论文部分内容阅读
目的探讨自噬抑制剂3-甲基腺嘌呤(3MA)对Ce6-PDT诱导结肠癌SW620细胞凋亡及自噬的影响。方法取对数生长期SW620细胞,分为空白对照组、单纯光照组、Ce6-PDT组和3MA-Ce6-PDT组,Ce6-PDT组加入不同浓度Ce6(0.125、0.25、0.5、1.0、2.0、4.0、8.0μg/m L),3MA-Ce6-PDT组给予1.0μg/m L的Ce6+5 mmol/m L的3MA。采用MTT法测算细胞存活率;Annexin V-PE/7-ADD染色及罗丹明123联合流式细胞仪检测细胞凋亡率和细胞线粒体膜电位变化;吖啶橙染色联合荧光显微镜观察细胞自噬体形成;蛋白免疫印迹法检测细胞凋亡相关蛋白Caspase-3、B细胞淋巴瘤/白血病基因-2(Bcl-2)及自噬相关蛋白LC3Ⅱ/Ⅰ表达。结果 Ce6-PDT组细胞存活率与Ce6浓度浓度呈负相关(r=-0.820,P<0.01),细胞凋亡率与Ce6浓度呈正相关(r=0.845,P<0.01)。选取细胞存活率和细胞凋亡率约为50%时Ce6浓度1μg/m L为后续给药浓度。在Ce6浓度达到1μg/m L时,线粒体膜电位随Ce6浓度增加而降低(P<0.01)。空白对照组和单纯光照组几乎没有亮红色荧光产生,Ce6-PDT组中有大量亮红色荧光出现,3MA-Ce6-PDT组中出现亮红色荧光的数量较Ce6-PDT组减少。与Ce6-PDT组相比,3MA-Ce6-PDT组细胞存活率降低、细胞凋亡率升高、自噬相关蛋白表达降低、凋亡相关蛋白表达增加(P均<0.05)。结论Ce6-PDT可引起结肠癌SW620细胞发生凋亡和自噬,3MA会促进Ce6-PDT诱导的结肠癌细胞SW620凋亡。
Objective To investigate the effect of 3-methyladenine (3MA), an autophagy inhibitor, on the apoptosis and autophagy of colon cancer SW620 cells induced by Ce6-PDT. Methods SW620 cells in logarithmic growth phase were divided into blank control group, Ce6-PDT group, 3MA-Ce6-PDT group and Ce6-PDT group. Ce6 (0.125,0.25,0.5,1.0,2.0, 4.0, 8.0 μg / m L) and the 3MA-Ce6-PDT group was given 1.0 μg / m L of Ce6 + 5 mmol / m L of 3MA. Cell viability was measured by MTT method. Apoptosis rate and mitochondrial membrane potential were measured by Annexin V-PE / 7-ADD staining and rhodamine 123 combined with flow cytometry. Acridine orange staining and fluorescence microscopy were used to observe the changes of cell autophagosome The expressions of Caspase-3, Bcl-2 and LC3Ⅱ / Ⅰ were detected by Western blotting. Results The cell viability in Ce6-PDT group was negatively correlated with Ce6 concentration (r = -0.820, P <0.01). The cell apoptosis rate was positively correlated with Ce6 concentration (r = 0.845, P <0.01). Select the cell survival rate and apoptosis rate of about 50% Ce6 concentration 1μg / m L for the subsequent dose concentration. Mitochondrial membrane potential decreased with the increase of Ce6 concentration when Ce6 concentration reached 1μg / ml (P <0.01). There was almost no bright red fluorescence in the blank control group and the pure light group, and a large number of bright red fluorescence appeared in the Ce6-PDT group. The number of bright red fluorescence in the 3MA-Ce6-PDT group was lower than that in the Ce6-PDT group. Compared with Ce6-PDT group, 3MA-Ce6-PDT group had lower cell survival rate, higher apoptosis rate, lower expression of autophagy-related protein and higher apoptosis-related protein expression (all P <0.05). Conclusion Ce6-PDT can induce apoptosis and autophagy in SW620 colon cancer cells, and 3MA can promote the apoptosis of colon cancer cell line SW620 induced by Ce6-PDT.