苦龙胆酯苷诱导肝癌细胞凋亡的机制研究

发布时间：2018-05-11 08:43

  本文选题：獐芽菜 + 苦龙胆酯苷　； 参考：《重庆医科大学》2016年博士论文

【摘要】：目的:为探明苦龙胆酯苷(amarogentin)是否对肝癌细胞有抑制作用,以及抑制作用的机制如何?本课题研究了苦龙胆酯苷对HepG2和SMMC~7721肝癌细胞系增殖、凋亡和迁移的影响;苦龙胆酯苷对裸鼠p53相关细胞凋亡信号通路的影响,探索了苦龙胆酯苷诱导肝癌细胞凋亡的分子机制,为研究苦龙胆酯苷的药理作用奠定了基础。方法:采用乙醇法正丁醇部位萃取苦龙胆酯苷成分。分别用梯度浓度的苦龙胆酯苷作用于人正常肝细胞(LO2)和人肝癌细胞(HepG2和SMMC~7721)后,利用细胞计数试剂盒检测三种细胞在12、24和48小时的增殖情况;利用异硫氰酸荧光素标记的膜联蛋白/碘化丙啶,结合流式细胞仪检测苦龙胆酯苷成分在作用这三种细胞48小时后的凋亡率;利用Transwell法检测HepG2和SMMC~7721人肝癌细胞的迁移情况。在细胞水平和裸鼠肝癌模型中同时观察苦龙胆酯苷对p53相关细胞凋亡信号通路的影响,利用实时荧光定量聚合酶链反应(realtime PCR)和蛋白质印记法(western blot)分别检测人肝癌细胞p53、AKt、NF~κB、hTERT和p38的mRNA和蛋白表达水平。结果:我们用改良过的提取方法,从獐芽菜提取物中获得苦龙胆酯苷粉末,其苦龙胆酯苷的获得率为18.33±1.20%,用高压液相仪检测最终纯化的苦龙胆酯苷,其纯度大于99.21%。通过体外细胞实验发现苦龙胆酯苷能够有效地抑制HepG2和SMMC~7721的增殖,并存在时间和剂量依赖性。当苦龙胆酯苷浓度达到110μg/mL,作用于肝癌细胞48小时后,不仅能够抑制人肝癌细胞的迁移,而且对人肝癌细胞的凋亡有促进作用。其中,p53的mRNA和蛋白表达明显增高,同时Akt、NF~κB和hTERT的mRNA和蛋白表达明显被抑制,和裸鼠肝癌模型组织中观察到的结果一致,而p38的mRNA和蛋白水平均无明显变化(P0.05),差异具有统计学意义。说明苦龙胆酯苷通过p53凋亡相关信号通路促进肝癌细胞的凋亡,且与p38途径无关。结论:苦龙胆酯苷上调了肝癌细胞p53的mRNA和蛋白质表达,下调了hTERT、Akt、NF~κB亚基RelA(P65)的mRNA和蛋白质表达水平,促进了肝癌细胞的凋亡,对肝癌模型的治疗有一定的效果,为临床辅助治疗肝癌提供了新的思路。
[Abstract]:Objective: to investigate whether amarogentin has inhibitory effect on hepatoma cells and the mechanism of inhibition. In this study, we studied the effects of gentian on the proliferation, apoptosis and migration of HepG2 and SMMC~7721 hepatoma cell lines, and the effects of gentian on the apoptotic signaling pathway of p53 related cells in nude mice, and explored the molecular mechanism of the apoptosis of hepatoma cells induced by gentian in nude mice. It lays a foundation for the study of the pharmacological action of gentian glucoside. Methods: the components of gentian glucoside were extracted from n-butanol by ethanol method. The proliferation of three kinds of cells at 1224 and 48 hours was detected by cell counting kit after they were treated with gradient gentian glucoside on human normal hepatocytes (LO2) and human hepatoma cells (HepG2 and SMMC-7721) respectively. Using fluorescein isothiocyanate labeled binding protein / propanidime iodide, flow cytometry was used to detect the apoptosis rate of the three kinds of cells treated with gentian glucoside for 48 hours, and the migration of HepG2 and SMMC~7721 human hepatoma cells was detected by Transwell assay. The effects of gentian glucoside on the apoptotic signaling pathway of p53 related cells were observed at the cell level and in nude mice liver cancer model. The expression levels of mRNA and protein in human hepatoma cell line p53 ~ 魏 B ~ 魏 B hTERT and p38 were detected by real-time fluorescent quantitative polymerase chain reaction (PCR) and protein imprinting method (Western blot), respectively. Results: with the improved extraction method, we obtained the gentian glucoside powder from the extract of roe. The yield of gentian was 18.33 卤1.20. The purity of the purified gentian was more than 99.21 by high performance liquid chromatography. In vitro cell experiments showed that gentian glucoside could effectively inhibit the proliferation of HepG2 and SMMC~7721 in a time-and dose-dependent manner. When the concentration of gentian glucoside reached 110 渭 g / mL for 48 hours, it not only inhibited the migration of human hepatoma cells, but also promoted the apoptosis of human hepatoma cells. The expression of mRNA and protein of p53 was significantly increased, and the expression of mRNA and protein of hTERT and NF 魏 B were inhibited obviously, which was consistent with the results observed in nude mice liver cancer model. The level of mRNA and protein of p38 had no significant change (P 0.05), and the difference was statistically significant. The results showed that gentian glucoside promoted apoptosis of hepatoma cells through p53 apoptosis-related signaling pathway, and was not related to p38 pathway. Conclusion: gentian glucoside upregulated the expression of mRNA and protein of p53, down-regulated the expression of mRNA and protein of hTERTttAkB / NF-kappa B subunit Rela P65, promoted the apoptosis of hepatoma cells, and had a certain effect on the treatment of HCC model. It provides a new idea for clinical adjuvant treatment of liver cancer.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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