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目的以草鱼(Ctenopharyngodon idellus)肝细胞为实验对象,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。方法采用温胰蛋白酶消化法和红细胞裂解液分离、纯化肝细胞,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。结果采用0.25%浓度的温胰蛋白酶消化法,消化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%。结论在含10%胎牛血清、10μg/mL胰岛素的M199培养基中,以接种浓度1.7×106cell/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。
Objective To investigate the optimal conditions and culture methods for hepatocytes of Ctenopharyngodon idellus under different conditions and to study the effects of dietary nutrients and non-nutritive substances on the metabolism of hepatocytes of grass carp , Injury mechanism of action. Methods The hepatocytes were isolated and purified by trypsin digestion and erythrocyte lysis. The cell proliferation rate was determined by MTT assay. The contents of LDH, Alb and BUN in supernatants were measured at different periods, and the growth of hepatocytes was analyzed. Results The cells were digested with 0.25% trypsin digestion for 20min, and the hepatocytes were collected in steps. The number of viable cells was ≥99% by trypan blue staining and counting with hemocytometer. Conclusion M199 medium containing 10% fetal bovine serum and 10μg / mL insulin is suitable for inoculation at a concentration of 1.7 × 106cell / mL and can be successfully cultured in a thermostatic incubator at 27 ℃ and 4.5% CO2. Primary hepatocytes.