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目的探讨原代大鼠肝细胞分离及培养的简易方法,并对其纯度及生物活性进行鉴定。方法采用改良原位两步非循环灌流法及多次过滤低速离心法分离原代大鼠肝细胞,常规DMEM高糖培养基培养原代大鼠肝细胞,锥虫蓝拒染法检测接种时肝细胞的存活率,倒置显微镜动态观察肝细胞形态变化。过碘酸-Schiff反应检测肝细胞糖原合成能力,并利用免疫细胞化学方法检测肝细胞角蛋白18(CK-18)的表达联合判断肝细胞纯度。结果每只大鼠可获取(1.38～1.74)×108个肝细胞,接种瞬时肝细胞活性>90%。倒置显微镜下观察肝细胞在接种后4 h基本完成贴壁,贴壁的细胞胞体变大变平,随后细胞相互靠拢呈岛状或条索状连接。糖原染色发现肝细胞内糖原被染成红色颗粒或片状。抗CK-18免疫细胞化学染色发现CK-18在肝细胞内均匀分布,被染成棕黄色。糖原染色及细胞CK-18联合鉴定肝细胞纯度达95%以上。结论用改良原位两步非循环灌注法及多次过滤低速离心法成功分离并培养原代大鼠肝细胞,可操作性强,所培养肝细胞数量、活力和纯度高,可在具备基本细胞培养条件的实验室推广。
Objective To explore a simple method for isolation and culture of primary rat hepatocytes and to identify their purity and biological activity. Methods Primary rat hepatic cells were isolated by modified two-step non-perfusion method and multiple filtration low-speed centrifugation. Primary rat hepatocytes were cultured in DMEM high glucose medium. Trypan blue exclusion assay The survival rate of cells, inverted microscope dynamic observation of liver cell morphological changes. The periodic acid-Schiff reaction was used to detect the glycogen synthesis ability of hepatocytes, and the hepatocyte cytoprotection was evaluated by the immunocytochemical method to detect the expression of cytokeratin 18 (CK-18). Results Each rat obtained (1.38 ~ 1.74) × 108 hepatocytes, transient hepatocyte activity was> 90%. Under inverted microscope, the hepatocytes were almost completely adhered 4 h after inoculation, and the adherent cells became larger and flattened. Subsequently, the cells approached each other and became island-like or cord-like connections. Glycogen staining found that glycogen in liver cells is dyed red particles or flaky. Anti-CK-18 immunocytochemical staining found that CK-18 evenly distributed in the liver cells, was dyed brown. Glycogen staining and cell CK-18 identification of hepatocytes purity of more than 95%. Conclusion The improved primary two-step non-circulatory perfusion method and multiple filtration low-speed centrifugation successfully isolated and cultured primary rat hepatocytes, maneuverability, the number of cultured hepatocytes, high activity and purity, with basic cells Laboratory conditions of culture promotion.