犊牛肝细胞的分离与原代培养  

The Isolation and Primary Culture of Calf Hepatocytes

作　　者：张才[1] 王利民[2] 刘国文[1] 苏蔚[1] 黄建龙[1] 谢光洪[1] 王哲[1] 

机构地区：[1]吉林大学畜牧兽医学院动物营养代谢病与中毒研究室,长春130062 [2]四川农业大学动物科学与技术学院,雅安625014

出　　处：《细胞生物学杂志》2007年第6期880-884,共5页Chinese Journal of Cell Biology

基　　金：国家自然科学基金资助项目(No.30230260)~~

摘　　要：以初生犊牛作肝细胞供者,采用稍加改良的两步胶原酶灌流法和一步灌流结合组织块消化法分离获取肝细胞,并进行原代培养;以台盼蓝染色法测细胞活力,在倒置显微镜下观察肝细胞形态变化,采用Beckman全自动生化分析仪检测较好培养体系不同时间培养上清液中白蛋白、乳酸脱氢酶(LDH)、尿素的含量。结果显示,相比较于一步灌流结合组织块消化法,胶原酶消化法所获取的肝细胞形态完整、贴壁良好、活性高、功能强;LDH漏出量、白蛋白分泌及尿素合成等指标在1周内呈现规律性变化,第3和第4天时LDH漏出量最低,白蛋白分泌及尿素合成功能正常,表明所分离的肝细胞在培养第3￣4天功能最佳。Calf hepatocytes were isolated by modified two steps collagenase perfusion and one step perfusion combined with tissue blocks collagenase digestion respectively, and cultured in hepatoZYME-SFM. The viability of cultured hepatocytes was assessed by trypan blue exclusion. The morphologic change of cultured hepatocytes was observed, and the concentrations of albumin, urea and lactate dehydrogenase （LDH） in the supernatant collected from different cultural period of better isolated system were examined. The results were as follows： hepatocytes obtained by modified two steps collagenase perfusion were intact and had a prosperous viability and an active function. The fluctuated changes of LDH leakage, albumin synthesis and urea level were displayed in one week, and the lesser LDH leakage and higher concentrations of albumin and urea were observed on the third day. It is concluded that the collagenase digestion method is feasible for isolation of hepatocytes. Calf hepatocytes have an optimal function on the third days of the orimarv culture.

关 键 词：犊牛 肝细胞 分离 原代培养 

分 类 号：S823[农业科学—畜牧学] Q813.1[农业科学—畜牧兽医]