广西瑶山亚种树鼩原代肝细胞的分离、纯化、培养及鉴定  

Isolation,purification,culture and identification of primary hepatocytes in Tupaia belangeri yaoshanensis from Guangxi

作　　者：刘金花[1] 农微[1] 黄正团[1] 运晨霞[1] 夏钰淇 冷静[1] 

机构地区：[1]广西中医药大学,南宁530001

出　　处：《黑龙江畜牧兽医》2017年第4期42-45,290,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：广西科技攻关课题项目(桂科攻1347003-6);广西科学研究与技术开发计划项目(桂科能14123006-1);广西特色实验动物病证模型重点实验室建设项目(桂教科研[2014]14号);广西壮族自治区卫生厅中医药科技专项(GZPT13-02)

摘　　要：为了探讨瑶山亚种树原代肝细胞分离、培养及鉴定方法,试验对传统的两步原位胶原酶灌注法进行了优化,先将持续灌注法改为间断夹闭下腔静脉快速灌注法,再通过严格控制胶原酶的消化时间来分离提取树肝细胞,最后应用percoll分离液梯度低速离心纯化树肝细胞,采用PAS染色法和免疫细胞化学方法对树肝细胞进行综合鉴定。结果表明:分离得到的树肝细胞产量高、活力好;经PAS染色鉴定,发现树肝细胞内糖原被染成紫红色颗粒或片状;免疫细胞化学染色观察到树肝细胞胞浆内被染成棕黄色。说明成功分离培养树鼩肝细胞并对其进行鉴定,可用于进一步研究。To investigate the methods of isolation, cultivation and identification of primary hepatocytes in Tupaia belangeri yaoshanensis, the conventional two - step collagenase perfusion in situ method was optimized. A continuous perfusion was replaced by a rapid perfusion of intermittent occlusion of inferior vena cava,and then the collagenase digestion time was strictly controlled to isolate and extract the hepatocytes in Tupaia belangeri yaoshanensis. The hepatocytes were purified by percoll gradient centrifugation at low speed, and then were comprehensively identified using PAS staining and immunocytochemistry. The results showed that the hepatocytes were isolated and obtained, which had high yield and good viability; it was found that the glycogens in the hepatocytes were stained purple granules or flakes by PAS staining, and the cytoplasm in the hepatocytes was stained brown by immunocytochemistry staining. The results indicate that the primary hepatocytes in Tupaia belangeri yaoshanensis, which can be used for further research.

关 键 词：树鼩 肝细胞 分离 纯化 培养 PAS染色 免疫细胞化学 

分 类 号：Q27[生物学—细胞生物学]