黄柏苷及绿原酸诱导A549细胞凋亡机制的研究

发布时间：2018-06-07 06:45

本文选题：黄柏苷 + 绿原酸　；参考：《吉林大学》2017年硕士论文

【摘要】：癌症一直是人类健康的杀手,近年来,肺癌发病率仅次于乳腺癌和前列腺癌,已成为导致癌症死亡的主要原因。黄柏苷和绿原酸是黄檗树叶中的有效成分,作为天然产物,二者毒性较低且副作用少。本研究旨在探究黄柏苷和绿原酸的抗肿瘤活性及其分子机制,为癌症治疗寻找新型方案。利用不同浓度的黄柏苷和绿原酸分别处理A549细胞48小时,通过MTT法检测两种药物对细胞生长的抑制率,并利用流式细胞术分析两种药物处理后A549细胞周期分布及细胞凋亡情况。同时,通过比色法检测并比较了用药前后A549细胞内SOD活力、GSH含量、MDA含量以及caspase 3活性的变化;利用JC-1染色法,通过荧光显微镜观察了不同时间内黄柏苷和绿原酸对A549细胞线粒体膜电位的影响。此外,还通过免疫印迹分析了A549细胞内凋亡相关蛋白Bax及Bcl-2的表达水平。通过MTT实验结果发现,药物浓度为2000μM时,黄柏苷对A549细胞的生长抑制率为70.87%,绿原酸对细胞的抑制率为29.01%。流式细胞术检测结果表明,黄柏苷可以诱导细胞周期停滞在G2/M期,并引起84.32%的细胞发生凋亡,而绿原酸将A549细胞周期阻滞在了S期,并造成30.10%的细胞发生凋亡。荧光显微镜观察结果显示,在24小时内,黄柏苷和绿原酸均能够降低A549细胞线粒体膜电位。此外,黄柏苷可以导致A549细胞内SOD活力下降50%,GSH含量降低30%,MDA含量增加60%,但绿原酸对此并无显著影响。而且,黄柏苷和绿原酸分别将A549细胞内caspase 3活性提高了31%和25%。免疫印迹结果表明,黄柏苷将细胞内Bax/Bcl-2水平上调为对照组的33倍,绿原酸则将Bax/Bcl-2水平上调为对照组的1.8倍。本研究发现,黄柏苷和绿原酸均可以通过阻滞细胞周期,损伤细胞遗传物质,上调Bax因子的表达,同时下调Bcl-2因子的表达,并且激活caspase 3进而诱导A549细胞凋亡。
[Abstract]:Cancer has always been the killer of human health. In recent years, the incidence of lung cancer is second only to breast cancer and prostate cancer, which has become the main cause of cancer death. Yellow cyperidin and Lv Yuan acid are effective components in the leaves of Berberis berberis. As a natural product, they are less toxic and have less side effects. The aim of this study was to explore the anti-tumor activity and molecular mechanism of berberine and Lv Yuan acid in order to find a new regimen for cancer treatment. A549 cells were treated with different concentrations of yellow cyperidin and Lv Yuan acid for 48 hours. The inhibition rate of the two drugs on A549 cell growth was detected by MTT assay. The cell cycle distribution and apoptosis of A549 cells were analyzed by flow cytometry. At the same time, the changes of SOD content and caspase 3 activity in A549 cells before and after treatment were detected and compared by colorimetry, and JC-1 staining was used to detect and compare the changes of GSH content and caspase 3 activity in A549 cells. The effects of flavin and Lv Yuan acid on mitochondrial membrane potential of A549 cells were observed by fluorescence microscope. In addition, the expression of apoptosis-related protein Bax and Bcl-2 in A549 cells was analyzed by Western blot. The results of MTT assay showed that when the concentration was 2000 渭 M, the growth inhibition rate was 70.87 and the inhibition rate of Lv Yuan acid on A549 cells was 29.01 渭 M. The results of flow cytometry showed that flavin could induce cell cycle arrest in G _ 2 / M phase and induce apoptosis in 84.32% of the cells, while Lv Yuan acid blocked A549 cell cycle in S phase and caused 30.10% of cell apoptosis. Fluorescence microscopy showed that both flavin and Lv Yuan acid could decrease mitochondrial membrane potential in A549 cells within 24 hours. In addition, the SOD activity of A549 cells decreased by 50%, and the content of Lv Yuan increased by 60%, but Lv Yuan acid had no significant effect on it. Moreover, the activity of caspase 3 in A549 cells was increased by 31% and 25% respectively by flavin and Lv Yuan acid. The results of Western blotting showed that the intracellular Bax/Bcl-2 level was 33 times higher than that of the control group, and the Bax/Bcl-2 level of Lv Yuan acid was 1.8-fold higher than that of the control group. In this study, it was found that both flavin and Lv Yuan acid could inhibit cell cycle, damage genetic material, up-regulate the expression of Bax factor, down-regulate the expression of Bcl-2 factor, and activate caspase 3 to induce apoptosis of A549 cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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