小鼠肝实质细胞的分离、鉴定及原代培养

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英文篇名：Isolation, Identification and Primary Culture of Mouse Hepatocytes 作者：陈阳洁 ; 高柯欣 ; 王智豪 ; 李峰 ; 张琳琳 ; 刘娇 ; 陈晓光 英文作者：CHEN Yang-jie;GAO Ke-xin;WANG Zhi-hao;LI Feng;ZHANG Lin-lin;LIU Jiao;CHEN Xiao-guang;College of Animal Science and Technology,Henan University of Science and Technology; 关键词：小鼠 ; 肝实质细胞分离 ; 原代培养 ; 细胞鉴定 英文关键词：mouse;;hepatocyte isolation;;primary culture;;cell identification 中文刊名：NMXK 英文刊名：Animal Husbandry and Feed Science 机构：河南科技大学动物科技学院; 出版日期：2019-05-07 08:51 出版单位：畜牧与饲料科学 年：2019 期：v.40;No.253 基金：河南科技大学大学生研究训练计划(SRTP)(2018368) 语种：中文; 页：NMXK201904004 页数：5 CN：04 ISSN：15-1228/S 分类号：13-17

摘要

为分离、培养高纯度原代小鼠肝实质细胞,并鉴定其纯度及生物活性,试验采用原位两步循环灌流法及多次低速差速离心法分离纯化肝实质细胞,促贴壁培养基原代培养,台盼蓝检测其存活率,PAS反应检测其糖原合成能力,免疫荧光化学检测细胞中CK18表达情况。结果表明:每只小鼠可获取约1.5×10~6个细胞,存活率>97%;镜下发现细胞在接种后6 h开始贴壁,72 h贴壁细胞生长状态良好,胞体变大、不规则,细胞间相互靠拢呈岛状或条索状连接;肝细胞出现成片紫红色糖原颗粒,CK18在细胞中均匀分布;糖原反应联合CK18免疫荧光显示细胞纯度在90%以上。说明该试验所用方法分离出肝实质细胞数量和存活率高,促贴壁培养基培养的肝实质细胞纯度高,为细胞代谢、细胞毒性等的研究奠定了基础。
        The aim of the present study was to establish the methods for isolation and identification of mouse hepatocytes with high purity and viability, and to evaluate their purity and biological activity. The mouse hepatocytes were isolated and purified by using two-step in-situ-circulating perfusion method in combination with multi-round of low speed differential centrifugation.Hepatocytes were primarily cultured in adherent culture medium and their viability were evaluated by trypan blue exclusion. PAS reaction and immunofluorescence assay were used to examine their glycogen synthesis ability and CK18 expression level,respectively. The results showed that a total of 1.5×10~6 hepatocytes with >97% of motility rate could be harvested from individual mice in this study. The primary adherence of hepatocytes was observed after 6 h of inoculation under inverted microscope. The adherent cells grew well 72 h post inoculation. The size of the hepatocytes was enlarged with irregular shape, and they were connected with the surrounding cells in island or strip-like junctions. Glycogen deposits in form of purple-red particles were found in hepatocytes in which CK18 was uniformly distributed. PAS reaction in combination with immunofluorescence staining for CK18 demonstrated the high-purity(>90%) of hepatocytes. In conclusion, the mouse hepatocytes isolated by using the established method in this study had high harvest yield and viability, and the cells cultured in adherent culture medium had high purity, providing a foundation for further investigation on metabolism and cytotoxicity of hepatocytes.

引文

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