小鼠肝脏多种类型细胞同步分离法

Synchronization isolation method for multiple types of cells from mouse liver

目的探索小鼠肝细胞、肝星状细胞(HSC)和淋巴细胞分离、纯化的简易可行方案。方法雄性C57bl/6小鼠采用经肝门静脉灌注消化法获取细胞悬液,随后percoll非连续密度梯度离心法分离、纯化细胞。台盼蓝拒染法检测细胞存活率,糖原染色、细胞角蛋白18和透射电子显微镜鉴定肝细胞,α-平滑肌肌动蛋白联合结蛋白免疫荧光鉴定HSC,流式细胞术分析肝脏淋巴细胞亚群。结果体质量约22 g的小鼠其肝脏经分离纯化后可获得约2.7×10^(7)个肝细胞,约5.7×10^(5)个HSC,约4.6×10^(6)个肝单个核细胞,各组分中细胞存活率均>95%。肝细胞中可见成片紫红色糖原颗粒和细胞角蛋白18,电镜结果显示肝细胞内存在丰富的细胞器,细胞间存在紧密连接;HSC表达α-平滑肌肌动蛋白和结蛋白;流式细胞术显示肝单个核细胞中包括CD4、CD8、自然杀伤细胞和自然杀伤T细胞等淋巴细胞亚群。结论肝门静脉灌注消化法可同步分离小鼠肝脏中多种原代细胞,具有简便、高效的特点。

Objective To explore a simple and feasible method for the isolation and purification of hepatocytes,hepatic stellate cells(HSC),and lymphocytes from mice.Methods The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation.Trypan blue exclusion was used to determine cell viability.Glycogen staining,cytokeratin 18,and transmission electron microscopy were used to identify hepatic cells.Immunofluorescence was used to detectα-smooth muscle actin combined with desmin in HSCs.Flow cytometry was used to analyze lymphocyte subsets in the liver.Results After isolation and purification,about 2.7×10^(7) hepatocytes,5.7×10^(5) HSCS,and 4.6×10^(6) hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g.The cell survival rate in each group was>95%.Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18.Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells.HSC had expressedα-smooth muscle actin and desmin.Flow cytometry showed hepatic mononuclear cells,including lymphocyte subsets such as CD4,CD8,NKs,and NKTs.Conclusion The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.