醋酸铅对大鼠胰岛瘤细胞INS-1自噬与凋亡的影响

Effects of Lead Acetate on the Autophagy and Apoptosis of the Rat Insuloma Cells INS 1

为了研究环境中常见重金属污染物铅对大鼠胰岛瘤细胞INS-1自噬与凋亡的影响及其可能的分子机制,使用不同浓度醋酸铅暴露大鼠胰岛瘤细胞INS-1,利用磺酰罗丹明B(sulforhodamine B,SRB)法检测细胞存活率;Western blot法检测不同浓度醋酸铅对细胞中自噬与凋亡标志蛋白及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路标志分子表达的影响;利用活性氧(reactive oxygen species,ROS)试剂盒检测细胞内ROS水平;用ROS清除剂N-乙酰-L-半胱氨酸(N-acetylcysteine,NAC)预处理细胞,检测醋酸铅引起的细胞自噬与凋亡是否受到影响。使用统计学软件SPSS 17.0对数据进行统计学分析后,与对照组相比,醋酸铅导致INS-1细胞的存活率下降(p<0.05),且呈时间-剂量依赖性;随醋酸铅浓度增大,自噬和凋亡标志蛋白表达增加,mTOR信号途径标志分子没有明显变化;NAC预处理后醋酸铅引起的细胞自噬和凋亡减少。实验结果表明,醋酸铅可通过mTOR-非依赖途径诱导INS-1细胞的自噬与凋亡,其可能机制为刺激细胞内ROS的产生。

To study the effect of the environmental heavy metal pollutant lead on the autophagy and apoptosis of the rat insuloma cells INS 1 and their probable molecular mechanisms, the INS 1 cells were exposed to lead acetate with different concentration, the viabilities of the cells were determined by sulforhodamine B （SRB） assay. Western blot analysis was performed to detect the expression of the markers of autophagy and apoptosis and the related proteins of mammalian target of rapamycin （mTOR）. Reactive oxygen species （ROS） kit was used to detect the intra- cellular ROS. ROS scavenger N acetylcysteine （NAC） was used to pretreat the cells to determine the effects of lead on the autophagy and apoptosis of INS 1 cells. By analyzing the results with statistical software SPSS 17.0, cornpared with the control group, the viability of INS 1 cells which were treated with lead acetate, decreased in a time and dose dependent manner （p 〈0.05）. The expression of autophagy and apoptosis markers were enhanced with the concentration of lead acetate increasing. However, the markers of mTOR signal chain were no significant change. It was interesting to found that the effect of lead on the autophagy and apoptosis of INS 1 cells were decreased after they were pretreated with NAC. Thus, it was deduced that lead acetate may induce autophagy and apoptosis in INS 1 cells via an mTOR independent pathway. The probable mechanism was stimulating an increase of ROS.