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目的:研究苦参碱(matrine)对肺癌A549细胞增殖及自噬的影响。方法:应用matrine作用肺癌A549细胞后,MTT法检测细胞增殖活性;吖啶橙染色(AO)法在荧光显微镜下观察细胞自噬状态;蛋白质印迹法检测自噬相关蛋白LC3-Ⅱ的表达情况;GFP-LC3质粒转染实验观察细胞胞质中绿色点状聚集的自噬泡。结果:matrine可以显著抑制肺癌A549细胞的增殖(P<0.01),随着给药时间和剂量的增加,matrine对肺癌细胞的生长抑制率显著上升,并呈一定的时间-剂量依赖关系。matrine作用于肺癌A549细胞48 h后,细胞内酸性滤泡染成亮红色荧光比例增多,其中以0.5 mg/m L和1.0 mg/m L浓度较为明显,蛋白质印迹法检测实验即选择0.5mg/m L和1.0 mg/m L作为实验浓度。与对照组相比,matrine0.5 mg/m L和1.0 mg/m L组LC3-Ⅱ/GAPDH蛋白表达水平均显著升高(P<0.01);与matrine0.5 mg/m L组相比,matrine0.5 mg/m L+3-MA组LC3-Ⅱ/GAPDH蛋白表达水平显著下降(P<0.01);与matrine1.0 mg/m L组相比,matrine1.0 mg/m L+3-MA组LC3-Ⅱ/GAPDH蛋白表达水平也显著下降(P<0.01)。GFP-LC3转染实验选择matrine1.0 mg/m L于肺癌A549细胞中进行验证,结果显示转染GFP-LC3质粒的细胞在1.0 mg/m L处理后,细胞胞质中出现绿色点状聚集的自噬泡,而正常培养组极少细胞有自噬泡形成;同时转染GFP-Control对照质粒的细胞在1.0 mg/m L处理及正常培养条件下均未观察到点状聚集的自噬泡产生。结论:matrine抑制肺癌A549细胞的增殖并诱导细胞发生自噬。
Objective: To study the effects of matrine on the proliferation and autophagy of lung cancer A549 cells. Methods: The proliferation of A549 cells was detected by MTT assay. The autophagy was observed by acridine orange staining (AO) and the expression of autophagy-related protein LC3-Ⅱ was detected by Western blotting. GFP-LC3 plasmid transfection experiments observed in the cytoplasm of green dot-like aggregation of autophagic vacuoles. Results: Matrine could significantly inhibit the proliferation of lung cancer A549 cells (P <0.01). The growth inhibition rate of matrine on lung cancer cells increased significantly with the time and dose of matrine increasing. The results showed that matrine could inhibit the growth of lung cancer A549 cells in a time-and dose-dependent manner. After 48 h of matrine treatment, the percentage of bright red fluorescence increased with the intracellular acid follicular staining. The concentrations of 0.5 mg / mL and 1.0 mg / mL of matrine were significantly higher than those of the control group (0.5 mg / mL and 1.0 mg / mL as experimental concentrations. Compared with the control group, LC3-Ⅱ / GAPDH protein expression in matrine 0.5 mg / m L and 1.0 mg / m L groups were significantly increased (P <0.01); compared with matrine0.5 mg / m L group, The levels of LC3-Ⅱ / GAPDH protein in matrine0.5 mg / m L + 3-MA group were significantly decreased (P <0.01). Compared with matrine1.0 mg / m L group, matrine1.0 mg / The expression of LC3-Ⅱ / GAPDH in MA group also decreased significantly (P <0.01). GFP-LC3 transfection experiments selected matrine1.0 mg / m L in lung cancer A549 cells were verified, the results showed that cells transfected with GFP-LC3 plasmid 1.0mg / m L treatment, the cells appear cytoplasm green dot-like aggregation Of autophagic vacuoles, and few cells in normal culture group had autophagic vacuoles. At the same time, cells with GFP-Control control plasmid were not treated with 1.0 mg / mL of LPS and normal culture conditions, Bubble generation. Conclusion: Matrine inhibits the proliferation of lung cancer A549 cells and induces autophagy.