多氯联苯(PCB1254)诱导胰岛细胞凋亡的机制研究

Apoptosis induced by polychlorinated biphenyls(PCB1254) in islet cells

目的 :本研究探讨多氯联苯(PCB1254)诱导体外培养胰岛β细胞株INS-1凋亡的可能机制。方法 :用不同浓度的PCB1254刺激INS-1细胞后,使用CCK-8法检测细胞活性,选择适当浓度的PCB1254进行INS-1细胞凋亡机制的研究;PCB1254(5μg/ml)刺激INS-1细胞后,倒置显微镜下观察细胞的形态学变化,并以流式细胞仪检测细胞凋亡;Western blot检测Cleaved Caspase-3、Caspase-3、Bim、Bcl-2、c-Fos、p-JNK、p-P38、p-ERK蛋白的表达;二氢乙啶(dihydroethidium,DHE)荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;PCB1254诱导INS-1细胞凋亡后,使用ERK抑制剂PD98059进行干预,倒置显微镜下观察细胞凋亡情况。结果:随着浓度的增加,PCB1254对细胞活性的抑制逐渐增强,当浓度≥5μg/ml时,与对照组差异有统计学意义(P<0.01);倒置显微镜下观察PCB1254(5μg/ml)能引起INS-1细胞凋亡,凋亡细胞脱落,漂浮于培养基中;流式细胞术检测结果显示PCB1254能诱导INS-1细胞凋亡;Western blot检测凋亡细胞中凋亡相关蛋白Cleaved Caspase-3、Bim表达上调,抗凋亡蛋白Bcl-2表达下调,氧化应激相关蛋白c-Fos表达上调,ROS荧光增强;MAPK信号通路中p-ERK蛋白表达上调;ERK抑制剂PD98059干预后细胞凋亡无明显变化。结论:PCB1254可能通过氧化应激信号通路引起INS-1细胞的凋亡,此过程中伴有ERK信号通路的激活。

Objective：To investigate the possible mechanisms of apoptosis occurred in islet β-cell line（INS-1） which was induced by PC B1254 in vitro. Methods：After the treatment with different concentrations of PCB1254,the cell viability of INS-1 was assayed by CCK-8. After the treatment with PCB1254（5 μg/ml）,the morphological change and apoptosis situation of INS-1 were observed by inverted microscope. The apoptosis of INS-1 was further detected by flow cytometry. The expression of Caspase-3,Bim,Bcl-2,C-Fos, P-JNK,P-P38 and P-ERK were measured by Western blot. Reactive oxygen species （ROS） level was detected by dihydroethidium （DHE） fluorescence probe. The apoptosis level of INS-1 was obsarved under inverted microscope after ERK inhibitor PD98059 intervening the PCB1254 treated INS-1 cells. Results：With the increasing of concentration of PCB1254,the cell viability of INS-1 declined. When the concentration was higher than 5 μg/ml,there were significant differences compared with the control group（P 〈 0.01）. INS-1 cells apoptosis, which was induced by PCB1254 （5μg/ml）, was observed through inverted microscope. The apoptosis cells turned dark and floated in the medium. Flow cytometry assay showed that PCB1254 could induce the apoptosis of INS-1 cells. Western blot showed that the expressions of apoptosis related Cleaved Caspase-3 and Bim proteins were up-regulated,while the expression of anti-apoptotic protein Bcl-2 was down-regulated. The expression of oxidative stress related protein C-Fos was up-regulated and the ROS fluorescence was enhanced. The expression of p-ERK in MAPK signal pathway was up-regulated. No obvious change of apoptosis was found under the intervention of ERK inhibitor PD98059. Conclusion：PCB1254 may induce the apoptosis of INS-1 cells through oxidative stress signaling pathway,and this process may be accompanied with the activation of ERK signaling pathway.