1,2-二氯乙烷通过线粒体通路诱导人星形胶质细胞凋亡研究

1,2-Dichloroethane-induced apoptosis in human astrocytes through mitochondrial pathway

目的通过筛选细胞凋亡差异表达蛋白,探讨1,2-二氯乙烷(1,2-DCE)诱导人星形胶质细胞(HAs)凋亡的相关分子机制。方法以终浓度分别为0~80 mmol/L或0~40 mmol/L的1,2-DCE染毒HAs,培养24 h后,采用磷脂结合蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶流式细胞技术检测细胞凋亡,采用AAH-APO-1-2蛋白芯片筛选差异表达蛋白,采用实时荧光定量-聚合酶链式反应(qRT-PCR)验证相关蛋白的差异表达基因。结果在1,2-DCE浓度为0~80 mmol/L时,随着染毒剂量增加,HAs细胞总凋亡率升高,呈剂量-效应关系(P<0.01)。凋亡蛋白芯片筛选出7个差异表达蛋白,其中,与对照组比较,胰岛素样生长因子结合蛋白(IGFBP)-1、IGFBP-4和细胞色素C(Cyto C)表达均上调,P27、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2相互作用的细胞死亡中介物(BIM)和BH3交叉域死亡受体激动剂(BID)表达均下调。差异表达基因的qRT-PCR验证结果显示,40 mmol/L剂量组HAs中IGFBP-1、IGFBP-4和Cyto C的mRNA表达上调,与蛋白芯片的表达趋势一致;该组HAs中Caspase-3、BIM、BID的mRNA表达也上调。结论 1,2-DCE可能通过线粒体通路诱导HAs凋亡。

Objective To explore the molecular mechanism underlying 1,2-dichloroethane（1,2-DCE） induced apoptosis by screening differentially expressed proteins in human astrocytes（ HAs）. Methods HAs were cultured in complete medium with 1,2-DCE at various concentrations of 0-80 or 0-40 mmol/L. After 24 hours,apoptosis of HAs was evaluated using flow cytometry and staining with annexin Ⅴ-fluoresce in isothiocyanate and propidium iodide. An AAH-APO-1-2 protein chip was used to screen differentially expressed proteins and quantitative real-time polymease chain reaction（qRT-PCR） was used to verify related differentially expressed genes（DEGs）. Results At 1,2-DCE concentrations of0-80 mmol/L,the total apoptosis rate of HAs increased with 1,2-DCE concentrations in a dose-dependent manner（ P 0. 01）. Seven different kinds of proteins were screened out by apoptotic protein chip. Among them,the expression of insulin-like growth factor-binding protein（ IGFBP）-1,IGFBP-4 and cytochrome C（ Cyto C） were up-regulated,while the expression of P27,cysteine aspartic acid specific protease-3（ Caspase-3）,B-cell lymphoma-2 interacting mediator of cell death（ BIM） and BH3 interacting domain death agonist（ BID） were down-regulated compared with the control group. The result of DEGs verified by qRT-PCR showed that the expression of mRNA of IGFBP-1,IGFBP-4 and Cyto C at 1,2-DCE concentrations of 40 mmol/L was up-regulated. This result was in consistent with the trend of target expression in the protein chip. The mRNA expression of Caspase-3,BIM and BID was also up-regulated. Conclusion 1,2-DCE induces apoptosis of HAs through mitochondrial pathway.