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目的探讨伊马替尼(STI571)、十字孢碱(STS)对非小细胞肺癌A549细胞凋亡的影响。方法经STI571和STS处理后,通过Western印迹检测A549细胞内凋亡诱导因子(apoptosis-inducing factor,AIF)的变化,然后利用膜联蛋白(annexin)Ⅴ-FITC PI凋亡试剂盒,借助流式细胞仪,检测A549细胞凋亡;利用Hoechst染色检测A549细胞凋亡现象并通过免疫荧光技术检测细胞内AIF和线粒体的变化。结果 Western印迹检测结果显示,STI571和STS处理能够提高AIF在A549细胞内以相对分子质量为57×103的形式表达;流式细胞仪检测结果显示,STS处理组膜联蛋白ⅤFITC染色的细胞数随着STS处理时间的增加呈现先增后降的趋势,溴化乙锭PI染色的细胞数随着处理时间增加到20 h呈现增多的趋势;STI571处理组膜联蛋白ⅤFITC的染色结果和溴化乙锭的染色结果与对照组相比,变化均不显著;STI571与STS共同作用组膜联蛋白ⅤFITC染色细胞数少于STS单独处理组结果,溴化乙锭染色的细胞数明显多于STS单独处理组;Hoechst染色结果显示,STS处理组和STI571、STS共同处理组细胞核内染色质发生凝集;间接免疫荧光结果显示,AIF发生聚集且细胞内线粒体染色亮度减弱。结论 STS和STI571处理能够引起细胞内AIF分布形式上的变化,STS可引起A549细胞发生细胞凋亡,STI571处理不能引起细胞发生凋亡,但能引起细胞内AIF发生聚集并对STS引起的细胞凋亡有促进作用。
Objective To investigate the effects of imatinib (STI571) and staurosporine (STS) on the apoptosis of non-small cell lung cancer A549 cells. Methods After treated with STI571 and STS, the changes of apoptosis-inducing factor (AIF) in A549 cells were detected by Western blotting, and then analyzed by flow cytometry with Annexin Ⅴ-FITC PI apoptosis kit. Apoptosis of A549 cells was detected by flow cytometry. The apoptosis of A549 cells was detected by Hoechst staining and the changes of AIF and mitochondria were detected by immunofluorescence. Results Western blotting results showed that STI571 and STS treatment could increase the expression of AIF in A549 cells with relative molecular mass of 57 × 103. Flow cytometry showed that the number of annexin ⅤFITC stained with STS treatment group was STS treatment time increased first and then decreased, the number of cells stained with ethidium bromide PI increased with the treatment time increased to 20 h; STI571 treatment group annexin Ⅴ FITC staining results and Bromide B The staining results of ingot were not significantly different from those of the control group. The number of Annexin Ⅴ FITC staining cells was less than that of STS alone treated with STI571 and STS. The number of cells stained with ethidium bromide was significantly more than STS alone Hoechst staining showed that chromatin condensation occurred in nuclei of STS-treated group and STI571 and STS groups. Indirect immunofluorescence showed that AIF aggregated and mitochondrial staining decreased. Conclusion STS and STI571 treatment can induce the change of intracellular AIF distribution. STS can induce apoptosis of A549 cells. STI571 treatment can not induce cell apoptosis, but it can cause intracellular accumulation of AIF and cause STS-induced apoptosis Death has a catalytic role.