小鼠原代肝星状细胞的分离、鉴定及生物学功能分析  

Isolation, Identification and Evaluation of Hepatic Stellate Cells from C57.BL/6J Mouse

作　　者：韩聚强 杜静华[3] 徐小洁[2] 刘文鹏[4] 梁吉光[5,2] 李佳睿 叶棋浓[2] HAN Ju-Qiang;DU Jing-Hua;XU Xiao-Jie;LIU Wen-Peng;LIANG Ji-Guang;LI Jia-Rui;YE Qi-Nong(Department of Liver Disease, PLA Army General Hospital, Beijing 100700,China;Department of Traditional and Western Medical Hepatology,Third Hospital of Hebei Medical University, Shijiazhuang 050051,China;Department of Infectious Disease,Third Hospital of Hebei Medical University, Shijiazhuang 050051,China;Beijing Institute of Biotechnology, Beijing 100850,China;Department of Interventional Radiography, First Hospital of Jilin University, Changchun 130021,China)

机构地区：[1]陆军总医院肝病科,北京100700 [2]军事医学科学院生物工程研究所,北京100850 [3]河北医科大学第三医院中西医结合肝病科,河北石家庄050051 [4]河北医科大学第三医院感染科,河北石家庄050051 [5]吉林大学第一医院介入科,吉林长春130021

出　　处：《生物技术通讯》2017年第5期600-603,708,共5页Letters in Biotechnology

基　　金：首都医学特色发展基金(z141107002514057)

摘　　要：目的:探索C57.BL/6J小鼠肝星状细胞(HSCs)分离、纯化的可行方案,并评价该法所得HSCs的生物学特性。方法:经肝门静脉,先用不含钙镁离子的D-Hanks液充分灌注肝脏,再以适宜浓度的链霉蛋白酶和Ⅳ型胶原酶顺序灌注,随后Percoll非连续密度梯度离心分离、纯化HSCs,台盼蓝拒染法检测HSCs活性,光镜观察体外培养的HSCs的形态学变化,免疫细胞化学鉴定α-平滑肌肌动蛋白(α-SMA)和结蛋白的表达,Western印迹检测α-SMA和Ⅰ型胶原蛋白的表达。结果:纯化后每只小鼠获得HSCs约5.5×105个,纯度及存活率均大于95%;免疫细胞化学技术证实培养的HSCs分别表达α-SMA和结蛋白;Western印迹显示原代HSCs体外培养7 d时,α-SMA和Ⅰ型胶原蛋白的表达量达到峰值。结论:建立的分离、纯化方案可获得高纯度、高活率、功能正常的小鼠原代HSCs,为进一步研究肝纤维化的发病机制提供了技术保障。Objective:To investigate and evaluate the feasible methods of isolating and purifying hepatic stellatecells(HSCs)from C57.BL/6J mouse in vitro.Methods:Mouse's liver was infused with D-Hank fluids via portalvein in situ.Subsequently,HSCs were isolated by discontinuous Percoll gradient centrifugation after liver was digested with pronase E,Ⅳtype collgenase and DNase,the cell viability was determined by trypan blue exclusion test.The morphological character of HSCs was observed by microscope.Bothα-SMA and desmin were identifiedby immunocytochemistry staining.Bothα-SMA andⅠtype collagen was detected by Western blot.Results:Theyield amount of HSCs was about5.5×105per mouse's liver.Both viability and purity were more than95%.Immunocytochemistry staining demonstrated thatα-SMA and desmin were positive in HSCs cultured in vitro.Westernblot assay showed thatα-SMA andⅠtype collagen were expressed extremely at7th day.Conclusion:The established protocol is so successful in isolating and culturing the primary mouse HSCs that provides a technical support for research of relevant liver fibrogenesis in the future.

关 键 词：肝星状细胞 原代培养 细胞分离 小鼠 

分 类 号：Q24[生物学—细胞生物学]