氯化两面针碱对肺癌A549细胞凋亡及细胞周期影响的研究

发布时间：2018-02-27 04:33

本文关键词： 氯化两面针碱肺癌细胞凋亡细胞周期 Wnt通路　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的:由于环境及生活方式的改变,肺癌的发生率逐年升高,目前已成为全世界急需攻克的难题。大部分患者在就诊时已处于进展期,使肺癌的五年生存率处于低水平。因此,我们急切寻找更多治疗肺癌的有效药物。近几年天然产品氯化两面针碱被认为具有抗肿瘤作用。已有多项实验证明氯化两面针碱对乳腺癌、肝癌、结肠癌、前列腺癌等细胞具有抑制增殖作用,但尚未见对肺癌细胞作用的研究。本实验以肺癌A549细胞为研究对象,观察氯化两面针碱对其抑制作用并讨论相关机制。1.观察氯化两面针碱对人肺癌细胞生长和增殖的影响;2.观察氯化两面针碱对人肺癌细胞凋亡及细胞周期的影响,并探讨其机制。方法:1.不同浓度的氯化两面针碱作用于A549细胞,体外培养48h后,应用MTT法检测A549细胞的生长和增殖情况;2.不同浓度的氯化两面针碱作用于A549细胞后,应用Hoechst33258染色法观察细胞凋亡的形态学变化;应用Annexin V-FITC/PI双染流式细胞术检测各组细胞凋亡率,PI单染法检测各组细胞周期比例;应用Western blot方法检测各组细胞凋亡通路、Wnt通路及细胞周期相关分子的表达情况。结果:1.MTT结果显示,氯化两面针碱显著抑制A549细胞的增殖,其抑制增殖作用呈时间和剂量依赖性。单因素方差分析,各时间段内实验组与对照组比较,差异有统计学意义(**P0.05,***P0.01);2.应用Hoechst33258染色法,可见经氯化两面针碱处理过的A549细胞核或细胞质内可见浓染致密的颗粒状、块状荧光,形成大小不等、内含细胞器和细胞核的凋亡小体;3.Annexin V-FITC/PI双染流式细胞术检测结果显示,不同浓度的氯化两面针碱对A549细胞具有诱导凋亡作用,与对照组相比,差异具有统计学意义(*P0.05,**P0.01);4.PI单染法结果显示,经氯化两面针碱处理后的A549细胞,G2/M期细胞显著增多,呈剂量依赖性,提示氯化两面针碱具有阻滞细胞周期的作用;5.Western blot检测结果显示,随着氯化两面针碱浓度的升高,A549细胞的凋亡通路相关分子Bcl-xl、Bid、PARP、p-β-catenin的表达水平下调;Cyto-C、β-catenin的表达水平上调;Caspase-9、Caspase-3、Caspase-8活性增强。提示氯化两面针碱能诱导A549细胞凋亡,呈剂量依赖性。细胞周期相关蛋白Cyclin D1表达水平逐渐下调。提示氯化两面针碱可阻滞细胞周期,呈剂量依赖性。结论:1.氯化两面针碱可抑制肺癌A549细胞的生长和增殖;2.氯化两面针碱可通过线粒体通路和死亡受体通路诱导肺癌A549细胞的凋亡,可能有Wnt/β-catenin信号转导通路的参与,并受Bcl-2蛋白家族的调节;3.氯化两面针碱可阻滞肺癌A549细胞于G2/M期。
[Abstract]:Background and objective: the incidence of lung cancer has increased year by year due to changes in environment and lifestyle, and has become an urgent problem in the world. The five-year survival rate for lung cancer is low. We are eager to find more effective drugs for the treatment of lung cancer. In recent years, the natural product, dianethine chloride, has been considered to have an anti-tumor effect. There have been many experiments to prove that chlorinated nitidine is effective in breast cancer, liver cancer, colon cancer, and so on. Prostate cancer and other cells have inhibitory effect on proliferation, but no study has been done on lung cancer cell line A549. To observe the inhibitory effect of nitidine chloride on the growth and proliferation of human lung cancer cells and to discuss the related mechanism. 2. To observe the effect of nitidine chloride on apoptosis and cell cycle of human lung cancer cells. Methods 1. A549 cells were treated with different concentrations of nitidine chloride. After cultured for 48 hours, the growth and proliferation of A549 cells were detected by MTT assay. The morphological changes of apoptosis were observed by Hoechst33258 staining and the cell cycle ratio was detected by Annexin V-FITC / Pi double staining flow cytometry and Pi single staining. Western blot method was used to detect the expression of Wnt pathway and cell cycle related molecules in A549 cells. Results: 1. The results showed that the proliferation of A549 cells was significantly inhibited by A549. Univariate analysis of variance (ANOVA) showed that the difference between the experimental group and the control group was statistically significant. The Hoechst33258 staining method was used. It can be seen that the nucleus or cytoplasm of A549 treated with nitidine chloride can be seen in dense granular, massive fluorescence, and different size. 3. Annexin V-FITC / Pi double staining flow cytometry results showed that the A549 cells contained organelles and nuclear apoptotic bodies. The apoptosis of A549 cells was induced by different concentrations of nitidine chloride. Compared with the control group, the difference was statistically significant. 4. The results of Pi single staining showed that the number of G 2 / M phase cells in A549 cells treated with dianethine chloride was significantly higher than that in the control group. In a dose-dependent manner, it was suggested that nitidine chloride could block the cell cycle. 5. Western blot analysis showed that, With the increase of the concentration of nitidine chloride, the expression level of Bcl-xlbup- 尾 -catenin was down-regulated in A549 cells. The expression level of 尾 -catenin up-regulated the activity of Caspase-9, Caspase-3 and Caspase-8 in A549 cells, suggesting that nitidine chloride could induce apoptosis of A549 cells. The expression of cell cycle associated protein Cyclin D1 was down-regulated in a dose-dependent manner. Conclusion: 1. Dianethine chloride can inhibit the growth and proliferation of lung cancer A549 cells. The apoptosis of lung cancer A549 cells can be induced by nitidine chloride through mitochondrial pathway and death receptor pathway, which may be involved in Wnt/ 尾 -catenin signal transduction pathway. It was also regulated by Bcl-2 protein family. Nitidine chloride could block A549 cells in G _ 2 / M phase.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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