二氯化钴诱导A549细胞凋亡及机制的研究

目的探讨二氯化钴(CoCl2)诱导人非小细胞肺癌A549细胞凋亡的作用机制。方法应用不同浓度CoCl2作用A549细胞不同时间,建立化学性缺氧诱导细胞凋亡的实验模型;应用MTT法检测细胞生长抑制率;应用AO/EB荧光染色法检测细胞凋亡情况;应用Western blotting法检测总Akt(total-Akt)、磷酸化Akt(p-Akt)和survivin蛋白表达情况。结果 300、400μmol/L CoCl2作用24、36、48h后A549细胞的生长受到不同程度的抑制,并呈现比较明显的剂量和时间依赖关系;通过荧光显微镜下能观察到细胞呈新月形、核质体绿色、染色质浓缩,呈现凋亡显著特征,200、300、400μmol/L CoCl2处理24、48h后的细胞凋亡率显著高于对照组(P<0.01);Western blotting检测结果显示300、400μmol/L CoCl2作用48h后细胞内p-Akt、survivin蛋白表达量较对照组显著减少(P<0.01),300μmol/L CoCl2作用36、48h较对照组能显著下调p-Akt、survivin蛋白表达量(P<0.01),而总Akt蛋白表达量均无显著变化。结论 CoCl2可能是通过抑制p-Akt和survivin的蛋白表达,从而诱导A549细胞凋亡。

Mechanism of Apoptosis in A549 Cells Induced by CoCl2

Objective To explore the mechanism of apoptosis in A549 cell line of non-small lung cancer induced by CoCl2.Mothods The hypoxia model was set up by using different concentration of cobalt chloride(CoCl2) to affect A549 cell line at different times.The growth inhibition was detected by MTT assay and the morphological transformation of cell was observed by fluorescent microscope.The change expression of Akt,p-Akt and survivin were quantified by Western bolotting.Recults The growth inhibition rate of A549 was significantly increased in the groups that were treated by CoCl2(300,400μmol/L) in 24,36,48h respectively(P<0.01).From the results of morphology,after treatment of 24,48h with CoCl2(200,300,400μmol/L),we demonstrated that A549 cell line was characterized by apoptosis of crescent-shaped cells,the green nuclear plastid and nuclear fragmentation in this groups and the growth inhibition rate of A549 was significantly increased in the group that was treated by CoCl2(300,400μmol/L) in 48h when compared with the control group(P<0.01).After exposing to CoCl2(300μmol/L) in 36,48h,the expression of survivin and p-Akt protein in A549 cell line were apparently downregulated(P<0.01),however,the expression of Akt had no markable change.Conclusion CoCl2 induces apoptosis of A549 cell line through suppressing the expression of p-Akt and survivin protein.