大鼠肝细胞的分离及BRL细胞培养上清对原代肝细胞增殖的影响

Isolation of rat hepatocytes and effect of BRL cell culture supernatant on rat hepatocytes proliferation

目的 :研究大鼠肝细胞分离的方法及正常大鼠肝细胞来源的BRL细胞培养上清对原代大鼠肝细胞贴壁和增殖的影响。方法 :采用体外胶原酶 2步灌注法分离大鼠肝细胞 ,台盼兰染色法计算细胞数及细胞活率。以四甲基偶氮唑蓝 (MTT)法检测不同浓度的BRL细胞培养上清及含体积分数 15 %胎牛血清的普通培养液对肝细胞贴壁和增殖的影响。结果 :平均每只大鼠可获 2× 10 8个肝细胞 ,平均活力 94 .3%。用普通培养液培养肝细胞 ,肝细胞贴壁率低 ;BRL细胞培养上清能明显促进肝细胞的贴壁和生长 ,且有量效关系 (P <0 .0 5 )。结论 :用本法分离的大鼠肝细胞有较高的获取率和活力 。

Aim: To study a simplified method to isolation of rat hepatocytes and to observe the stimulation effect of supernatant from BRL cell culture on rat hepatocytes proliferation. Methods: Rat hepatocytes were isolated by a single two-step perfusion method. The yield and viability were assessed by trypan blue exclusion. [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide](MTT) was used to detect the effect of different supernatant concentration from BRL cell culture on the proliferation of rat hepatocytes.Results: The average yield of hepatocytes was 2×10 8 cells, with an average viability of 94.3%. The supernatant from BRL cell culture had strong biological activity to stimulate the proliferation of rat hepatocytes. And there was a close relationship between the different supernatant concentration from BRL cell culture and proliferation effect.Conclusion: The Rat hepatocytes isolated by the method mentioned above could have high yield and viability. Stimulation effect of the supernatant from BRL cell culture on the proliferation of rat hepatocytes might be attributed to some unknown factor(s) in the supernatant.