and compared.

[0085] The experimental results are as follows Figure 1 As shown in Table 1:

[0086] Table 1 Absorbance difference of cells after different treatments

[0087] Experimental treatment groups Absorbance (450nm) Control group 0.724±0.675 Treatment Group 1 0.727±0.063 Treatment Group 2 0.729±0.072 Treatment Group 3 0.735±0.065 Treatment Group 4 1.016±0.081 Treatment Group 5 1.032±0.076 Treatment Group 6 1.210±0.085 Treatment Group 7 1.345±0.071

[0088] By observation Figure 1 From the data in Table 1, we can draw the following conclusions: the absorbance of treatment group 1, treatment group 2 and treatment group 3 did not show significant differences compared with the control group. This indicates that simply adding different concentrations of gentian glycosides (1μM, 25μM and 50μM) to the culture medium has no significant effect on improving the proliferation capacity of bone marrow mesenchymal stem cells (BM-MSCs), and also proves that it will not cause obvious toxicity to cells at the above concentrations.

[0089] Compared with the control group, the absorbance of treatment group 3 increased. This result shows that the use of extracellular matrix (ECM)-coated cell culture dishes for culturing BM-MSCs can enhance the proliferation ability of cells. This may be because ECM provides cell attachment and proliferation conditions that are closer to the physiological environment.

[0090] In treatment group 5, even with the addition of 1 μM gentianin, the absorbance did not change significantly compared with treatment group 4. This suggests that the combined use of 1 μM gentianin and ECM-coated culture dishes did not further enhance the proliferation effect of BM-MSCs.

[0091] However, in treatment groups 6 and 7, the addition of 25 μM and 50 μM gentian resulted in a significant increase in absorbance, and this increase was statistically significant. This suggests that the combination of higher concentrations of gentian and ECM-coated culture dishes can significantly enhance the proliferation capacity of BM-MSCs. This synergistic effect may be due to the activation of certain cell signaling pathways by gentian at higher concentrations, which complements the cell proliferation-promoting effect of ECM.

[0092] Example 5

[0093] (1) extracting protein samples from cells cultured in the manner of the control group, treatment group 3, treatment group 4 and treatment group 7 in Example 4;

[0094] (2) Prepare 5% upper gel and 12% lower gel, and carefully assemble the electrophoresis tank, ensuring that all parts are properly installed and well sealed;

[0095] (3) Add the sample and marker to the corresponding electrophoresis wells and add the newly prepared electrophoresis buffer;

[0096] (4) Initially, the electrophoresis voltage was set to 80 V for 20 min to allow the sample to enter the gel; the voltage was then increased to 120 V for 90 min to complete the protein separation;

[0097] (5) Assemble the transfer cassette, make sure the PVDF membrane is correctly placed in the cassette, place the transfer cassette in the electroporation tank, and add enough electroporation solution;

[0098] (6) Set the current to 200 mA for 1.5 h to transfer the protein from the gel to the PVDF membrane;

[0099] (7) After the transfer, remove the PVDF membrane, soak it in 5% skim milk powder solution, and block it on a shaker at room temperature for 1 hour to reduce nonspecific binding;

[0100] (8) Remove the PVDF membrane and wash it with TBST buffer for 5 minutes each time, for a total of 3 times to remove the blocking solution;

[0101] (9) After the membrane is cut, it is mixed with the primary antibody dilution of Cyclin-D1 and GAPDH and incubated on a shaker at 4°C overnight to specifically bind to the target protein;

[0102] (10) Aspirate the primary antibody solution and wash the membrane again with TBST buffer for 5 minutes each time, for a total of 3 times;

[0103] (11) Mix the membrane with the secondary antibody and incubate on a shaker at room temperature for 1 hour to form a primary antibody-secondary antibody complex;

[0104] (12) In a dark room, quickly add a drop of luminescent liquid onto the PVDF membrane and develop the image to detect the expression of the target protein.

[0105] from Figure 2 From the data analysis, we can draw the following conclusions: when only 50 μM gentian glycosides were added to the culture medium, the gray value of the cell cycle protein Cyclin-D1 in treatment group 3 did not show a significant difference compared with the untreated control group. This observation suggests that the use of gentian glycosides alone may not be sufficient to significantly promote the expression of cell cycle protein Cyclin-D1 in bone marrow mesenchymal stem cells (BM-MSCs);

[0106] However, when we turn our attention to treatment group 4, the situation is different. In this treatment group, the gray value of the cell cycle protein Cyclin-D1 is increased compared with the control group, which suggests that the addition of extracellular matrix (ECM) may have a positive effect on the expression of Cyclin-D1 protein in BM-MSCs. This is consistent with the results of Example 4, further confirming the potential role of ECM in regulating stem cell function;

[0107] In addition, the experimental results of treatment group 7 were particularly striking. In this group, the expression level of Cyclin-D1 was not only increased compared with treatment group 4, but also significantly increased compared with the control group. This finding shows that although gentian glycosides have limited effects when used alone, when used in combination with ECM, they can significantly enhance the promotion effect of ECM on Cyclin-D1 expression in BM-MSCs. This synergistic effect helps to improve the proliferation capacity of stem cells, thus providing a new strategy for obtaining stem cells with higher proliferation potential.

[0108] Example 6

[0109] For stem cells, as the in vitro culture time increases, the stemness of the stem cells will gradually decrease until it disappears, which will affect its effectiveness in clinical practice. Therefore, the present invention determines whether the stem cells cultured with extracellular matrix and gentian glycosides have better stemness.

[0110] (1) Treat cells according to the following experimental groups:

[0111] Control group: Bone marrow mesenchymal stem cells were inoculated into commercially available common cell culture dishes, and after adding DMEM complete medium, the culture dishes were placed in a cell culture incubator. When the cell density reached about 80-90%, the cells were passaged at a ratio of 1:2 according to the same treatment, and the cells were passaged to the 9th generation for standby use;

[0112] Experimental group 1: Bone marrow mesenchymal stem cells were inoculated into commercially available ordinary cell culture dishes, and DMEM complete medium containing 50 μM gentian glycoside was added. The culture dishes were placed in a cell culture incubator. When the cell density reached about 80-90%, the cells were passaged at a ratio of 1:2 according to the same treatment, and the cells were passaged to the 9th generation for standby use;

[0113] Experimental group 2: Bone marrow mesenchymal stem cells were inoculated into a cell culture dish coated with an extracellular matrix according to the method of Example 1, and after adding DMEM complete medium, the culture dish was placed in a cell culture incubator. When the cell density reached about 80-90%, the cells were passaged at a ratio of 1:2 according to the same treatment, and the cells were passaged to the 9th generation for standby use;

[0114] Example 3: Bone marrow mesenchymal stem cells were inoculated into a cell culture dish coated with an extracellular matrix according to the method of Example 1, and after adding DMEM complete medium containing 50 μM gentian glycoside, the culture dish was placed in a cell culture incubator. When the cell density reached about 80-90%, the cells were passaged at a ratio of 1:2 according to the same treatment, and the cells were passaged to the 9th generation for standby use;

[0115] (2) Take cells from the control group, experimental group 1, experimental group 2, and experimental group 3, add Trizol reagent, and extract RNA;

[0116] (3) According to the operating steps in the reverse transcription kit manual, reverse transcribe the RNA of each group into cDNA;

[0117] (4) Prepare the fluorescence quantitative PCR reaction system according to the following ratio:

[0118] Table 2 Fluorescence quantitative PCR reaction system

[0119]

[0120] (5) First, the initial thermal reaction phase of PCR was performed at 37°C for 2 minutes to activate the required enzymes;

[0121] Next, in the second step, the sample is heated to 95°C and maintained for 5 minutes for DNA denaturation;

[0122] This is followed by the PCR amplification phase, which requires the execution of 40 cycles. Each cycle consists of two steps: first, a brief heating at 95°C for 10 seconds to ensure complete denaturation of the DNA; then, the temperature is lowered to 60°C for 30 seconds;

[0123] Finally, after the PCR amplification cycle was completed, a final thermal cycle was performed, which included three temperature stages: first, the temperature was raised to 95°C for 15 seconds for final DNA denaturation; then, the temperature was lowered to 60°C for 1 minute to allow for primer extension; finally, the temperature was raised to 95°C again for 15 seconds;

[0124] (6) Calculate the expression levels of stem cell stemness genes Sox2 and Oct4 in different experimental groups.

[0125] The results obtained are as follows: Figure 4 As shown in the figure, it can be seen that when gentiana glycosides are added to the culture medium alone, it does not have a significant promoting effect on the expression levels of Sox2 and Oct4 genes, which is similar to the cell proliferation results observed previously. However, when gentiana glycosides are used in combination with ECM, the situation changes significantly. Experimental data show that in stem cells under co-culture conditions, the expression levels of Sox2 and Oct4 are not only higher than those in the control group, but also significantly higher than those cells cultured only with ECM. This result shows that the combined use of gentiana glycosides and ECM can produce a synergistic effect that can effectively increase the expression of stemness genes in stem cells.

[0126] Of particular note is that this synergistic effect helps to solve the problem of reduced stem cell gene expression during long-term culture. By combining gentianin and ECM, we may have found a new way to maintain or restore stem cell stemness, which is of great significance for the long-term expansion and application of stem cells.

Claims

1. A method for preparing stem cells with high proliferation ability, wherein the method comprises the following steps: (1) Inoculate stem cells into a cell culture dish coated with extracellular matrix; (2) adding a culture medium containing gentian glycoside to the cell culture dish; (3) placing the cell culture dish in a cell culture incubator and continuing to culture for a certain period of time to obtain stem cells with high proliferation ability; The stem cells are bone marrow mesenchymal stem cells; The preparation method of the extracellular matrix-coated cell culture dish comprises the following steps: (1) Bone marrow mesenchymal stem cells were seeded into a cell culture dish, culture medium containing 10% FBS was added, and the cell culture plate was placed in a cell culture incubator for culture; (2) When the cell density reaches more than 75%, replace the culture medium with 40-100 μg / mL ascorbic acid for 12-14 days; (3) Remove the extracellular matrix induction medium and add PBS containing 20 mmol / L NH4OH and 0.5-1% Triton X-100 for decellularization at room temperature for 30 minutes; (4) After washing with PBS 2-3 times, add PBS solution containing 100U / mL DNase and place in a cell culture incubator for 1-2h; (5) Remove the PBS solution containing DNase, wash with PBS 2-3 times, and obtain a cell culture dish coated with extracellular matrix; The concentration of gentioside in the gentioside-containing culture medium is 25 μM-50 μM.

2. A method for preparing stem cells with high proliferation ability according to claim 1, characterized in that: The extracellular matrix induction medium is an α-MEM medium containing 5 μmol / L forskolin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 200 μg / L recombinant human neuregulin β1, and 10% fetal bovine serum.

3. A method for preparing stem cells with high proliferation ability according to claim 2, characterized in that: The certain culturing time is culturing for more than or equal to 48 hours.

4. Use of a composition for enhancing stem cell proliferation and maintenance ability, characterized in that: The composition consists of a culture medium containing gentian glycoside and a cell culture dish coated with an extracellular matrix; The stem cells are bone marrow mesenchymal stem cells; The concentration of gentian glycoside in the gentian glycoside-containing culture medium is 25 μM-50 μM; The extracellular matrix-coated cell culture dish is prepared according to the method for preparing the extracellular matrix-coated cell culture dish described in claim 1.

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