AAVC-1诱导A549细胞凋亡的线粒体机制研究

Mitochondrial mechanism of AAVC-1 induced apoptosis in A549 cells

目的:探讨皖南五步蛇蛇毒抑瘤组分Ⅰ(AAVC-1)诱导人非小细胞型肺癌A549细胞凋亡的作用,并分析其可能的线粒体机制。方法:以不同作用浓度的AAVC-1处理A549细胞,采用Annexin V/PI双染流式细胞仪分析法检测A549细胞凋亡百分率;JC-1检测细胞线粒体的膜电位变化,免疫组化检测细胞色素C在线粒体内的分布。结果:不同浓度梯度AAVC-1 1.0、3.0、9.0、20.0μg/m L处理24 h后,与对照组相比,A549细胞早期凋亡率明显增加(P<0.05);线粒体膜电位绿色荧光百分比由(23.01±6.07)%上升至(87.26±9.54)%(P<0.01),荧光显微镜观察到绿色荧光增强;免疫组化显示,随着AAVC-1浓度增加,胞质内细胞色素C平均光密度值明显增加(P<0.05)。结论:AAVC-1可以通过降低线粒体膜电位和促进细胞色素C的释放,激活线粒体通路诱导人非小细胞型肺癌A549的凋亡。

Objective： To investigate the apoptosis pathway and mechanisms of human non-small cell lung cancer A549 cells induced by anticoagulant fraction from Agkistrodon acutus venom（ AAVC-1）.Methods： Different dose of AAVC-1 was used to treat A549 cells,and the apoptosis percentage of A549 cells was assessed by flow cytometry with annexin V / PI double staining. Mitochondrial trans-membrane potential（ Δψm） of A549 cells was measured by JC-1 fluorescence probe and microscopy,and distribution of cytochrome C in the mitochondria was detected by immunohistochemistry after AAVC-1 treatment.Results： The early apoptosis rate of A549 cells was significantly increased after treatment with different dose of AAVC-1 for 24 h as compared with the control group（ P〈0.05）.The fluorescence green intensity was increased from（ 20.13±1.6） % to（ 89.12± 1.2） %（ P〈0.01）,and fluorescence spectroscopy indicated intensified intracellular green fluorescence.Immunohistochemical test revealed markedly increased average light density of cytochrome C with added dose of AAVC-1 treatment（ P〈0.05）.Conclusion： AAVC-1 can induce the apoptosis of human non-small cell lung cancer A549 cells by reducing the mitochondrial membrane potential and promoting the release of cytochrome C.