大口黑鲈原代肝细胞的培养及其应用于CYP450活性的诱导.pdfVIP

第3O卷第1期 海 洋 渔 业 V0l_3O．No．1 2008年O2月 M撕ne Fisheries Feb．， 2008 文章编号：1004—2490(2008)01—0031—06 大口黑鲈原代肝细胞的培养及其 应用于CYP450活性的诱导 喻文娟，李 聃，王翔凌，张 宁，邱军强，杨先乐 (上海水产大学农业部渔业动植物病原库，上海 200090) 摘 要：体外培养大口黑鲈(Micropterus salmoides)原代肝细胞，观察其在培养过程中的形态变化，同时检测 培养上清液中的乳酸脱氢酶(LDH)、白蛋白(ALh)含量，从而判定肝细胞的增殖和代谢能力，进而应用于 CYP450的诱导研究。结果表明：培养5 d后，肝细胞连接成片，分裂增多；培养至7 d，细胞渐至单层；14 d后， 细胞停止生长，出现老化迹象。培养至第3—4天的细胞上清液中LDH的渗出量呈上升趋势，在第4天时达 到峰值，随着培养时间的延长，肝细胞的酶渗出量逐渐减少。ALh的分泌量在第3—5天呈上升的趋势，第4 — 8天维持在较高水平，在第5天达到最高，此后随着培养时间的延长，ALh的分泌量呈下降趋势，故选择培 养至第5天的大13黑鲈肝细胞应用于CYP450酶系中的氨基比林一N一脱甲基酶(ANDM)的诱导研究。40 IxM 利福平诱导24 h后，ANDM活性最高。ANDM的诱导研究为鱼类细胞药物代谢酶体外诱导奠定基础。 关键词：大口黑鲈；肝细胞；原代培养；乳酸脱氢酶；白蛋白；氨基比林．N一脱甲基酶 中图分类号：Q593．1 文献标识码：A Studies of Micropterus salmoides on the culture of primary hepatocytes and on the induction of CYP450 activity YU Wen-juan，LI Dan，WANG Xiang-ling，ZHANG Ning，QIU Jun-qiang，YANG Xian-le (Fishery Pathogen Pool of the Ministry of Agriculture，Shanghai Fisheries University，Shanghai 200090，China) Abstract：The present paper deals with the studies on the culture of primary hepatocytes of Micropterus salmoides and to be used to induce the activity of CYP450．The primary hepatocytes of Micropterus salmoides were cultured in vitro，the morphological changes of the cells in the culturing process were observed，the contents of LDH enzyme leakage and albumin secretion as well as activity of CYP450(aminopyrine-N- demethylase)were inspected．The results showed that the hepatocytes cultured in the plastic flask were extended，progressively spread and became confluent at 5th day．By 7th day of culture，the continuously increasing adherent herds had joined together to form a monolayer．With the passage of time，the viability of hepatocytes was gradual