牛磺酸对原代培养大鼠肝细胞的促凋亡作用研究

目的探索分离培养原代正常大鼠肝细胞的条件和方法,研究牛磺酸对原代培养的正常大鼠肝细胞的促凋亡作用。方法使用经典的两步灌注法分离原代肝实质细胞后贴壁培养,记录细胞生长状况,台盼蓝拒染法测定细胞活率、绘制生长曲线。经用不同浓度的牛磺酸培养24 h后,用TUNEL法测定牛磺酸对原代培养大鼠肝细胞致凋亡作用,测定细胞凋亡率(AI)。结果两步灌注分离取得的肝细胞活率>70%,贴壁生长良好,从生长曲线可看出培养第3天进入对数生长期;TUNEL实验3个给药组AI分别为:3.71%,29.72%,42.56%。结论两步灌注法是一种细胞产量高,损伤小,纯度高的分离原代正常大鼠肝细胞的方法;高浓度的牛磺酸对体外培养肝细胞有一定致凋亡作用。

Effect of Taurine on Apoptosis of Rat Hepatocytes in Primary Culture

OBJECTIVE To study the condition of the primary culture of adult rat hepatocytes and the promoted effect of taurine on apoptosis of the hepatocytes.METHODS The situ two-step portal vein perfusion was used to obtain primary hepatocytes from normal rat.The growth of cells was observed by the typan blue exclusion assay,and the cell growth curve was draw.After incubating the cells in the medium containing 3 different concentrations of taurine for 24 h,TUNEL apoptosis tests were done. RESULTS The survival rate of the new-isolated rat hepatocytes was greater than 70%.These cells were in exponential phase of growth at 3rd day according to the growth curve.The apoptosis index of 3 experimental groups in TUNEL tests were 3.71%,29.72%,and 42.56% respectively.CONCLUSION The situ two-step portal vein perfusion was a good method for obtaining hepatocytes of adult rat for in vitro primary culture.Large dose of taurine can promot the apoptosis of hepatocytes in the cellular experiments.