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目的观察顺铂联合自噬特异性增强剂beclin1和特异性抑制剂3-MA对食管癌细胞增殖抑制的影响,探讨自噬在顺铂诱导的食管癌TE1细胞死亡中的作用。方法 MTT法检测空白对照组、顺铂组、顺铂分别联合beclin1和3-MA组对食管癌TE1细胞增殖抑制作用的影响;Western blot法检测顺铂对食管癌TE1各组细胞中bcelin1蛋白表达的影响;MDC荧光染色检测各组细胞内自噬囊泡数量的变化;流式细胞仪检测各组细胞中细胞周期和凋亡的改变。结果 MTT检测各组细胞的增殖抑制率分别为:1.06%,31.59%,18.93%,51.44%。DDP组能有效抑制TE1细胞的增殖。与单独使用DDP组相比,beclin1+DDP组对TE1细胞的增殖抑制率明显降低(P<0.01);3-MA+DDP组对TE1细胞的增殖抑制率明显增高(P<0.01);Western blot检测各组细胞中beclin1蛋白表达量分别为0.319±0.029,1.175±0.06,1.537±0.076,0.91±0.047(P<0.01);荧光显微镜观察beclin1+DDP组中MDC标记的自噬荧光强度最高,细胞质内出现了明显的绿色荧光;FCM检测细胞周期分布结果显示,各组细胞中G0/G1期细胞比例分别为(44.82±0.437)%,(56.25±0.624)%,(27.553±0.085)%,(66.48±0.638)%;各组细胞中S期细胞比例分别为(34.85±0.235)%,(24.417±0.607)%,(51.8±0.721)%,(19.167±0.666)%。各组细胞的凋亡率分别为(1.237±0.015)%,(4.813±0.025)%,(1.32±0.01)%,(6.117±0.035)%(P<0.01)。结论顺铂可以诱导食管癌TE1细胞自噬和凋亡;反应性的自噬活性增强可能与顺铂耐药的形成有关,自噬水平的下调可能增加了食管癌细胞对化疗药物顺铂的敏感性,提示监测及调控肿瘤细胞的自噬水平可能为逆转食管癌顺铂耐药提供新的思路。
Objective To investigate the effect of autophagy-specific enhancers beclin1 and 3-MA on the proliferation of esophageal cancer cells and to explore the role of autophagy in cisplatin-induced esophageal cancer TE1 cell death. Methods MTT assay was used to detect the effects of blank control group, cisplatin group and cisplatin combined with beclin1 and 3-MA on the proliferation of esophageal cancer TE1 cells respectively. Western blot was used to detect the expression of bcelin1 protein in esophageal cancer TE1 cells The changes of the number of autophagic vesicles in each group were detected by MDC fluorescence staining. The changes of cell cycle and apoptosis in each group were detected by flow cytometry. Results The MTT assay showed that the inhibitory rates of proliferation were 1.06%, 31.59%, 18.93% and 51.44% respectively. DDP group can effectively inhibit the proliferation of TE1 cells. Compared with DDP group alone, the inhibition rate of TE1 cells was significantly decreased in beclin1 + DDP group (P <0.01); the inhibition rate of TE1 cells in 3-MA + DDP group was significantly increased (P <0.01); Western blot The mRNA expression of beclin1 in each group was 0.319 ± 0.029, 1.175 ± 0.06, 1.537 ± 0.076 and 0.91 ± 0.047, respectively (P <0.01). The fluorescence intensity of MDC-labeled autophagy in beclin1 + DDP group was the highest under fluorescence microscope, (44.82 ± 0.437)%, (56.25 ± 0.624)% and (27.553 ± 0.085)%, respectively. The percentage of cells in G0 / G1 phase in each group was 66.48 ± 0.638)%. The percentage of S phase cells in each group was (34.85 ± 0.235)%, (24.417 ± 0.607)%, (51.8 ± 0.721)% and (19.167 ± 0.666)%, respectively. The apoptotic rate of each group was (1.237 ± 0.015)%, (4.813 ± 0.025)%, (1.32 ± 0.01)%, (6.117 ± 0.035)%, respectively. Conclusion Cisplatin can induce esophageal cancer TE1 cell autophagy and apoptosis. The increased autophagy activity may be related to the formation of cisplatin resistance. The down-regulation of autophagy may increase the sensitivity of esophageal cancer cells to chemotherapeutic drug cisplatin It suggests that monitoring and regulating the autophagy level of tumor cells may provide a new idea for reversing cisplatin resistance in esophageal cancer.