大鼠原代肝细胞与枯否细胞同步分离的优化研究  

Simultaneous isolation and optimization of rat hepatocyte and Kupffer cells

作　　者：叶茜[1,2,3,4] 朱瑞[1,2,3] 何瑞曦 陈鑫[1,2,3] 李鑫[1,2,3] 陈卫东[1,2,3,4] 

机构地区：[1]安徽中医药大学药学院,合肥230012 [2]安徽中医药大学药物代谢研究所,合肥230012 [3]安徽省中医药科学院,合肥230012 [4]安徽道地中药材品质提升协同创新中心,合肥230012

出　　处：《中南药学》2017年第2期150-154,共5页Central South Pharmacy

基　　金：安徽省教育厅项目(No.gxbj ZD201660);安徽省教育厅项目(No.2014HZ010);2013安徽高校省级自然科学研究重点项目(产学研)(No.KJ2013A176)

摘　　要：目的优化分离培养高纯度的大鼠肝细胞与枯否细胞,为肝细胞与枯否细胞的共同培养提供实验细胞。方法 1原位胶原酶灌注法分离细胞;2 Percoll液不连续密度梯度离心法分离枯否细胞;3选择性贴壁纯化枯否细胞;4台盼蓝拒染法判定细胞活力,PAS染色法鉴定肝细胞,墨汁吞噬及CD68免疫染色法鉴定枯否细胞。结果通过改良的原位胶原酶灌注法,不连续密度梯度离心法以及选择性贴壁法等手段,成功实现了肝细胞与枯否细胞的同时分离,且所分离出的2种细胞的得率与纯度均达到后续实验所需细胞的要求。结论通过该创新方法同时分离肝细胞与枯否细胞是有效可行的。Objective To optimize the experimental method to simultaneously separate and cultivate high purity hepatocyte and Kupffer cells in rats. Methods （1） Situ collagenase perfusion method was used to separate cells; Percoll discontinuous density gradient centrifugation was adopted to harvest Kupffer cells; （3） Kupffer cells were purified according to selective adherence; （4） Trypan Blue exclusion was used to determine cell viability, PALS stain method identify hepatocytes, while ink phagocytic test and CD68 immunostaining were used to iden- tify Kupffer cells. Results Hepatocyte and Kupffer cells were successfully separated by situ collagenase perfu- sion method, discontinuous density gradient centrifugation and selective adherence, etc., and the receiving rate and purity of harvested hepatocyte and Kupffer cells met the experiment standards. Conclusion It is feasible to harvest hepatocyte and Kupffer ceils simultaneously via this method.

关 键 词：原代肝细胞 枯否细胞 原位灌注 密度梯度离心 选择性贴壁 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]