【作者】 秦洁； 叶元土； 冷向军； 蔡春芳； 宋亮； 许凡；

【Author】 QIN Jie1,YE Yuan-tu2,LENG Xiang-jun1,CAI Chun-fang2, SONG Liang2,XU Fan2 (1.College of Fisheries and Life Science of Shanghai Ocean University,Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Shanghai 201306,China 2.College of Basic Medical and Biological Sciences of Soochow University,Key Laboratory of Aquatic Animal Nutrition in Jiangsu,Jiangsu 215123,China)

【机构】 上海海洋大学水产与生命学院； 上海海洋大学省部共建水产种质资源发掘与利用教育部重点实验室； 苏州大学基础医学与生物科学学院江苏省水产动物营养重点实验室；

【摘要】 以草鱼肝细胞为试验对象,采用温胰蛋白酶消化法和红细胞裂解液分离、纯化肝细胞,并在不同条件下进行原代培养,以探讨适合草鱼肝细胞生长的最佳条件及培养方法,用于饲料营养与非营养物质对草鱼肝细胞代谢、损伤作用机制的研究。采用MTT法测定细胞增殖率,并测定不同时期培养液上清液中LDH、Alb和BUN的含量,分析肝细胞生长状况。结果表明:采用0.25%浓度的温胰蛋白酶消化法,消化时间20min,分步收集肝细胞,经台盼蓝染色检测和血球计数板计数,活细胞数≥99%。在含10%胎牛血清、10μg/mL胰岛素的M199培养基中,以接种浓度1.7×106 cell/mL左右为宜,置于27℃、4.5%CO2浓度的恒温培养箱中可成功培养草鱼原代肝细胞。更多还原

【Abstract】 In this study,the utilization of hepatocytes from grass carp after strengthen cultured used for experiment,using warm trypsin digestion and red blood cell lysate to separate and purify hepatocytes.Proliferation of hepatocytes was tested by MTT assay,function of hepatocytes were examined using the levels of lactic acid dehydrogenase(LDH),Albumin(ALB),and urea nitrogen(BUN) in supernatant at different times,respectively.The results showed that 0.25% concentration of trypsin to dissociate liver tissue for 20 minutes per time and collect the hepatocytes step by step,the initial viabilities routinely exceeding 99%which detected by trypan blue and blood cell counting chamber.The cells were cultivated in M199 medium supplement with 10%foetal bovine serum and 10μg/mL insulin,at 7×106 cell/mL ceiling concentration under the temperature of 28℃,concentration of CO2 being 4.5%for a long term.更多还原