不同分离介质对小鼠原代肝星状细胞分离纯化效果的影响

目的 比较不同的分离介质对小鼠原代肝星状细胞（hepatic stellate cells,HSCs）分离纯化效率的差异.方法 肝门静脉原位灌注,预灌注不含钙镁离子的D-Hanks液及含有Ⅳ型胶原酶的灌注液,取消化后的肝脏,分别采用Ficoll-Paque PLUS、Percoll、Nycodenz作为分层介质,密度梯度离心纯化原代HSCs.显微镜下观察细胞纯度并计算细胞得率.锥虫蓝染色判断细胞活率.激光共聚焦显微镜观察原代HSCs的固有荧光情况.油红O染色观察获得原代HSCs的纯度情况.结果 采用Ficoll-Paque PLUS、Percoll、Nycodenz分离细胞得率分别为（1＆#177;0.5）＆#215;106,（1＆#177;0.5）＆#215;105,（0.8＆#177;0.5）＆#215;106;纯度分别为75％ ～ 85％,91％ ～95％,91％ ～95％；活率均高于90％.结论 用Nycodenz作为分离介质分离肝星状细胞的方案最优.

The performance of different isolation regents for purification of mice primary hepatic stellate cells

Objective To compare the advantages and disadvantages of the three isolation regents for isolating primary hepatic stellate cells（HSCs） of mice.Methods The mice HSCs was isolated by in situ perfusion of per-perfusion solution,perfusion with collagenase Ⅳ.After digestion,Ficoll-Paque PLUS,perco11 and Nycodenz were use for the gradient centrifugation.To calculate the quantity,purity and the viability of the HSCs.The autofluorescence and Oil O staining were used to identify primary HSCs.Results The quantity of HSCs was （1.0 ＆#177; 0.5） ＆#215; 106,（1.0 ＆#177; 0.5） ＆#215; 105,（0.8 ＆#177; 0.5） ＆#215; 106/ g_liver,respectively.Using Ficoll-Paque PLUS,percoll and Nycodenz,the purity of primary HSCs were 75%-85%,91%-95%,91%-95%,respectively.The viability were all above 90%.Conclusion compared with Ficoll-Paque PLUS and percoll,the Nycodenz is the the best separating medium for isolation and purification of mice primary HSCs.