乙醇对胎鼠脑新皮质神经干细胞凋亡的影响

Effects of alcohol on apoptosis in neural stem cells from fetal rat cerebral neocortex

目的探讨乙醇对胎鼠神经干细胞凋亡的影响。方法将妊娠14 d胎鼠脑新皮质来源的神经干细胞暴露于终浓度分别为0(对照)、25、50、100 mmol/L的乙醇培养基溶液3 d。采用Annexin V/PI法检测细胞早期和晚期凋亡情况,采用JC-1探针法检测线粒体膜电位的改变,并检测凋亡相关基因Bcl-2、Bax、Caspase-3 mRNA和蛋白的表达。结果与对照组比较,50、100 mmol/L乙醇染毒组神经干细胞的早期凋亡细胞率明显升高,差异有统计学意义(P<0.05);而各浓度乙醇染毒组神经干细胞的晚期凋亡率无明显改变。且随着乙醇染毒浓度的升高,神经干细胞的早期凋亡细胞率呈上升趋势。仅100 mmol/L乙醇染毒组神经干细胞线粒体膜电位低于对照组,差异有统计学意义(P<0.05)。与对照组比较,各浓度乙醇染毒组神经干细胞Bax mRNA的表达水平均上升,差异有统计学意义(P<0.05);而Bcl-2和Caspase-3 mRNA的表达水平均无明显改变。且随着乙醇染毒浓度的升高,Bax mRNA表达量呈上升趋势。与对照组比较,50、100 mmol/L乙醇染毒组神经干细胞Bax蛋白的表达水平均升高,而Bcl-2/Bax蛋白的比值均降低,差异有统计学意义(P<0.05);各浓度乙醇染毒组神经干细胞Bcl-2、Caspase-3蛋白的表达水平均无明显改变。结论乙醇可引起神经干细胞早期凋亡,其机制可能与线粒体损伤和Bax表达上调有关。

Objective To understand the effects of ethanol on the apoptosis of neural stem cells. Methods Neural stem cells isolated from G14 fetal rat cerebral neocortex were exposed to ethanol medium at the doses of 0（control）,25,50,100 mmol/L respectively for three days. Cell early and late apoptosis were detected by Annexin V/PI assay. Mitochondrial membrane potential was detected by JC-1 probe method,and apoptosis related gene Bcl-2,Bax,Caspase-3 mRNA and protein expression were detected. Results The early apoptosis rates of neural stem cells in the 50 mmol/L and 100 mmol/L groups were significantly higher than that in the control group（P 0.05）. As exposure dose increased,the rate of early apoptotic cells increased accordingly, the late apoptosis cells rate had no significant changes in each exposure group. The mitochondrial membrane potential of neural stem cells only in 100 mmol/L group was significantly lower than that in control group（P0.05）.Compared with the control group,the expression levels of Bax mRNA in each exposure group increased significantly（P0.05）,with a dose dependent trend, the mRNA expression levels of Bcl-2 and Caspase-3 had no significant change. Compared with the control group, the expression of Bax protein increased significantly in the 50 mmol/L and 100 mmol/L groups,while the ratio of Bcl-2/Bax protein decreased, the difference was statistically significant（P0.05）. There was no significant change in the expression levels of Bcl-2 and caspase-3 protein in each exposure group. Conclusion Alcohol can cause early apoptosis of neural stem cells and mitochondrial injury and up-regulation of Bax may be involved in it.