草鱼肝细胞的分离与原代培养

摘要

Objective To explore the optimal methods of isolation and culture of hepatocytes from grass carp and serve the studies on nutritional metabolism and injury mechanisms in carp hepatocytes. Methods Proliferation of hepatocytes was tested by MTT assay. Function of THE hepatocytes was examined by measuring the levels of lactic acid dehydro-genase (LDH), albumin (ALB), and urea nitrogen (BUN) in the supernatant at different times, respectively. Results Using 0.25% warm trypsin digestion for 20 minutes each time, and to collect the hepatocytes step by step, the results of trypan blue test and blood cell counting chamber method showed that more than 99% of viable hepatocytes were obtained. Conclusion The cells were cultivated in M199 medium supplemented with 10% fetal bovine serum and 10μg/mL insulin, at 7 ×106 cell/mL concentration under the temperature of 28℃ , 4. 5% CO2 for a long term. Primary culture of carp hepa-tocytea can be successfully obtained by the following procedure: cells cultured in M199 medium supplemented with 10% fetal bovine serum and 10 μg/mL insulin, at 7 × 105 cell/mL concentration, under the temperature of 27℃ and 4.5% CO2.%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.