一种分离新生小鼠肝细胞的简单方法

A simple method for isolation and culture of hepatocvtes from neonatal mouse liver.

目的建立新生小鼠肝细胞体外分离培养方法。方法采用胶原酶消化组织块法分离新生小鼠肝细胞，倒置显微镜动态观察细胞形态特征，并进行鉴定：PAS（periodieacid-Schiff，PAS）染色观察细胞肝糖元的含量；免疫组化SP法检测细胞甲胎蛋白AFP（alpha fetal protein，AFP）的表达；生化法检测肝细胞培养上清白蛋白的含量。结果肝组织块经用胶原酶直接消化，获得的肝细胞成活率达80％以上。肝细胞培养一周后长满瓶底，呈集落生长，融合成片。培养的肝细胞PAS糖原染色阳性；AFP表达阳性；培养上清白蛋白含量至培养5～7d时迭峰值，随后又逐渐下降。结论胶原酶消化组织块法分离新生小鼠肝细胞，方法简单、高效，培养的原代肝细胞符合新生肝细胞的生物学特性。

Objective： To establish a method for isolation and culture of hepatocytes from neonatal mouse liver in vitro. Methods： The liver of neonatal mouse was digested by collagenase to isolate the hepatocytes. The biological characteristics of the hepatocytes were identified, including the dynamic observation of morphological features, the detection of hepatin and alpha fetal protein （AFP） in the hepatocytes and the measurement ofthe levels of albumin from the supernatant of the culture. Results ： The hepatocytes obtained by collagenase digestion had a viability of at least 80%. The expression of hepatin and AFP in the hepatocytes was positive. The levels of albumin from the supematant of the culture peaked at 5 - 7 days. Conclusion ： The method is simple, rapid and efficient.