Liver stem cell culture method

Abstract

The invention discloses a liver stem cell culture method which comprises the following steps: separating to obtain liver tissue cells, inoculating the cells into a culture medium, and culturing to obtain proliferated liver stem cells, and the culture medium comprises dihydromyricetin-7-glucoside and methyl ursolate. According to the application disclosed by the invention, experiments find that efficient in-vitro amplification and high-purity culture of liver stem cells are realized by scientifically compounding dihydromyricetin-7-glucoside, methyl ursolate, ergobenzyl ester, trehalose, EGF (Epidermal Growth Factor), bFGF (Basic Fibroblast Growth Factor), glutamine, albumin, taurine, vitamin C and liver-derived proliferation promoting polypeptide in an RPMI-1640 basal culture medium; and the cell activity of the target cells is effectively maintained.

Description

Technical field

[0001] The present invention relates to the technical field of cell culture, and in particular to a method for culturing liver stem cells. [Background Technology]

[0002] The liver is an extremely important metabolic and detoxification organ in the human body, responsible for regulating the body's glucose and lipid metabolism, protein synthesis, and the elimination of various toxic substances. Liver damage not only endangers the body's homeostasis, but is also closely related to a variety of common diseases such as viral hepatitis, fatty liver, and drug-induced liver injury. The incidence of liver-related diseases continues to rise worldwide, seriously affecting patients' health and quality of life. For patients with advanced liver damage, liver transplantation remains the main treatment option, but is limited by donor scarcity, immune rejection, and high medical costs.

[0003] With advances in regenerative medicine and tissue engineering, liver stem cells, with their multidirectional differentiation potential, are considered ideal seed cells for liver tissue repair and disease treatment. The in vitro expansion and functional maintenance of liver stem cells has become a research hotspot for alternatives to liver transplantation, promoting liver regeneration, exploring the mechanisms of liver disease, and developing new cell-based therapies.

[0004] However, there are multiple difficulties in the in vitro culture of liver stem cells using existing technologies, and the results of in vitro culture of liver stem cells are not satisfactory. [Summary of the invention]

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a method for culturing liver stem cells. To achieve the above-mentioned object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for culturing liver stem cells, comprising isolating and obtaining liver tissue cells, inoculating the cells into a culture medium, and obtaining proliferated liver stem cells after culture, wherein the culture medium components include dihydromyricetin-7-glucoside and ursolic acid methyl ester.

[0007] Preferably, the culture medium includes the basic culture medium RPMI-1640, and also includes the following components: dihydromyricetin-7-glucoside: 15-30 μM, methyl ursolic acid: 60-80 μM, ergobenzyl ester: 50-200 mg / L, trehalose: 20-50 mg / L, EGF: 5-15 ng / mL, bFGF: 3-10 ng / mL, glutamine: 20-50 mg / L, albumin: 20-40 g / L, taurine: 50-200 mg / L, vitamin C: 50-150 mg / L.

[0008] Preferably, the culture medium further comprises 5-10 μg / mL of a hepatic proliferation-promoting polypeptide, the amino acid sequence of the hepatic proliferation-promoting polypeptide being Ac-CLGSPYRKLCETNKC-NH2, and being prepared by solid phase synthesis.

[0009] Preferably, the culture conditions are: temperature of 37° C., CO 2 concentration of 5%, medium replacement every 2 to 3 days, and total culture for 8 to 10 days.

[0010] Preferably, the cell seeding density is 1×10 4 ~5×10 4 pieces / cm 2 .

[0011] The present invention is different from the prior art in that the present invention achieves the following technical effects:

[0012] This study experimentally demonstrated that by combining dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and a liver-derived proliferation-promoting polypeptide in RPMI-1640 basal culture medium, efficient in vitro expansion and high-purity culture of liver stem cells were achieved, while effectively maintaining the cell viability of the target cells.

Brief Description of the Drawings

[0013] Figure 1 This is a photograph of morphological observation of the liver stem cells cultured in Example 1 of the present invention at the early stage of proliferation.

[0014] Figure 2 This is a photograph of the morphological observation of the liver stem cells cultured in Example 1 of the present invention at the mid-stage of proliferation.

[0015] Figure 3 This is a photograph of the morphological observation of the liver stem cells cultured in Example 1 of the present invention at the late stage of proliferation. [Specific implementation method]

[0016] The technical solutions and technical effects of the present invention are described in detail below in conjunction with specific embodiments and accompanying drawings. Experimental methods for which specific conditions are not specified are generally performed under conventional conditions, such as those described in textbooks and experimental guides, or under conditions recommended by manufacturers, and are well known or easily known to those of ordinary skill in the art. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. The following examples are only preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0017] Source of raw materials: intact fetuses aborted 3 to 4 months old, donated voluntarily with informed consent from the providers; fetal hepatitis virus markers, syphilis, and HIV tests were all negative.

[0018] Example 1

[0019] Example 1 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, benzyl ergosterol: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0020] A method for culturing liver stem cells, wherein the specific culturing process is as follows:

[0021] (1) Cell separation:

[0022] Aseptically remove liver tissue, rinse with PBS 3-5 times, and mince. Add the tissue pieces to a mixture of 0.02% (w / v) type I collagenase and 0.02% type II collagenase in PBS and gently digest at 37°C for 30 minutes. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 minutes, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3×10 4 pieces / cm 2 .

[0023] (2) Cell inoculation and culture medium preparation:

[0024] Prepare a culture medium with the above components and proportions.

[0025] (3) Culture conditions:

[0026] Incubate at 37°C, 5% CO2 in a humidified atmosphere, changing the medium every 2 days for a total of 9 days.

[0027] Cell morphology diagram see Figure 1-3 .

[0028] Example 2

[0029] Example 2 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 15 μM, methyl ursolic acid: 60 μM, benzyl ergosterol: 50 mg / L, trehalose: 20 mg / L, EGF: 5 ng / mL, bFGF: 3 ng / mL, glutamine: 20 mg / L, albumin: 20 g / L, taurine: 50 mg / L, and vitamin C: 50 mg / L.

[0030] A method for culturing liver stem cells, wherein the specific culturing process is as follows:

[0031] (1) Cell separation:

[0032] Aseptically remove liver tissue, rinse with PBS 3-5 times, and mince. Add the tissue pieces to a mixture of 0.02% (w / v) type I collagenase and 0.02% type II collagenase in PBS and gently digest at 37°C for 30 minutes. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 minutes, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3×10 4 pieces / cm 2 .

[0033] (2) Cell inoculation and culture medium preparation:

[0034] Prepare a culture medium with the above components and proportions.

[0035] (3) Culture conditions:

[0036] Incubate at 37°C, 5% CO2 in a humidified atmosphere, changing the medium every 2 days for a total of 8 days.

[0037] Example 3

[0038] Example 3 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 30 μM, methyl ursolic acid: 80 μM, benzyl ergosterol: 200 mg / L, trehalose: 50 mg / L, EGF: 15 ng / mL, bFGF: 10 ng / mL, glutamine: 50 mg / L, albumin: 40 g / L, taurine: 200 mg / L, and vitamin C: 150 mg / L.

[0039] A method for culturing liver stem cells, wherein the specific culturing process is as follows:

[0040] (1) Cell separation:

[0041] Aseptically remove liver tissue, rinse with PBS 3-5 times, and mince. Add the tissue pieces to a mixture of 0.02% (w / v) type I collagenase and 0.02% type II collagenase in PBS and gently digest at 37°C for 30 minutes. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 minutes, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3×10 4 pieces / cm 2 .

[0042] (2) Cell inoculation and culture medium preparation:

[0043] Prepare a culture medium with the above components and proportions.

[0044] (3) Culture conditions:

[0045] Incubate at 37°C, 5% CO2 in a humidified atmosphere, changing the medium every 2 days for a total of 10 days.

[0046] Example 4

[0047] Example 4 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and a hepatic proliferation-promoting polypeptide. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, benzyl ergosterol: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, vitamin C: 94 mg / L, and 8 μg / mL of the hepatic proliferation-promoting polypeptide.

[0048] Among them, the amino acid sequence of the liver-derived proliferation-promoting polypeptide is Ac-CLGSPYRKLCETNKC-NH2, its N-terminus is acetylated, its C-terminus is amidated, and it contains a cysteine ​​residue that can form a disulfide bond. It is prepared by solid-phase synthesis.

[0049] A method for culturing liver stem cells, wherein the specific culturing process is as follows:

[0050] (1) Cell separation:

[0051] Aseptically remove liver tissue, rinse with PBS 3-5 times, and mince. Add the tissue pieces to a mixture of 0.02% (w / v) type I collagenase and 0.02% type II collagenase in PBS and gently digest at 37°C for 30 minutes. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 minutes, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3×10 4 pieces / cm 2 .

[0052] (2) Cell inoculation and culture medium preparation:

[0053] Prepare a culture medium with the above components and proportions.

[0054] (3) Culture conditions:

[0055] Incubate at 37°C, 5% CO2 in a humidified atmosphere, changing the medium every 2 days for a total of 9 days.

[0056] Example 5

[0057] Example 5 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 15 μM, methyl ursolic acid: 60 μM, benzyl ergosterol: 50 mg / L, trehalose: 20 mg / L, EGF: 5 ng / mL, bFGF: 3 ng / mL, glutamine: 20 mg / L, albumin: 20 g / L, taurine: 50 mg / L, vitamin C: 50 mg / L, and 5 μg / mL of a liver-derived proliferation-promoting polypeptide.

[0058] The amino acid sequence of the liver-derived proliferation-promoting polypeptide is Ac-CLGSPYRKLCETNKC-NH2, and it is prepared by solid-phase synthesis.

[0059] A method for culturing liver stem cells, wherein the specific culturing process is as follows:

[0060] (1) Cell separation:

[0061] Aseptically remove liver tissue, rinse with PBS 3-5 times, and mince. Add the tissue pieces to a mixture of 0.02% (w / v) type I collagenase and 0.02% type II collagenase in PBS and gently digest at 37°C for 30 minutes. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 minutes, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3×10 4 pieces / cm 2 .

[0062] (2) Cell inoculation and culture medium preparation:

[0063] Prepare a culture medium with the above components and proportions.

[0064] (3) Culture conditions:

[0065] Incubate at 37°C, 5% CO2 in a humidified atmosphere, changing the medium every 2 days for a total of 9 days.

[0066] Example 6

[0067] Example 6 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 30 μM, methyl ursolic acid: 80 μM, benzyl ergosterol: 200 mg / L, trehalose: 50 mg / L, EGF: 15 ng / mL, bFGF: 10 ng / mL, glutamine: 50 mg / L, albumin: 40 g / L, taurine: 200 mg / L, vitamin C: 150 mg / L, and 10 μg / mL of a liver-derived proliferation-promoting polypeptide.

[0068] The amino acid sequence of the liver-derived proliferation-promoting polypeptide is Ac-CLGSPYRKLCETNKC-NH2, and it is prepared by solid-phase synthesis.

[0069] A method for culturing liver stem cells, wherein the specific culturing process is as follows:

[0070] (1) Cell separation:

[0071] Aseptically remove liver tissue, rinse with PBS 3-5 times, and mince. Add the tissue pieces to a mixture of 0.02% (w / v) type I collagenase and 0.02% type II collagenase in PBS and gently digest at 37°C for 30 minutes. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 minutes, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3×10 4 pieces / cm 2 .

[0072] (2) Cell inoculation and culture medium preparation:

[0073] Prepare a culture medium with the above components and proportions.

[0074] (3) Culture conditions:

[0075] Incubate at 37°C, 5% CO2 in a humidified atmosphere, changing the medium every 2 days for a total of 9 days.

[0076] Comparative Example 1

[0077] Comparative Example 1 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, benzyl ergosterol: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0078] The culture medium of Comparative Example 1 used dihydromyricetin instead of dihydromyricetin-7-glucoside, and the rest was the same as that of Example 1.

[0079] Comparative Example 2

[0080] Comparative Example 2 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, ergobenzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, ergobenzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0081] The culture medium of Comparative Example 2 was the same as that of Example 1 except that methyl ursolic acid was not used.

[0082] Comparative Example 3

[0083] Comparative Example 3 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0084] The culture medium of Comparative Example 3 did not use ergobenzyl ester and trehalose, and the rest was the same as that of Example 1.

[0085] Comparative Example 4

[0086] Comparative Example 4 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 7 μM, methyl ursolic acid: 30 μM, benzyl ergosterol: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0087] The culture medium of Comparative Example 4 reduced the dosage of dihydromyricetin-7-glucoside and ursolic acid methyl ester, and the rest was the same as that of Example 1.

[0088] Comparative Example 5

[0089] Comparative Example 5 provides a culture medium for liver stem cells, comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, benzyl ergosterol, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 45 μM, methyl ursolic acid: 130 μM, benzyl ergosterol: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0090] The culture medium of Comparative Example 5 was increased in the amount of dihydromyricetin-7-glucoside and ursolic acid methyl ester, and the rest was the same as that of Example 1.

[0091] Comparative Example 6

[0092] Comparative Example 6 provides a culture medium for liver stem cells, the culture medium comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, ursolic acid methyl ester, ergobenzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and a hepatic proliferation-promoting polypeptide. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, ursolic acid methyl ester: 73 μM, ergobenzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, vitamin C: 94 mg / L, and 2 μg / mL of hepatic proliferation-promoting polypeptide.

[0093] The culture medium of Comparative Example 6 reduced the amount of liver-derived proliferation-promoting polypeptide used, and the rest was the same as that of Example 4.

[0094] Comparative Example 7

[0095] Comparative Example 7 provides a culture medium for liver stem cells, the culture medium comprising a basal medium RPMI-1640 and the following components added to the basal medium RPMI-1640: dihydromyricetin-7-glucoside, ursolic acid methyl ester, ergobenzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and a hepatic proliferation-promoting polypeptide. The contents of the above components in the basal medium RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, ursolic acid methyl ester: 73 μM, ergobenzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, vitamin C: 94 mg / L, and 24 μg / mL of hepatic proliferation-promoting polypeptide.

[0096] The culture medium of Comparative Example 7 was the same as that of Example 4 except that the amount of the liver-derived proliferation-promoting polypeptide was increased.

[0097] Experimental testing

[0098] (1) Cell proliferation rate

[0099] The number of liver stem cells obtained in Examples 1 to 6 and Comparative Examples 1 to 7 after the culture was completed was counted using a Countster cell counter, and the cell proliferation rate was calculated. All data were obtained from three independent experiments, each with three replicates. The data are expressed as mean ± standard deviation. The results are shown in Table 1 below.

[0100]

[0101]

[0102] Analysis of Table 1 shows that the cell proliferation multiples of Examples 4, 6 and 5 are significantly higher than those of all control groups, indicating that the combination of liver-derived proliferation-promoting polypeptide with dihydromyricetin-7-glucoside and ursolic acid methyl ester has a synergistic effect and can significantly enhance the in vitro expansion capacity of liver stem cells.

[0103] Although Examples 1, 3, and 2 do not contain liver-derived proliferation-promoting polypeptides, the proliferation multiples are significantly better than those of the traditional control examples, indicating that the combination of dihydromyricetin-7-glucoside and ursolic acid methyl ester, as well as dosage optimization, also plays a key role in promoting the proliferation of liver stem cells.

[0104] Although Comparative Example 6 contains a liver-derived proliferation-promoting polypeptide, the proliferation effect is significantly lower than that of the optimal embodiment due to the low dosage of the polypeptide. In Comparative Examples 3, 4, 5, and 7, the cell proliferation ability is inhibited or restricted to varying degrees due to the lack of key components of the present invention or unreasonable dosage settings, further illustrating that the key components are indispensable and that there is a window effect in dosage.

[0105] Comparative Example 1 and Comparative Example 2 lack the key components of the present invention and have the weakest proliferation capabilities.

[0106] Therefore, the comparison results further verified that the key ingredients and dosage ratios in the formula selected by the present invention are the best choices and cannot be arbitrarily reduced or increased blindly, otherwise the cell proliferation effect will decrease.

[0107] (2) Cell purity analysis

[0108] The liver stem cells of Example 4, Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 6 were collected and digested with 0.25% trypsin containing 1 mmol / L EDTA. The cells were centrifuged and washed twice with PBS. The cells were resuspended in PBS buffer and the cell density was adjusted to 1×10 6 The cells were diluted with 100 μL of PBS per tube. FITC- or PE-labeled monoclonal antibodies to CD34, CD44, CD90, CD105, CD13, HLA-ABC, and HLA-DR were added, mixed gently, and incubated at room temperature in the dark for 15–20 minutes. A negative control tube (no antibody added, an equal volume of PBS buffer) was set up. After washing once with PBS, the cells were resuspended in 400 μL of PBS and analyzed on a BD flow cytometer. Flow cytometric data were analyzed using software. Each group was repeated three times, and the mean ± standard deviation of the positive rate for each antigen was calculated. The results are shown in Table 2 below.

[0109]

[0110] The expression levels of typical liver stem cell surface markers CD44, CD90, CD105 and CD13 in Example 4 were the highest, and the expression levels of negative markers CD34 and HLA-DR were the lowest, which were significantly higher than those in other examples and comparative groups. This fully demonstrates that the culture method of the present invention not only expands the number of cells, but also significantly improves the phenotypic specificity and purity of stem cells. Although the key polypeptide was not added to Example 1, the stem cell phenotype of the cells was still high, significantly higher than that of all comparative groups, reflecting the importance of the core small molecule combination of the present invention (dihydromyricetin-7-glucoside + ursolic acid methyl ester); the stem cell specificity of Comparative Example 3 was In comparison example 1, the expression of stem cell markers was significantly reduced, while the expression of CD34 and HLA-DR was significantly increased, indicating that ergobenzyl ester and trehalose played an irreplaceable auxiliary role in maintaining cell stemness, and their deletion significantly reduced the cell purity; in comparison example 2, the positive stem cell markers were further reduced, while CD34 and HLA-DR were significantly increased, indicating that ursolic acid methyl ester was crucial for maintaining the stem cell phenotype; comparison example 1 performed the worst, with the lowest stem cell-specific markers and the highest differentiation or impurity cell markers CD34 and HLA-DR, indicating that dihydromyricetin-7-glucoside is a key component necessary for maintaining stem cell characteristics and cannot be replaced at will.

[0111] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for culturing liver stem cells, comprising isolating liver tissue cells, inoculating the cells into a culture medium, and culturing to obtain proliferated liver stem cells, characterized in that: The culture medium components include dihydromyricetin-7-glucoside and ursolic acid methyl ester.

2. The method for culturing liver stem cells according to claim 1, wherein: The culture medium includes the basic culture medium RPMI-1640, and also includes the following components: dihydromyricetin-7-glucoside: 15-30 μM, ursolic acid methyl ester: 60-80 μM, ergobenzyl ester: 50-200 mg / L, trehalose: 20-50 mg / L, EGF: 5-15 ng / mL, bFGF: 3-10 ng / mL, glutamine: 20-50 mg / L, albumin: 20-40 g / L, taurine: 50-200 mg / L, and vitamin C: 50-150 mg / L.

3. The method for culturing liver stem cells according to claim 2, wherein: The culture medium further comprises 5 to 10 μg / mL of a hepatic proliferation-promoting polypeptide, the amino acid sequence of the hepatic proliferation-promoting polypeptide being Ac-CLGSPYRKLCETNKC-NH2, and being prepared by a solid phase synthesis method.

4. The method for culturing liver stem cells according to claim 1, wherein The culture conditions are: temperature of 37° C., CO 2 concentration of 5%, medium replacement every 2 to 3 days, and total culture for 8 to 10 days.

5. The method for culturing liver stem cells according to claim 1, wherein The cell seeding density was 1×10 4 ~5×10 4 pieces / cm 2 .

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