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以强化培育后草鱼的肠道粘膜为试验材料,采用不同消化法和离心转速梯度分离肠道粘膜细胞团,分别试验不同培养液与不同CO2浓度组合、胎牛血清浓度及细胞接种浓度时细胞生长效果,并且观察草鱼原代肠道粘膜细胞生长过程,同时采用3种细胞形态观察法、MTT检测法及相关酶活力系统评价细胞培养效果。结果表明:采用机械刮取消化法,分离转速为400 r/min,在使用M199培养液、6%CO2、15%胎牛血清、接种浓度为2×103(个/孔)条件下可批量复制草鱼原代肠道粘膜上皮细胞;细胞增殖过程符合动物原代肠道粘膜上皮细胞生长分化规律,采用荧光倒置显微镜与Giemsa染色法相结合、MTT检测法、AKP酶活力及LDH/MTT OD值可系统评价细胞培养效果。
Intestinal mucosa of grass carp were used as experimental material to culture intestinal mucosa of different groups. The intestinal mucosal cell mass was separated by different digestion methods and centrifugation speed gradient. Cell growth was tested in different culture medium with different CO2 concentration, fetal bovine serum concentration and cell seeding concentration Effect, and observe the growth of primary intestinal mucosal cells of grass carp. Meanwhile, three kinds of cell morphology observation, MTT assay and related enzyme activity system were used to evaluate the cell culture effect. The results showed that the speed of separation was 400 r / min by mechanical scraping and digesting method, and could be batch-copied under the conditions of M199 culture medium, 6% CO2 and 15% fetal bovine serum at the inoculation concentration of 2 × 103 Grass carp primary intestinal mucosa epithelial cells; cell proliferation process in line with animal primary intestinal mucosa epithelial cell growth and differentiation, fluorescence inverted microscope and Giemsa staining, MTT assay, AKP enzyme activity and LDH / MTT OD value system Evaluation of cell culture effect.