大鼠肝实质细胞原代培养模型的研究及其功能鉴定

目的:为肝细胞功能及相关的研究提供一种客观的、简便的大鼠肝细胞原代培养模型,并鉴定肝细胞的功能。方法:在传统的两步灌流法的基础上进行改良分离培养肝实质细胞;流式细胞术测定原代肝细胞的细胞周期和细胞凋亡情况;SRB法测定不同培养基对原代肝细胞的生长和功能的影响。结果:分离得到的肝细胞成活率可达90%以上,纯度可达95%以上;流式细胞术结果表明原代肝细胞大多处于G0/G1期,且基本无凋亡;SRB法和白蛋白分泌量检测结果显示,低糖DMEM组原代肝细胞生长和白蛋白分泌功能在短期内(1~5 d)与高糖DMEM组没有明显差异;RPMI1640组肝细胞的生长和功能则明显低于前两组(P<0.05)。结论:体外培养的肝细胞活力和纯度均较高,体外培养后细胞功能正常,是一种实用的体外研究肝细胞良好的细胞学模型。

Development of reliable primary cultured hepatocytes model

AIM: To develop a reliable primary cultured hepatocytes model in vitro for liver metastasis research. METHODS: Hepatocytes were isolated by a modification of the two-step collagenase perfusion method. The apoptosis and cell cycle of hepatocytes were measured with flow cytometry. The proliferation of hepatocytes was detected by SRB method. RESULTS: The viability and purity of hepatocytes were 90% and 95%,respectively. The result of flow cytometry analysis showed that there was little apoptosis in hepatocytes and most of hepatocytes were in G_0/G_1 phase. The proliferation and albumin-secreting function of hepatocyte cultured by low glucose DMEM and high glucose DMEM were higher than that of cultured by RPMI1640 during 1 to 6 day, but there was no significant different between low glucose DMEM group and high glucose DMEM group. CONCLUSION: Hepatocytes have higher purity and viability with the normal biological activity for about 6 days by this method and it may be a cell model for the study of liver metastasis in vitro.