大鼠肝实质细胞原代培养模型的研究及其功能鉴定

Development of reliable primary cultured hepatocytes model

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.

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 G0/G1 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.