硒酸壳聚糖通过线粒体途径诱导A549细胞凋亡的初步研究

Preliminary Study of Seleno-chitosan-induced Apoptosis in A549 Cells through a Mitochondrial Signaling Pathway

本文研究了硒酸壳聚糖(SC)对非小细胞肺癌A549增殖抑制及诱导凋亡作用,并进一步研究了SC对其凋亡前后蛋白差异表达。采用MTT法检测了SC对A549细胞的增殖抑制作用;并用激光共聚焦分别观察了Hoechst33342/PI染色后A549细胞形态的变化以及钙黄绿素染色后细胞线粒体MPTP的开放状况;流式细胞仪检测了SC对A549细胞线粒体跨膜电位的影响;利用2-DE结合MALDI-TOF质谱技术检测了细胞凋亡相关蛋白的差异表达。结果表明:SC对A549细胞具有显著的增殖抑制作用,并在一定范围内呈现时间和浓度依赖性;SC可使A549细胞发生形态改变,使细胞变得不规则,染色质固缩和边集;经SC处理后,细胞的线粒体膜通道逐渐开放;经MALDI-TOF鉴定发现了2个差异表达的蛋白,即抗增殖蛋白(prohibitin isoform 1),细胞内热休克蛋白(heat shock protein)。

The effects of seleno-chitosan（SC） on cell proliferation inhibition and apoptosis in human non-small-cell lung cancer A549 cells were investigated, and the differential expression of proteins in SC-treated and untreated cells were determined. The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay was employed to determine the inhibitory effect of SC on the proliferation of A549 cells. Morphological changes in the A549 cells after Hoechst 33342/PI staining and the opening of the mitochondrial permeability transition pore（MPTP） after calcein staining were observed by laser scanning confocal fluorescence microscopy. The effect of SC on mitochondrial membrane potential（ΔΨm） was analyzed by flow cytometry（FCM）. Furthermore, two-dimensional gel electrophoresis（2-DE） combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF） was used to identify the differential expression of apoptosis-related proteins. The results showed that SC could significantly suppress the proliferation of A549 cells in a time-and concentration-dependent manner. Morphological changes of A549 cells could be induced by SC; the cell shape became irregular and chromatin condensation and margination occurred. After SC treatment, the MPTP of A549 cells was gradually opened. Additionally, two differentially expressed proteins, prohibitin isoform 1 and heat shock protein, were identified by MALDI-TOF.