WO2021184408A1 - 胃癌原代细胞的培养基及培养方法 - Google Patents

胃癌原代细胞的培养基及培养方法 Download PDF

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WO2021184408A1
WO2021184408A1 PCT/CN2020/081414 CN2020081414W WO2021184408A1 WO 2021184408 A1 WO2021184408 A1 WO 2021184408A1 CN 2020081414 W CN2020081414 W CN 2020081414W WO 2021184408 A1 WO2021184408 A1 WO 2021184408A1
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cells
gastric cancer
medium
concentration
primary
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刘青松
黄涛
陈程
王文超
任涛
王黎
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合肥中科普瑞昇生物医药科技有限公司
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Definitions

  • the invention relates to the field of biotechnology, in particular to a culture medium and a culture method for rapid expansion of gastric cancer primary cells, and its application in the evaluation and screening of drug efficacy.
  • Gastric cancer is the most common malignant gastrointestinal tumor. Patients with early gastric cancer have no obvious symptoms. Therefore, most gastric cancer patients have developed to the middle and late stages when they are diagnosed. Gastric cancer cells are less sensitive to chemotherapy drugs, so their chemotherapy effects are poor, resulting in a low five-year survival rate. In recent years, with the development of science and technology, gastric cancer diagnosis and treatment technology has been continuously improved, especially the progress of comprehensive treatment methods such as surgery, endoscopy, radiotherapy and chemotherapy, and targeted therapy. For patients with gastric cancer with metastatic lesions, the prognosis of comprehensive treatment is still unsatisfactory.
  • Xuefeng Liu et al. used irradiated mouse fibroblasts and Rho-related kinase inhibitor (Y-27632) to amplify epithelial-derived cells. This system has the ability to achieve unlimited growth of epithelial-derived cells without genetic manipulation.
  • Xuefeng Liu et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nat.Protoc.2017,12,439
  • the method established by Xuefeng Liu et al. has a long culture period and cannot achieve rapid cell expansion, which limits the application of this technology.
  • the present invention provides a culture medium and a culture method for rapidly expanding gastric cancer primary cells in vitro.
  • One aspect of the present invention is to provide a culture medium for primary gastric cancer cells, the culture medium comprising an initial medium, Rho protease inhibitors, antibiotics, insulin, non-essential amino acids, hydrocortisone, cholera toxin, and glutamine , Fetal Bovine Serum (FBS), and at least one additive selected from B27 additives and N2 additives.
  • the culture medium comprising an initial medium, Rho protease inhibitors, antibiotics, insulin, non-essential amino acids, hydrocortisone, cholera toxin, and glutamine , Fetal Bovine Serum (FBS), and at least one additive selected from B27 additives and N2 additives.
  • FBS Fetal Bovine Serum
  • the initial medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640.
  • At least one additive selected from the group consisting of B27 additives and N2 additives is preferably B27 additives, or B27 additives and N2 additives.
  • Rho protein kinase inhibitor is selected from one or more of Y27632, hydroxyfasudil and GSK429286A, when selected from Y27632, the concentration range is 2.5-40 ⁇ M, preferably 10-20 ⁇ M; when selected from hydroxyfasudil In the case of diltiazem, the concentration range is 2 to 32 ⁇ M, preferably 4 to 16 ⁇ M; when selected from GSK429286A, the concentration range is 2 to 32 ⁇ M, preferably 4 to 16 ⁇ M;
  • the antibiotic is selected from one or more of streptomycin/penicillin, amphotericin B and Primocin.
  • streptomycin/penicillin the concentration of streptomycin ranges from 25 to 400 ⁇ g/mL, preferably 50 ⁇ 200 ⁇ g/mL, more preferably 200 ⁇ g/m
  • the penicillin concentration range is 25 ⁇ 400U/mL, preferably 50 ⁇ 200U/mL, more preferably 200U/mL
  • the concentration range is 0.25 ⁇ 4 ⁇ g/mL, preferably 0.5-2 ⁇ g/mL
  • Primocin is selected, the concentration range is 25-400 ⁇ g/mL, preferably 50-200 ⁇ g/mL;
  • the concentration of insulin ranges from 5 to 20 ⁇ g/mL, preferably 10 to 20 ⁇ g/mL;
  • Non-essential amino acids are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the total concentration of non-essential amino acids is 50 ⁇ 800 ⁇ M, preferably 100 ⁇ 400 ⁇ M;
  • the concentration range of hydrocortisone is 0.1-1.6 ⁇ g/mL, preferably 0.2-0.8 ⁇ g/mL;
  • the concentration of cholera toxin ranges from 0.1 to 0.4 nM, preferably 0.1 to 0.2 nM
  • the concentration of glutamine ranges from 1 to 8 mM, preferably 1 to 4 mM;
  • the volume ratio of fetal bovine serum (FBS) to the culture medium is 1:5 ⁇ 1:40;
  • the concentration range of the B27 additive is in a volume ratio of 1:25 to 1:400, preferably 1:25 to 1:200;
  • the volume ratio of the N2 additive to the medium is 1:25 to 1:400, preferably 1:25 to 1:100.
  • the invention also provides a method for culturing gastric cancer primary cells.
  • the gastric cancer primary cell culture medium of the present invention is used to culture the gastric cancer primary cells.
  • trophoblast cells are added at a cell density of 2 to 3 ⁇ 10 4 cells/cm 2.
  • the trophoblast cells are irradiated NIH-3T3 cells, the irradiation source is X-ray or ⁇ -ray, and the irradiation dose is 20-50Gy.
  • the gastric cancer primary cell culture method of the present invention further includes the following steps:
  • tissue samples such as endoscopic specimens
  • tissue cleaning solution for digestion
  • tissue digestion solution for example, use 8-14ml, preferably 12ml tissue digestion
  • the digestion temperature range is 4 degrees Celsius to 37 degrees Celsius, preferably 37 degrees Celsius
  • the digestion speed range is 200 rpm to 350 rpm, preferably 300 rpm;
  • the digestion time range is 4-8 hours, preferably 6 hours;
  • the centrifugal speed range is 1200-1600rpm, preferably 1500rpm; the centrifugal time range is 2-6 minutes, preferably 5 minutes Among them, the serum-containing initial medium can be, for example, DMEM/F12 medium containing 10% fetal bovine serum.
  • the gastric cancer primary cells obtained in the above step 1 are resuspended and counted in the gastric cancer primary cell culture medium of the present invention, and planted in a culture dish according to the cell density of 5-10 ⁇ 10 4 cells/cm 2 , and at the same time according to the cell density 2 Add trophoblast cells to ⁇ 3 ⁇ 10 4 cells/cm 2 and add trophoblast cells at a cell density of 0.5-1 ⁇ 10 4 cells/cm 2 after culturing for 5-7 days, until the cells in the culture dish are more than 85% and can be digested and passaged .
  • the formula of the tissue cleaning solution described in step 1 is: DMEM/F12 basal medium containing 100-200 ⁇ g/mL Primocin, 2% penicillin/streptomycin solution; the preparation method of tissue digestion solution described in step 1 For: 1 ⁇ 2mg/mL collagenase II, 1 ⁇ 2mg/mL collagenase IV, 50 ⁇ 100U/mL deoxyribonucleic acid I, 0.5 ⁇ 1mg/mL hyaluronidase, 1 ⁇ 3mM calcium chloride, 1 ⁇ 2% bovine serum albumin is dissolved in HBSS and RPMI-1640 at a volume ratio of 1:1; the trophoblast cells described in step 2 can be, for example, irradiated NIH-3T3 cells, and the irradiation source is X-ray or ⁇ -ray , Preferably gamma rays, and the irradiation dose is 20-50 Gy, preferably 30 Gy.
  • the present invention also provides a drug screening method for gastric cancer disease, which includes the following steps:
  • the cultured primary gastric cancer cells are not interfered by interstitial cells such as fibroblasts and adipocytes;
  • the amplified primary gastric cancer cells can also be serially passaged;
  • the culture cost is controllable: the culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10 and other factors, which is a simplification and improvement of the existing gastric cancer primary cell culture medium;
  • Figure 1 is a graph showing the effects of different additive factor combinations in the gastric cancer primary cell culture medium on the proliferation of gastric cancer primary cells.
  • Figure 2 is a graph showing the effect of the concentration of added factors in the gastric cancer primary cell culture medium on the proliferation of gastric cancer primary cells.
  • Fig. 3 is a photograph of gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention under a microscope.
  • Fig. 4 is the result of Wright Giemsa staining identification on gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention.
  • Fig. 5 is the result of cellular immunofluorescence staining of gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention.
  • Fig. 6 is a cell growth curve of gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention.
  • Fig. 7 is a comparison result of culturing gastric cancer primary cells using the gastric cancer primary cell culture medium of the present invention and the existing culture medium.
  • Figure 8 shows the results of culturing gastric cancer cells using the gastric cancer primary cell culture medium of the present invention and using cells of different generations for drug screening.
  • Example 1 The effect of each added factor in the gastric cancer primary cell culture medium on the proliferation of gastric cancer primary cells
  • the formula of the basic medium is: DMEM/F12 medium (purchased from Corning) + 10 ⁇ M Y27632 (purchased from MCE) + 100 ⁇ g/mL Primocin (purchased from InvivoGen).
  • Intraoperative/endoscopy were obtained from patients by professional medical staff in professional medical institutions, and all patients signed an informed consent form. Intraoperative samples were 2 soybean grains in size and endoscopic samples were rice grains; commercial tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.
  • NIH 3T3 cells purchased from ATCC irradiated with a dose of 30Gy gamma rays; disinfected surfaces such as 15mL sterile centrifuge tubes, pipettes, 10mL pipettes, sterile tips, etc., and put them in the ultra-clean workbench for 30 minutes of ultraviolet irradiation; Take out the basal medium from the refrigerator at 4°C 30 minutes in advance, and remove the tissue digestion solution from the refrigerator at -20°C 30 minutes in advance, where:
  • Tissue digestion fluid DMEM/F12 medium, collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75mg/mL), calcium chloride (3.3mM), BSA (10mg/mL).
  • the above-mentioned collagenase II was purchased from Sigma; Collagenase IV was purchased from Sigma; DNase was purchased from Sigma; Hyaluronidase was purchased from Sigma; Calcium chloride was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd. ; BSA was purchased from Biofroxx.
  • “+” means that compared with the basal medium, the medium added with the additive can promote the proliferation of three cases of gastric cancer primary cells isolated from gastric cancer tissue; “-” means the medium added with the additive It has an inhibitory effect on at least two cases of gastric cancer primary cells isolated from gastric cancer tissue; “ ⁇ ” means that the medium with the additive has an effect on at least two cases of gastric cancer primary cells isolated from gastric cancer tissue. Proliferation has no obvious effect.
  • Example 2 The effect of the combination of different added factors in the gastric cancer primary cell culture medium on the proliferation of gastric cancer primary cells
  • the gastric cancer primary cell culture medium with different additive factor combinations was prepared according to the ingredients in Table 2, and the proliferation-promoting effect of different additive factor combinations on gastric cancer primary cells was investigated.
  • the FBS mentioned above was purchased from Excell Company.
  • Example 4 Obtain gastric cancer primary cells from gastric cancer tissue (sample 4) according to the method of step (2) of Example 1, and divide the obtained cell suspension into 9 parts. Centrifuge at 1500 rpm for 4 minutes. After centrifugation, use 200 microliters of cells respectively.
  • BM, No.1 ⁇ 7 and GM-1 medium were resuspended and seeded in a 48-well plate (10,000 cells per well) at a viable cell density of 1 ⁇ 10 4 cells/cm 2, and then at a cell density of 2 ⁇ 10 4 cells/cm 2 were added to NIH-3T3 cells irradiated with gamma rays (irradiation dose 30Gy), and finally the volume of each well in the 48-well plate was supplemented to 500 microliters with the corresponding medium, and mixed thoroughly. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. Passage is performed until the cells in the 48-well plate grow to more than 85%.
  • the No. 1 to No. 7 and GM-1 medium can promote the proliferation of gastric cancer primary cells to varying degrees.
  • gastric cancer primary cell culture medium ie GM-1 medium
  • Primocin, insulin, hydrocortisone, non-essential amino acids, glutamine, cholera toxin, FBS, and B27 additives to culture gastric cancer primary cells .
  • the proliferation effect is significantly improved.
  • Example 3 The effect of different concentrations of added factors on the proliferation of primary gastric cancer cells
  • gastric cancer primary cells from endoscopic tissues (sample 5, sample 6, sample 7, sample 8 and sample 9) according to the method of step (2) of the 3 in Example 1, and use the GM- in Example 2 1
  • the primary gastric cancer cells obtained by culture in the medium are seeded in a 12-well plate (45,000 cells per well) at a viable cell density of 1 ⁇ 10 4 cells/cm 2 at a cell density of 2 ⁇ 10 4 cells/cm 2
  • Add NIH-3T3 cells irradiated with gamma rays (irradiation dose 30Gy) After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation.
  • Formulation 4 The GM-1 medium component in Example 2 does not contain insulin
  • Formulation 5 The GM-1 medium component in Example 2 does not contain cholera toxin;
  • Formulation 7 The GM-1 medium component in Example 2 does not contain Y27632;
  • Formulation 8 The GM-1 medium component in Example 2 does not contain FBS;
  • the ratio is the ratio of the number of cells obtained by culturing for one generation with each medium to the number of cells obtained by culturing for one generation in the corresponding control wells.
  • a ratio greater than 1 indicates that the proliferation-promoting effect of the formulated medium containing different concentrations of factors is better than that of the control well medium, and the ratio is less than 1, indicating that the proliferation-promoting effect of the formulated medium containing different concentrations of factors is weaker than that of the control well medium. .
  • the concentration of non-essential amino acids ranges from 50 to 800 ⁇ M, and the concentration of 400 ⁇ M is the most effective for cell proliferation; the concentration of glutamine ranges from 1 to 8 mM, and the concentration of 4 mM is the most effective for cell proliferation; the concentration range of B27 is 1:25 ⁇ 1:400, the concentration of 1:50 is the most obvious for cell proliferation; the insulin concentration range is 5 ⁇ 20 ⁇ g/mL, the concentration of 10 ⁇ g/mL is the most obvious cell proliferation effect; the cholera toxin concentration range is 0.1 ⁇ 0.4nM, the concentration of 0.1nM is the most obvious cell proliferation effect; the concentration of hydrocortisone is 0.1 ⁇ 1.6 ⁇ g/mL, the concentration of 0.8 ⁇ g/mL is the most obvious cell proliferation effect; the concentration of Y27632 is 2.5 ⁇ 40 ⁇ M, the concentration Adding 10 ⁇ M to the cell proliferation effect is the most obvious; the FBS concentration range is 1:5 ⁇ 1:40, the concentration is 1:10, the cell
  • Example 10 Obtain primary gastric cancer cells from the endoscopic tissue sample (sample 10) according to the method of step (2) of Example 1, and use the GM-1 medium in Example 2 for culture.
  • the obtained primary gastric cancer cells according to the viable cell density 1 ⁇ 10 4 pieces / cm 2 seeded in 12 well plates (45,000 cells per well), in accordance with a cell density of 2 ⁇ 10 4 pieces / cm 2 by the addition of ⁇ -ray irradiation (30Gy irradiation dose ) NIH-3T3 cells, mix well. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation.
  • Figure 3 (A) and (B) are photos taken under a 4x objective lens and a 10x objective lens, respectively.
  • the cells are in the microscope.
  • the bottom is closely arranged, and the shape is slightly irregular.
  • the cultured cells were rinsed with 200 microliters of 0.05% trypsin (purchased from Gibco) for 1 minute, and after aspiration, 500 microliters of 0.05% trypsin (purchased from Gibco) was added to each well. Company), placed in a 37°C, 5% CO 2 incubator for 10 minutes until the cells have been completely digested can be observed under the microscope (Invitrogen’s EVOS M500). Resuspend in 500 microliters of GM-1 medium to identify the primary gastric cancer cells obtained from the culture.
  • the dyeing method is as follows.
  • Figure 4 shows the results of Wright Giemsa staining identification of gastric cancer primary cells cultured in vitro.
  • (A) and (B) of Figure 4 are photos taken with a 4x objective lens and a 10x objective lens, respectively.
  • the cell nucleus is large and deeply stained, which is in line with the characteristics of cancer cells.
  • Immunofluorescence staining was performed on the primary gastric cancer cells after the sample was cultured.
  • the dyeing method is as follows.
  • the cultured primary gastric cancer cells were seeded on cell slides (purchased from Thermo Fisher), and placed in a 37°C, 5% CO 2 incubator for culture, and waited for the cells to adhere to the wall.
  • PBS+0.3% Triton X-100 was used to prepare 5% BSA (purchased from Shanghai Shenggong) for blocking, and blocking at 37°C for 30 minutes.
  • Dilute DAPI (purchased from Sigma) in 1:1000 PBS, stain for 5 minutes at room temperature and avoid light, and wash with PBS for 5 minutes x 3 times.
  • Image under microscope Invitrogen EVOS M500, take pictures and record.
  • Figure 5 shows the results of immunofluorescence staining identification of primary gastric cancer cells cultured in vitro, respectively, which are pictures taken by fluorescence under a 10x objective lens.
  • Pan-keratin, MUC1, and P63 are highly expressed, and Napsina is low, indicating that the sample is gastric squamous cell carcinoma. The result is consistent with the hospital pathological diagnosis.
  • Example 5 Primary culture cycle and cell number statistics of primary gastric cancer cells and calculation of Population Doubling (PD) value
  • the primary gastric cancer cells were obtained from 5 samples of gastric cancer tissue samples (samples 11-15) according to the method of step (2)-3 of Example 1.
  • the obtained primary gastric cancer cells were cultured using the GM-1 medium in Example 2.
  • the cells were seeded in a 6-well plate at a viable cell density of 1 ⁇ 10 4 cells/cm 2 and cultured, and the cells were to be expanded After reaching 85%, digest and count. At the same time, record the number of days of culture until digestion, and the number of days of culture until digestion is regarded as a culture cycle. Continue to culture under this experimental condition.
  • the amplified cells are expanded in different generations. After each generation is digested, count and record the corresponding culture period.
  • Population Doubling (PD) 3.32*log10 (total number of cells after digestion/ The initial number of implanted cells) calculate the PD.
  • the abscissa represents the number of days the cells have been cultured
  • the ordinate represents the cumulative multiple of cell proliferation, which represents the multiple of cell expansion during the culture cycle. The larger the value, the more the number of cell expansion in a certain cycle. That is, the more cells can be expanded, and the slope represents the rate of cell expansion.
  • the average culture period obtained by culturing 5 samples was 14.4 days, and the average number of cells obtained by expansion was 868,700.
  • the cumulative cell doubling number 3.32*log10 (the total number of cells after digestion/the number of initial seeded cells)
  • FM medium is a commonly used medium in the prior art, and its formula is: DMEM/F12 medium + 10% FBS + 5 ⁇ g/ml insulin + 250ng/ml amphotericin B (purchased from Selleck) + 10 ⁇ g/ml Qingda Mycin (purchased from MCE)+0.1nM cholera toxin+0.125ng/ml EGF+25ng/ml hydrocortisone+10 ⁇ M Y27632;
  • the primary gastric cancer cells were obtained from the intraoperative tissue sample (sample 16) according to the method of step (2) of Example 1, and were cultured under trophoblast conditions and culture without trophoblasts.
  • trophoblast cells use the above FM medium and the GM-1 medium in Example 2 to inoculate a 48-well plate (10,000 cells per well) at a viable cell density of 1 ⁇ 10 4 cells/cm 2 respectively. Subsequently, NIH-3T3 cells irradiated with gamma rays (irradiation dose 30Gy) were added according to the cell density of 2 ⁇ 10 4 cells/cm 2 , and the corresponding medium was used to supplement the volume of each well in the 48-well plate to 500 microliters. , Mix well. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. Passage is performed until the cells in the 48-well plate grow to more than 85%.
  • Example 2 In the absence of trophoblasts, use the above FM medium and the GM-1 medium in Example 2 to inoculate a 48-well plate (10,000 cells per well) at a viable cell density of 1 ⁇ 10 4 cells/cm 2 respectively. Then use the corresponding medium to make up the volume of each well in the 48-well plate to 500 microliters, and mix well. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. Passage is performed until the cells in the 48-well plate grow to more than 85%.
  • GM-1 can significantly promote the expansion of primary gastric cancer cells regardless of the presence or absence of trophoblast cells, and its effect is better than that of FM medium used in the prior art. Under the condition of trophoblasts, the effect of promoting the expansion of gastric cancer primary cells is more obvious.
  • Example 7 Gastric cancer primary cells amplified using the culture medium of the present invention are used for drug screening
  • Example 17 From the obtained gastric cancer endoscopic sample (sample 17), the primary gastric cancer cells were isolated in the same manner as in Example 1 and cultured using GM-1 medium. After the cells were expanded to 85%, they were digested and passaged as the first generation. The 1st, 2nd, 3rd, 4th and 5th generation cells were cultured for drug screening.
  • the cells were digested and counted according to the steps in Example 1.
  • GM-1 medium the cells were thoroughly mixed in a sample tank (purchased from Corning) at a viable cell density of 4 ⁇ 10 4 cells/mL, and then mixed well in the sample tank (purchased from Corning).
  • the wells were cultured in an opaque white cell culture plate (purchased from Corning), with a volume of 50 ⁇ L per well, and the number of cells was 2000 cells/well.
  • the surface was disinfected with 75% alcohol (purchased from Lierkang), placed in a 37°C, 5% CO 2 incubator, and added 24 hours later.
  • cell survival rate (%) chemiluminescence value of dosing hole/ chemiluminescence value of control hole*100%, calculate the cell survival rate after different drugs are applied to cells, and use graphpad prism software to calculate the half inhibition rate of drugs on cells (IC50). The results are shown in Figure 8.
  • gastric cancer cells cultured with the gastric cancer primary cell culture medium of the present invention are used for drug screening, and the inhibitory effect of the same drug on the cultured cells of different generations is basically the same (the inhibition curve is basically the same).
  • a culture medium and a culture method for realizing the rapid expansion of gastric cancer primary cells in vitro can be achieved.
  • the gastric cancer primary cell culture medium of the present invention and adopting the conditional reprogramming culture technology, the effective and rapid expansion of gastric cancer primary cells can be realized, so that the expanded cells maintain the pathological characteristics of the patient and improve the primary gastric cancer.
  • the success rate of cell culture and the rate of cell expansion can provide a research basis for personalized treatment of patients.

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Abstract

一种实现胃癌原代细胞体外快速扩增的培养基和培养方法。该培养基包含选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基、Rho蛋白酶抑制剂、抗生素、胰岛素、非必需氨基酸、氢化可的松、霍乱毒素、谷氨酰胺、胎牛血清、以及选自B27添加剂和N2添加剂中的至少一种的添加剂。

Description

胃癌原代细胞的培养基及培养方法 技术领域
本发明涉及生物技术领域，具体涉及一种用于胃癌原代细胞的快速扩增的培养基及培养方法，及其在药物的疗效评估和筛选中的应用。
背景技术
胃癌是最常见的恶性消化道肿瘤，早期胃癌患者无明显症状，因此多数胃癌患者确诊时已发展至中晚期。胃癌细胞对化疗药物敏感性较差，因此其化疗效果差，导致五年生存率低。近年来，随着科学技术的发展，胃癌诊疗技术不断提高，尤其是手术、内镜技术、放化疗、靶向治疗等综合治疗方式的进步，胃癌的治疗效果有所提升，但对于晚期及伴转移病灶的胃癌患者，综合治疗的预后仍不理想。流行病学调查分析显示，在我国胃癌发病例数和死亡例数分别占全球胃癌发病和死亡的42.6％和45.0％，在全球183个国家中位于发病率第5位、死亡率第6位。胃癌仍是严重危害中国居民健康的主要疾病之一，降低我国胃癌的发病率和死亡率越来越成为我们必须直面的重大公共卫生问题。
现有的体外培养的胃癌细胞系主要通过将正常细胞长期培养而自发永生化或者转染促使正常细胞永生化的癌基因获得。传统方法建立的细胞系依然是细胞、分子和癌生物学研究的主要支柱。但是，这些方法改变了细胞的遗传背景，长期培养的细胞系，也容易造成基因组不稳定，可能导致肿瘤细胞系的表型和体内肿瘤细胞发生人为的改变。这些细胞系中通常缺乏原发肿瘤的复杂异质性，从而限制了这些细胞系对于预测肿瘤细胞反应的应用，影响胃癌的科学研究和药物研发的准确性。另外，从胃癌组织获得的细胞培养成癌细胞的过程中，常规培养方法较难得到癌细胞，培养过程中存在容易被成纤维细胞干扰，形成的克隆无法传代等问题，限制了人胃癌原代细胞的应用。
2017年Xuefeng Liu等人使用辐照的小鼠成纤维细胞和Rho相关激酶抑制剂(Y-27632)来扩增上皮来源的细胞，该体系具有无需基因操作就能实现上皮来源细胞无限增长的能力(Xuefeng Liu等， Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nat.Protoc.2017,12,439)。但是，Xuefeng Liu等人建立的方法的培养周期较长，不能实现细胞的快速扩增，限制了该技术的应用。
发明内容
为了解决上述技术问题，本发明提供了一种用于在体外快速扩增胃癌原代细胞的培养基及培养方法。
本发明的一个方面在于提供一种胃癌原代细胞的培养基，所述培养基包含初始培养基、Rho蛋白酶抑制剂、抗生素、胰岛素、非必需氨基酸、氢化可的松、霍乱毒素、谷氨酰胺、胎牛血清(FBS)、以及选自B27添加剂和N2添加剂中的至少一种的添加剂。
所述初始培养基选自DMEM/F12、DMEM、F12或RPMI-1640。
其中，选自B27添加剂和N2添加剂中的至少一种的添加剂优选为B27添加剂、或者B27添加剂和N2添加剂。
(1)Rho蛋白激酶抑制剂选自Y27632、羟基法舒地尔和GSK429286A中的一种或多种，当选自Y27632时，浓度范围为2.5～40μM，优选为10～20μM；当选自羟基法舒地尔时，浓度范围为2～32μM，优选为4～16μM；当选自GSK429286A时，浓度范围为2～32μM，优选为4～16μM；
(2)抗生素选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种，当选自链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，优选为50～200μg/mL，更优选为200μg/m，青霉素浓度范围为25～400U/mL，优选为50～200U/mL，更优选为200U/mL；当选自两性霉素B时，浓度范围为0.25～4μg/mL，优选为0.5～2μg/mL；当选自Primocin时，浓度范围为25～400μg/mL，优选为50～200μg/mL；
(3)胰岛素的浓度范围为5～20μg/mL，优选为10～20μg/mL；
(4)非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，非必需氨基酸的总浓度范围为50～800μM，优选为100～400μM；
(5)氢化可的松的浓度范围为0.1～1.6μg/mL，优选为0.2～0.8 μg/mL；
(6)霍乱毒素的浓度范围为0.1～0.4nM，优选为0.1～0.2nM
(7)谷氨酰胺的浓度范围为1～8mM，优选为1～4mM；
(8)胎牛血清(FBS)相对于培养基的体积比为1:5～1:40；
(9)B27添加剂的浓度范围体积比为1:25～1:400，优选为1:25～1:200；
(10)N2添加剂相对于培养基的体积比为1:25～1:400，优选为1:25～1:100。
本发明还提供一种胃癌原代细胞的培养方法。本发明的胃癌原代细胞的培养方法中，使用本发明的胃癌原代细胞培养基对胃癌原代细胞进行培养。
本发明的胃癌原代细胞的培养方法中，按照细胞密度2～3×10 4个/cm 2加入滋养细胞。滋养细胞为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，辐照剂量为20～50Gy。
本发明的胃癌原代细胞培养方法还包括以下步骤：
1.胃癌原代细胞的分离
1.1对组织样本、例如内镜标本，用组织清洗液冲洗后加入组织消化液置于恒温摇床(知楚仪器ZQLY-180N)中进行消化，例如，利用8～14毫升、优选12毫升组织消化液进行消化；消化温度范围为4摄氏度～37摄氏度，优选为37摄氏度；消化转速范围为200rpm～350rpm，优选为300rpm；
1.2消化后取出观察，若未见明显组织块即可终止消化，否则继续消化，直至消化充分，消化时间范围为4～8小时，优选为6小时；
1.3消化完成后取出，离心后弃去上清液，加入含血清初始培养基重悬终止消化，离心转速范围为1200～1600rpm，优选为1500rpm；离心时间范围为2～6分钟，优选为5分钟，其中，含血清初始培养基例如可以使用含10％胎牛血清的DMEM/F12培养基。
2.使用本发明的胃癌原代细胞培养基进行培养
将上述步骤1中获得的胃癌原代细胞用本发明的胃癌原代细胞培养基重悬并计数，按照细胞密度5～10×10 4个/cm 2种入培养皿中，同时按照细胞密度2～3×10 4个/cm 2加入滋养细胞，培养5～7天后按照 细胞密度0.5～1×10 4个/cm 2补充加入滋养细胞，直至培养皿中细胞长满85％以上可进行消化传代。
其中，步骤1中所述的组织清洗液的配方为：DMEM/F12基础培养基含100～200μg/mL Primocin、2％青霉素/链霉素溶液；步骤1中所述的组织消化液的配制方法为：将1～2mg/mL胶原酶Ⅱ、1～2mg/mL胶原酶Ⅳ、50～100U/mL脱氧核糖核酸Ⅰ、0.5～1mg/mL透明质酸酶、1～3mM氯化钙、1～2％牛血清白蛋白溶于体积比1:1的HBSS和RPMI-1640中；步骤2中所述的滋养细胞例如可以为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，优选为γ射线，辐照剂量为20～50Gy，优选为30Gy。
本发明还提供一种胃癌疾病的药物筛选方法，其包括以下步骤：
(1)使用本发明的胃癌原代细胞的培养方法培养胃癌原代细胞，用于药物筛选；
(2)选定需要检测的药物并按照所需浓度梯度进行稀释；
(3)对(1)中培养得到的细胞添加稀释后的所述药物；
(4)进行细胞活性测试。
发明效果
(1)提高胃癌原代细胞培养的成功率，成功率达到85％以上；
(2)保证体外原代培养的胃癌原代细胞能够保持病人的病理特性；
(3)所培养的原代胃癌细胞不受成纤维细胞、脂肪细胞等间质细胞的干扰；
(4)扩增效率高，只要有10 4级别的细胞数量就可在两周左右时间内成功扩增出10 6数量级的胃癌原代细胞，扩增出的胃癌原代细胞还可以连续传代；
(5)培养成本可控：培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白、BMP抑制剂、FGF10等因子，是对已有胃癌原代细胞培养基的简化和改进；
(6)所述技术培养获得的胃癌原代细胞数量大，均一化程度高，适合高通量筛选新候选化合物和为病人提供高通量药物体外敏感性功 能测试。
附图说明
图1为表示胃癌原代细胞培养基中不同添加因子组合对胃癌原代细胞增殖的影响的图。
图2为表示胃癌原代细胞培养基中添加因子浓度对胃癌原代细胞增殖的影响的图。
图3为利用显微镜观察使用本发明的胃癌原代细胞培养基培养得到的胃癌原代细胞的照片。
图4为对使用本发明的胃癌原代细胞培养基培养得到的胃癌原代细胞进行瑞氏吉姆萨染色鉴定的结果。
图5为对使用本发明的胃癌原代细胞培养基培养得到的胃癌原代细胞进行细胞免疫荧光染色的结果。
图6为使用本发明的胃癌原代细胞培养基对胃癌原代细胞进行培养的细胞生长曲线。
图7为使用本发明的胃癌原代细胞培养基和现有培养基对胃癌原代细胞进行培养的比较结果。
图8为使用本发明的胃癌原代细胞培养基培养胃癌细胞，并将不同代数细胞用于药物筛选的结果。
具体实施方式
为更好理解本发明，下面结合实施例及附图对本发明作进一步描述，以下实施例仅是对本发明进行说明而非对其加以限定。
实施例1 胃癌原代细胞培养基中各添加因子对胃癌原代细胞增殖的影响
(1)胃癌原代细胞培养基的配制
首先配制基础培养基。基础培养基的配方为：DMEM/F12培养基(购自Corning公司)+10μM Y27632(购自MCE公司)+100μg/mL Primocin(购自InvivoGen公司)。
在基础培养基内分别加入不同种类的添加剂(参见表1)配制成含 有不同添加成分的胃癌原代细胞培养基。
(2)胃癌原代细胞的分离
1样品选择
胃癌实体瘤组织样品(术中/内镜)由专业医疗机构的专业医务人员从患者获取，患者均签署了知情同意书。术中样本2个黄豆粒大小，内镜样本米粒大小；采用商品化组织保存液(生产厂家：Miltenyi Biotec)存储运输。
2材料准备
准备：30Gy剂量γ射线照射过的NIH 3T3细胞(购自ATCC)；15mL无菌离心管、移液枪、10mL移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30min；提前30min从4℃冰箱取出基础培养基，提前30min从-20℃冰箱取出组织消化液，其中：
组织消化液：DMEM/F12培养基、胶原酶Ⅱ(2mg/mL)、胶原酶Ⅳ(2mg/mL)、DNA酶(50U/mL)、透明质酸酶(0.75mg/mL)、氯化钙(3.3mM)、BSA(10mg/mL)。
以上提及的胶原酶Ⅱ购自Sigma公司；胶原酶Ⅳ购自Sigma公司；DNA酶购自Sigma公司；透明质酸酶购自Sigma公司；氯化钙购自生工生物工程(上海)股份有限公司；BSA购自Biofroxx公司。
3样品分离
3.1超净台中取组织于培养皿中，去除带血液的组织，用基础培养基冲洗2次，将组织转移至另一培养皿中用无菌手术刀进行机械分离，使组织块至1*1*1mm 3大小；
3.2将切割后的术中或内镜组织吸至15mL离心管中，加入5mL基础培养基，混匀，于1500rpm离心4min；
3.3弃上清，按1:1比例加入基础培养基和消化酶(注：消化酶的加入量是1g肿瘤组织使用约10mL消化酶)，标记样品名称及编号，用封口膜密封，在37℃下于300rpm摇床中进行消化，期间每1h观察消化是否完成，判断依据无肉眼可见的颗粒物；
3.4消化完成后，经100μm滤网过滤掉未消化的组织团块，滤网上的组织团块用基础培养基冲洗入离心管中以减少细胞损失，于25℃下1500rpm离心4min；
3.5弃上清，观察是否有血细胞，若有血细胞，加10mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解20min，期间颠倒混匀一次，25℃，1500rpm离心4min；
3.6弃上清，加入2mL基础培养基重悬细胞，备用。
4细胞计数及处理
4.1镜下观察：移取少量重悬细胞平铺于培养皿中，显微镜下观察癌细胞密度和形态；
4.2活细胞计数：取重悬的细胞悬液10μL，加入80μLGM-1培养基，10μL台盼兰染液(生产厂家：生工生物工程(上海)股份有限公司)充分混合后，取10μL加入血球计数板(生产厂家：上海求精生化试剂仪器有限公司，规格：79mm*39mm*13mm)，显微镜下数四个大方格内的细胞总数，细胞浓度(细胞/mL)＝4个大方格内细胞总数/4*10 4*10(稀释倍数)。死细胞可被染成蓝色，活细胞不着色。计数200个细胞，计算出活的大细胞(细胞粒径>10μm)百分率＝活细胞数/总细胞数*100％。
(3)胃癌原代细胞的培养
将表1中不同成分的培养基按500μl/孔体积加入48孔板内。将按照上述步骤(2)从三例胃癌组织(样本1、样本2、样本3)分离得到胃癌原代细胞以1×10 4个/孔的细胞密度接种在48孔培养板内，同时按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞。以37℃、5％CO 2浓度的条件进行培养。培养开始后每4天进行一次培养基的更换。培养10天后，进行细胞计数。其中，作为实验对照，使用未添加任何添加剂的基础培养基，将实验结果示于表1。
表1
序号 培养基添加剂种类 供应商 终浓度 促细胞增殖程度分级
1 N2 Gibco 1:50 +
2 表皮生长因子 R&D 20ng/ml ○
3 肝细胞生长因子 Peprotech 20ng/ml ○
4 碱性成纤维细胞生长因子 R&D 20ng/ml ○
5 R-spondin1 R&D 250ng/ml ○
6 R-spondin3 R&D 250ng/ml ○
7 前列腺素E2 Tocris 1μM ○
8 胰岛素 Peprotech 20μg/mL +
9 人白血病抑制因子 Peprotech 100ng/ml ○
10 三碘甲状腺原氨酸 MCE 5nM －
11 B27 Gibco 1:50 +
12 A8301 MCE 500nM ○
13 SB202190 MCE 500nM ○
14 烟酰胺 Sigma 10mM ○
15 N-乙酰半胱氨酸 Sigma 1.25mM －
16 谷氨酰胺 GIBCO 4mM +
17 霍乱毒素 Sigma 0.1nM +
18 氢化可的松 Sigma 0.8μg/mL +
19 非必需氨基酸 Corning 400μM +
20 Noggin R&D 100ng/ml ○
21 FBS Excell 10％ +
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对从胃癌组织分离出的胃癌原代细胞中的三例有促进增殖的作用；“－”表示添加该添加剂的培养基对从胃癌组织分离出的胃癌原代细胞中的至少两例显示有抑制增殖的作用；“○”表示添加该添加剂的培养基对从胃癌组织分离出的胃癌原代细胞中的至少两例的增殖没有明显的影响。
实施例2 胃癌原代细胞培养基中不同添加因子的组合对胃癌原代细胞增殖的影响
根据表2中的成分配制不同添加因子组合的胃癌原代细胞培养基，考察不同添加因子组合对胃癌原代细胞的促增殖作用。
表2 不同组分培养基的配制(浓度为终浓度)
培养基 组分
基础培养基(BM) DMEM/F12+10μM Y27632+100μg/mL Primocin
No.1 BM+20μg/mL胰岛素
No.2 No.1+0.8μg/mL氢化可的松
No.3 No.2+400μM非必需氨基酸
No.4 No.3+4mM谷氨酰胺
No.5 No.4+0.1nM霍乱毒素
No.6 No.5+10％FBS
No.7 No.6+1:50 N2添加剂
GM-1 No.6+1:50 B27添加剂
以上提及的FBS购自Excell公司。
按照实施例1的步骤(2)之3的方法从胃癌组织(样本4)获得胃癌原代细胞，将所获得的细胞悬液平均分成9份，1500rpm离心4分钟，离心后分别使用200微升BM、No.1～7号以及GM-1培养基重悬，分别按照活细胞密度1×10 4个/cm 2接种于48孔板中(每孔1万细胞数)，随后按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞，最后分别使用对应的培养基补齐48孔板中各孔体积至500微升，充分混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。直至48孔板中细胞长至85％以上，进行传代。
在培养第7天，取出48孔板，使用200微升0.05％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入250微升0.05％胰蛋白酶(购自Gibco公司)，置于37℃、5％CO 2培养箱中反应10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全即终止消化，1500rpm离心4分钟后，弃上清，加入1毫升基础培养基重悬，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。将由分离自内镜组织样本50的胃癌原代细胞得到的结果示于图1。
根据图1的结果可知，与基础培养基相比，在使用上述No.1～No.7及GM-1培养基时，均能够不同程度地促进胃癌原代细胞的增殖。在使用含有Y27632、Primocin、胰岛素、氢化可的松、非必需氨基酸、谷氨酰胺、霍乱毒素、FBS、B27添加剂的胃癌原代细胞培养基(即 GM-1培养基)培养胃癌原代细胞时，增殖效果明显提高。
实施例3 所添加的因子的不同浓度对胃癌原代细胞增殖的影响
按照实施例1中步骤(2)之3的方法从内镜组织(样本5、样本6、样本7、样本8和样本9)分离并获得胃癌原代细胞，并使用实施例2中的GM-1培养基培养所获得的胃癌原代细胞，按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数)，按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。待细胞扩增至85％，取出12孔板，使用200微升0.05％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500微升0.05％胰蛋白酶(购自Gibco公司)，置于37℃、5％CO 2培养箱中反应10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全即终止消化，1500rpm离心4分钟后，弃上清，加入1毫升基础培养基重悬，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。所得细胞用于以下培养实验。
接着，配制以下9种配方培养基进行实验：
配方1：实施例2中的GM-1培养基组分中不含非必需氨基酸；
配方2：实施例2中的GM-1培养基组分中不含谷氨酰胺；
配方3：实施例2中的GM-1培养基组分中不含B27；
配方4：实施例2中的GM-1培养基组分中不含胰岛素；
配方5：实施例2中的GM-1培养基组分中不含霍乱毒素；
配方6：实施例2中的GM-1培养基组分中不含氢化可的松；
配方7：实施例2中的GM-1培养基组分中不含Y27632；
配方8：实施例2中的GM-1培养基组分中不含FBS；
配方9：实施例2中的GM-1培养基。
分别使用上述配方1～9和实施例2中GM-1培养基来稀释上述消化后的细胞悬液，按照每孔2万细胞，500微升体积种入24孔板中。
在使用配方1的培养基时，按照终浓度分别为800μM、400μM、200μM、100μM、50μM配制5个浓度梯度的非必需氨基酸，在接种有原代细胞的24孔板中分别添加配制好的非必需氨基酸每孔500微 升；并使用配方1的培养基设置对照孔(BC)。
在使用配方2的培养基时，按照终浓度分别为8mM、4mM、2mM、1mM、0.5mM配制5个浓度梯度的谷氨酰胺，在接种有原代细胞的24孔板中分别添加配制好的谷氨酰胺每孔500微升；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，按照终浓度分别为1:400、1:200、1:100、1:50、1:25配制5个浓度梯度的B27添加剂，在接种有原代细胞的24孔板中分别添加配制好的B27添加剂每孔500微升；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，按照终浓度分别为40μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL配制5个浓度梯度的胰岛素，在接种有原代细胞的24孔板中分别添加配制好的胰岛素每孔500微升；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，按照终浓度分别为0.4nM、0.2nM、0.1nM、0.05nM、0.025nM配制5个浓度梯度的霍乱毒素，在接种有原代细胞的24孔板中分别添加配制好的霍乱毒素每孔500微升；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，按照终浓度分别为1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL配制5个浓度梯度的氢化可的松，在接种有原代细胞的24孔板中分别添加配制好的氢化可的松每孔500微升；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，按照终浓度分别为40μM、20μM、10μM、5μM、2.5μM配制5个浓度梯度的Y27632，在接种有原代细胞的24孔板中分别添加配制好的Y27632每孔500微升；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，按照终浓度分别为1:40、1:20、1:10、1:5、1:2.5配制5个浓度梯度的FBS，在接种有原代细胞的24孔板中分别添加配制好的FBS每孔500微升；并使用配方8的培养基设置对照孔(BC)。
在使用配方9的培养基时，按照终浓度分别为1:400、1:200、1:100、1:50、1:25配制5个浓度梯度的N2添加剂，在接种有原代细胞的24 孔板中分别添加配制好的N2添加剂每孔500微升；并使用配方9的培养基设置对照孔(BC)。
同时上述各孔加入1万个辐照后的NIH-3T3细胞作为滋养细胞。
待细胞扩增至24孔的85％左右消化计数，分别参比对照孔(BC)细胞数计算比值，将结果分别示于图2的(A)～(I)。图2中，比值为使用各培养基培养一代得到的细胞数与对应的对照孔培养一代得到的细胞数的比。比值大于1说明配制的含不同浓度因子的培养基促增殖效果优于对照孔培养基，比值小于1，则说明配制的含不同浓度因子的培养基促增殖效果较对照孔培养基促增殖效果弱。
如图2所示，非必需氨基酸浓度范围为50～800μM，浓度为400μM加入细胞增殖效果最明显；谷氨酰胺浓度范围为1～8mM，浓度为4mM加入细胞增殖效果最明显；B27浓度范围为1:25～1:400，浓度为1:50比例加入细胞增殖效果最明显；胰岛素浓度范围为5～20μg/mL，浓度为10μg/mL加入细胞增殖效果最明显；霍乱毒素浓度范围为0.1～0.4nM，浓度为0.1nM加入细胞增殖效果最明显；氢化可的松浓度范围为0.1～1.6μg/mL，浓度为0.8μg/mL加入细胞增殖效果最明显；Y27632浓度范围为2.5～40μM，浓度为10μM加入细胞增殖效果最明显；FBS浓度范围为1:5～1:40，浓度为1:10比例加入细胞增殖效果最明显；N2浓度范围为1:25～1:400，浓度为1:50比例加入细胞增殖效果最明显。
实施例4 胃癌原代细胞培养及鉴定
按照实施例1的步骤(2)之3的方法从内镜组织样本(样本10)获得胃癌原代细胞，并使用实施例2中的GM-1培养基进行培养，所获得的胃癌原代细胞，按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数)，按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
在第12天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的胃癌原代细胞，图3的(A)和(B)分别是4倍物镜和10倍物镜下拍摄得到的照片，细胞在镜下呈紧密排列，形态略不规则。
并且，在第12天，对所培养的细胞，使用200微升0.05％胰蛋白 酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500微升0.05％胰蛋白酶(购自Gibco公司)，置于37℃、5％CO 2培养箱中反应10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全即终止消化，1500rpm离心4分钟后，弃上清，加入500微升GM-1培养基重悬，对培养得到的胃癌原代细胞进行鉴定。
取100微升上述胃癌原代细胞悬液涂片进行瑞氏-吉姆萨染色鉴定。染色方法如下所述。
(1)细胞悬液涂片晾干，滴入1滴瑞氏-吉姆萨A液(购自贝索公司)，随后滴入3滴瑞氏-吉姆萨B液(购自贝索公司)，混匀后染色3分钟；
(2)流水冲洗，冲洗时不能先倒掉染液，应以流水冲去，以防有沉渣沉淀在标本上；
(3)干燥，显微镜(奥林巴斯公司CX41)下观察拍照。
图4表示体外培养后的胃癌原代细胞进行瑞氏吉姆萨染色鉴定的结果。图4的(A)和(B)分别为4倍物镜和10倍物镜下拍摄得到的照片，细胞核大且深染，符合癌细胞特征。
将该样本培养后的胃癌原代细胞进行免疫荧光染色。染色方法如下所述。
(1)爬片
将培养后的胃癌原代细胞种于细胞玻片(购自赛默飞公司)上，置于37℃、5％CO 2培养箱培养，待细胞贴壁。
(2)固定
①PBS(购自上海生工)配制4％甲醛(购自Sigma公司)，4℃冰箱保存备用。
②细胞贴壁后，弃培养液，4％甲醛冰上固定细胞30分钟。PBS(购自上海生工)洗5分钟x3次。
(3)透明(避光)
①配制透明液：PBS+0.3％H 2O 2(购自上海生工)+0.3％Triton X-100(购自上海生工)。
②透明：弃PBS，加入透明液，避光下，摇床(100rpm左右)透明30分钟，PBS洗5分钟x3次。
(4)封闭
使用PBS+0.3％Triton X-100配制5％BSA(购自上海生工)用于封闭，37℃封闭30分钟。
(5)一抗孵育
①配制PBS+0.3％Triton X-100用于稀释抗体，按照1:50比例稀释特异性抗体Pan-keratin、MUC1、P63和Napsina(抗体均购自CST公司)，弃封闭液，加入配制好的一抗稀释液，至4℃冰箱孵育过夜，其中，Pan-keratin上皮组织中高表达，MUC1在胃肠癌症中高表达，P63在鳞癌中高表达，Napsina在腺癌细胞中高表达。
②4℃取出，平衡至室温，37℃继续孵育1小时，PBS洗5分钟x3次。
(6)二抗孵育(避光)
配制PBS+0.3％Triton X-100用于二抗稀释，按照1:1000比例稀释激发光为488且种属为兔的荧光二抗(购自赛默飞公司)，常温避光孵育1h，PBS洗5分钟x3次。
(7)DAPI染色(避光)
1:1000PBS稀释DAPI(购自Sigma公司)，常温避光染色5分钟，PBS洗5分钟x3次。显微镜(Invitrogen公司EVOS M500)下成像，拍照记录。
图5为体外培养后的胃癌原代细胞进行免疫荧光染色鉴定的结果，分别为10倍物镜下荧光拍照的图片。如图所示，Pan-keratin、MUC1和P63高表达，Napsina低表达，提示该样本为胃鳞癌，该结果和医院病理诊断结果一致。
实施例5 胃癌原代细胞初次培养周期和细胞数统计及Population Doubling(PD)值计算
按照实施例1步骤(2)之3的方法从5例样本胃癌组织样本(样本11～15)获得胃癌原代细胞。对于所获得的胃癌原代细胞，使用实施例2中的GM-1培养基培养，按照活细胞密度1×10 4个/cm 2将细胞接种在6孔板中并进行培养，待细胞扩增至85％后消化并计数，同时记录直至消化时培养的天数，将该直至消化时培养的天数作为一个培 养周期。在该实验条件下持续培养，将扩增所得的细胞进行不同代数扩增，每一代进行消化后计数并记录相应培养的周期，根据公式Population Doubling(PD)＝3.32*log10(消化后细胞总数/初始种入细胞数)计算PD。如图6所示，横坐标表示细胞培养的天数，纵坐标是累计的细胞增殖倍数，表示细胞在培养周期内扩增的倍数，数值越大表示细胞在一定周期内扩增的次数越多，即扩增得到的细胞数也就越多，斜率代表的是细胞扩增的速率。
从图6可以看出，使用本发明的胃癌原代细胞培养基对上述5例样本进行培养时，扩增至少至80天时细胞扩增速率基本保持不变仍具有继续扩增的能力。
如表1所示，培养5例样本所得平均培养周期是14.4天，扩增得到的平均细胞数是86.87万。
表3 胃癌样本组织培养周期
Figure PCTCN2020081414-appb-000001
注：累加细胞倍增数＝3.32*log10(消化后细胞总数/初始种入细胞数)
实施例6 与现有培养基培养效果的比较
(1)培养基配制
FM培养基是现有技术常用的培养基，其配方为：DMEM/F12培养基+10％FBS+5μg/ml胰岛素+250ng/ml两性霉素B(购自Selleck公司)+10μg/ml庆大霉素(购自MCE公司)+0.1nM霍乱毒素+0.125ng/ml EGF+25ng/ml氢化可的松+10μM Y27632；
(2)胃癌原代细胞的获取和培养
按照实施例1的步骤(2)之3的方法从术中组织样本(样本16)获得胃癌原代细胞，分别进行有滋养细胞条件的培养和无滋养细胞条件的培养。
在有滋养细胞时，分别使用上述FM培养基和实施例2中的GM-1培养基按照活细胞密度1×10 4个/cm 2接种于48孔板中(每孔1万细胞数)，随后按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞，最后分别使用对应的培养基补齐48孔板中各孔体积至500微升，充分混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。直至48孔板中细胞长至85％以上，进行传代。
在无滋养细胞时，分别使用上述FM培养基和实施例2中的GM-1培养基按照活细胞密度1×10 4个/cm 2接种于48孔板中(每孔1万细胞数)，再分别使用对应的培养基补齐48孔板中各孔体积至500微升，充分混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。直至48孔板中细胞长至85％以上，进行传代。
在培养第7天，取出48孔板，使用200微升0.05％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入250微升0.05％胰蛋白酶(购自Gibco公司)。置于37℃、5％CO 2培养箱中反应10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全即终止消化。1500rpm离心4分钟后，弃上清，加入1毫升基础培养基重悬，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数，将计数结果示于图7。
根据图7的结果可知，与FM培养基相比，不管有无滋养细胞，GM-1能显著促进胃癌原代细胞扩增，其效果优于现有技术采用的FM培养基，并且，在有滋养细胞的条件下，对于胃癌原代细胞的扩增促进效果更为明显。
实施例7 使用本发明培养基扩增得到的胃癌原代细胞用于药物筛选
1、细胞培养和铺板
从得到的胃癌内镜样本(样本17)与实施例1同样地分离得到胃癌原代细胞并使用GM-1培养基进行培养，待细胞扩增至85％，进行消化传代，作为一代。分别取培养第1代、第2代、第3代、第4代、第5代细胞进行药物筛选。
按照实施例1中步骤将细胞消化计数，使用GM-1培养基，将细胞按照活细胞密度4×10 4个/mL细胞于加样槽(购自康宁公司)中充分混匀后，在384孔不透明白色细胞培养板(购自康宁公司)进行培养，每孔体积50μL，细胞数目为2000个/孔。从孔板边缘加入GM-1培养基封板，板上标注样品名称及CellTiter-Glo(购自Promega公司)检测时间。表面75％酒精(购自利尔康)消毒，置37℃、5％CO 2培养箱培养，24小时后加药。
2、筛选药物配制
按照下表配制7个浓度梯度的2种药物(阿糖胞苷、硼替佐米；均购自MCE公司)，在384孔药板(购自赛默飞公司)每孔中添加30μL，-20℃保存待用。
表4 阿糖胞苷、硼替佐米药物添加液的配制
终浓度(μM) 配制浓度(μM)
10 5000
3.33 1666.7
1.11 555.6
0.37 185.2
0.12 61.7
0.04 20.6
0.01 6.9
3、高通量加药
取出配制好的药板，置于室温，待完全融化后置于离心机(Sigma公司3-18K)中室温1000rpm离心1分钟后取出。采用高通量自动化加样系统(Perkin Elmer公司JANUS)进行高通量加药。对培养有胃癌细胞的384孔板在每孔加入0.1μL对应浓度的筛选药物，加药结束后，384孔板表面消毒后移至培养箱中继续培养，72小时后测定细胞活性。
4、细胞活性测试
4℃冰箱取出CellTiter-Glo发光试剂(购自Promega公司)，取10毫升试剂于加样槽中，培养箱中取出待检测384孔板，每孔加入10μL CellTiter-Glo发光试剂，静置10min后使用多功能酶标仪(Perkin Elmer公司Envision)检测。
5、数据处理
按照公式细胞存活率(％)＝加药孔化学发光数值/对照孔化学发光数值*100％，计算得到不同药物作用细胞后的细胞存活率，使用graphpad prism软件计算药物对细胞作用的半数抑制率(IC50)。将结果示于图8。
由图8可以确认，使用本发明的胃癌原代细胞培养基培养得到的胃癌细胞进行药物筛选，相同药物对于培养的不同代数细胞抑制效果基本保持一致(抑制曲线基本保持一致)。
工业上的可利用性
根据本发明，能够一种实现胃癌原代细胞体外快速扩增的培养基和培养方法。通过使用本发明的胃癌原代细胞培养基，采用条件重编程的培养技术，能够实现胃癌原代细胞的有效快速扩增，这样扩增得到的细胞保持了病人的病理特性，提高了胃癌原代细胞的培养成功率和细胞扩增速率，可以为患者的个性化治疗提供研究基础。

Claims (6)

  1. 一种胃癌原代细胞培养基，其特征在于，包含：
    初始培养基、Rho蛋白酶抑制剂、抗生素、胰岛素、非必需氨基酸、氢化可的松、霍乱毒素、谷氨酰胺、胎牛血清、以及选自B27添加剂和N2添加剂中的至少一种的添加剂，
    所述初始培养基选自DMEM/F12、DMEM、F12或RPMI-1640。
  2. 如权利要求1所述的胃癌原代细胞培养基，其特征在于：
    Rho蛋白激酶抑制剂选自Y27632、羟基法舒地尔和GSK429286A中的一种或多种，当选自Y27632时，浓度范围为2.5～40μM，优选为10～20μM；当选自羟基法舒地尔时，浓度范围为2～32μM，优选为4～16μM；当选自GSK429286A时，浓度范围为2～32μM，优选为4～16μM；
    抗生素选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种，当选自链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，优选为50～200μg/mL，更优选为200μg/m，青霉素浓度范围为25～400U/mL，优选为50～200U/mL，更优选为200U/mL；当选自两性霉素B时，浓度范围为0.25～4μg/mL，优选为0.5～2μg/mL；当选自Primocin时，浓度范围为25～400μg/mL，优选为50～200μg/mL；
    胰岛素的浓度范围为5～20μg/mL，优选为10～20μg/mL；
    非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，非必需氨基酸的总浓度范围为50～800μM，优选为100～400μM；
    氢化可的松的浓度范围为0.1～1.6μg/mL，优选为0.2～0.8μg/mL；
    霍乱毒素的浓度范围为0.1～0.4nM，优选为0.1～0.2nM
    谷氨酰胺的浓度范围为1～8mM，优选为1～4mM；
    胎牛血清相对于培养基的体积比为1:5～1:40；
    B27添加剂相对于培养基的体积比为1:25～1:400，优选为1:25～1:200；
    N2添加剂相对于培养基的体积比为1:25～1:400，优选为1:25～ 1:100。
  3. 一种胃癌原代细胞的培养方法，其特征在于：
    使用权利要求1或2所述的胃癌原代细胞培养基对胃癌原代细胞进行培养。
  4. 如权利要求3所述的培养方法，其特征在于：
    在所述培养中，按照细胞密度2～3×10 4个/cm 2加入滋养细胞。
  5. 如权利要求4所述的培养方法，其特征在于：
    滋养细胞为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，辐照剂量为30～50Gy。
  6. 一种胃癌疾病的药物筛选方法，其特征在于，包括以下步骤：
    (1)使用权利要求3～5中任一项所述的胃癌原代细胞的培养方法培养胃癌原代细胞；
    (2)选定需要检测的药物并按照所需浓度梯度进行稀释；
    (3)对(1)中培养得到的细胞添加稀释后的所述药物；
    (4)进行细胞活性测试。
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