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目的:探讨5-氮杂-2’-脱氧胞苷对乳腺癌细胞自噬的影响及其可能的机制。方法:用依托泊苷、顺铂和5-氮杂-2’-脱氧胞苷处理乳腺癌细胞后,采用彗星实验测定细胞DNA损伤,蛋白质印迹法检测p53和p21蛋白表达;细胞自噬测定用3种方法:(1)蛋白质印迹法检测微管相关蛋白1轻链3-Ⅱ(microtubule-associated protein 1 light chain3-Ⅱ,LC3-Ⅱ)蛋白的表达;(2)用单丹磺酰戊二胺对乳腺癌细胞染色后,在荧光显微镜下计数自噬泡阳性细胞,并计算自噬泡阳性细胞百分比;(3)将pEGFP-LC3质粒转染至乳腺癌细胞后,在荧光显微镜下计数含绿色荧光蛋白的阳性细胞,并计算绿色荧光蛋白阳性细胞的百分比。结果:依托泊苷和顺铂均可诱导乳腺癌细胞DNA损伤和自噬,与对照组比较,药物处理组细胞的彗星长度增长(P<0.01),p53、p21和LC3-Ⅱ蛋白表达上调。5-氮杂-2’-脱氧胞苷也可诱导乳腺癌细胞DNA损伤和自噬,与对照组比较,处理组细胞的彗星长度增长(P<0.01),p53、p21和LC3-Ⅱ蛋白表达上调,自噬泡阳性细胞百分比和绿色荧光蛋白阳性细胞百分比上升,差异均有统计学意义(P<0.05和P<0.01)。结论:5-氮杂-2’-脱氧胞苷可诱导乳腺癌细胞自噬,其机制可能与其所诱导的DNA损伤有关。
Objective: To investigate the effect of 5-aza-2’-deoxycytidine on autophagy in breast cancer cells and its possible mechanism. Methods: After treatment of breast cancer cells with etoposide, cisplatin and 5-aza-2’-deoxycytidine, the DNA damage of cells was detected by comet assay and the expressions of p53 and p21 protein were detected by Western blot. Three methods: (1) Western blotting was used to detect the expression of microtubule-associated protein 1 light chain3-Ⅱ (LC3-Ⅱ) protein; (2) After staining the breast cancer cells with amines, autophagic vacuole positive cells were counted under a fluorescence microscope and the percentage of autophagy-positive cells was calculated. (3) The plasmid pEGFP-LC3 was transfected into breast cancer cells and counted under a fluorescence microscope Green fluorescent protein positive cells, and calculate the percentage of green fluorescent protein positive cells. Results: Both etoposide and cisplatin induced DNA damage and autophagy in breast cancer cells. Compared with the control group, the length of the comet was increased (P <0.01) and the expressions of p53, p21 and LC3-Ⅱ were up-regulated. 5-Aza-2’-deoxycytidine can also induce DNA damage and autophagy in breast cancer cells. Compared with the control group, the length of the comet of the treated group increased (P <0.01) and the expression of p53, p21 and LC3-Ⅱ The percentage of autophagic bubble-positive cells and the percentage of green fluorescent protein positive cells increased, the difference was statistically significant (P <0.05 and P <0.01). CONCLUSION: 5-Aza-2’-deoxycytidine can induce autophagy in breast cancer cells, and its mechanism may be related to the DNA damage induced by it.