乙醇对小鼠胰岛瘤细胞凋亡的影响及其分子机制

目的观察乙醇对小鼠胰岛瘤细胞(NIT-1细胞)凋亡的影响及Bcl-2、Bax和Caspase-3表达的变化,探讨乙醇诱发小鼠胰岛瘤细胞凋亡的分子机制。方法体外培养的小鼠NIT-1细胞,不同剂量乙醇(0、50、100、2004、00 mmol/L)作用24 h,单细胞凝胶电泳实验(SCGE),AnnexinV/PI双标记法流式细胞仪检测细胞凋亡情况。不同剂量的乙醇作用6、122、4 h,RT-PCR法检测与凋亡有关的基因Bcl-2,Bax和Caspase-3 mRNA表达水平。结果SCGE实验结果显示,低剂量乙醇损伤不明显,高剂量(2004、00 mmol/L)组DNA迁移率、DNA损伤程度分级、DNA平均迁移长度与对照组比较,差异均有显著性。AnnexinⅤ/PI检测结果表明,2004、00 mmol/L乙醇组NIT-1细胞凋亡率升高,与对照组相比,差异有非常显著性。乙醇作用6 h,随着乙醇剂量增加,Bcl-2/Bax mRNA表达先升高后降低;乙醇作用12 h,400 mmol/L剂量Bcl-2/Bax比值下降;作用24 h,各剂量组Bcl-2/Bax比值均下降。Caspase-3 mRNA表达水平与乙醇剂量和作用时间相关。400 mmol/L剂量组作用24 h,Caspase-3 mRNA表达增加。结论乙醇可诱发体外培养的NIT-1细胞凋亡,凋亡的发生很可能与Bcl-2家族和Caspase-3激活有关。

The study on apoptosis and its molecular mechanism in mouse insulinoma cells induced by ethanol

Objective To investigate the effect of ethanol on apoptosis in mouse insulinoma cells(NIT-1 cells) and to evaluate the pathway associated with the regulation of Bcl-2,Bax and Caspase-3.Methods After NIT-1 cells were treated with ethanol at concentrations of 0,50,100,200 and 400 mmol/L for 24 h,single cell gel electrophoresis(SCGE) and AnnexinV/PI methods were performed to detect the apoptosis.After NIT-1 cells were exposed to various concentrations of ethanol (same as above) for 6,12 and 24 h,the levels of Bcl-2,Bax and Caspase-3 mRNA expression were determined by RT-PCR method.Results SCGE results showed that the low concentration of ethanol induced no obviously DNA damage in NIT-1 cells,but the rate of DNA migration,degree of DNA damage and average length of DNA migration of 200 and 400 mmol/L ethanol-treated groups were significantly changed compared to those of untreated group.The results of Annexin V/PI assay indicated that the apoptosis rates of NIT-1 cells in 200 and 400 mmol/L ethanol-treated groups were higher than that of untreated group.At 6 h,the ration of Bcl-2/Bax mRNA expression was increased first,then decreased with the increasing of ethanol dosage.At 12 h and 24 h,the ration was decreased.By treatment at 400 mmol/L,the decrease was dramatically compared to control group.Ethanol increased the caspase-3 mRNA expression at high concentrations and long exposure time.At 24 h,the caspase3 mRNA expression was increased significantly by treatment at 400 mmol/L.Conclusion Ethanol induced apoptosis in NIT-1 cells and the potential molecular mechanism probably relates with the Bcl-2 family and Caspse-3.