【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。更多还原

【Abstract】 Objective: To investigate and evaluate the feasible methods of isolating and purifying hepatic stellate cells(HSCs) from C57.BL/6 J mouse in vitro. Methods: Mouse’s liver was infused with D-Hank fluids via portal vein in situ. Subsequently, HSCs were isolated by discontinuous Percoll gradient centrifugation after liver was digested with pronase E, Ⅳ type collgenase and DNase, the cell viability was determined by trypan blue exclusion test. The morphological character of HSCs was observed by microscope. Both α-SMA and desmin were identified by immunocytochemistry staining. Both α-SMA andⅠtype collagen was detected by Western blot. Results: The yield amount of HSCs was about 5.5×105 per mouse’s liver. Both viability and purity were more than 95%. Immunocytochemistry staining demonstrated that α-SMA and desmin were positive in HSCs cultured in vitro. Western blot assay showed that α-SMA andⅠtype collagen were expressed extremely at 7 th 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.更多还原