本文選題：獐芽菜 + 苦龍膽酯苷�。� 參考：《重慶醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:為探明苦龍膽酯苷(amarogentin)是否對(duì)肝癌細(xì)胞有抑制作用,以及抑制作用的機(jī)制如何?本課題研究了苦龍膽酯苷對(duì)HepG2和SMMC~7721肝癌細(xì)胞系增殖、凋亡和遷移的影響;苦龍膽酯苷對(duì)裸鼠p53相關(guān)細(xì)胞凋亡信號(hào)通路的影響,探索了苦龍膽酯苷誘導(dǎo)肝癌細(xì)胞凋亡的分子機(jī)制,為研究苦龍膽酯苷的藥理作用奠定了基礎(chǔ)。方法:采用乙醇法正丁醇部位萃取苦龍膽酯苷成分。分別用梯度濃度的苦龍膽酯苷作用于人正常肝細(xì)胞(LO2)和人肝癌細(xì)胞(HepG2和SMMC~7721)后,利用細(xì)胞計(jì)數(shù)試劑盒檢測(cè)三種細(xì)胞在12、24和48小時(shí)的增殖情況;利用異硫氰酸熒光素標(biāo)記的膜聯(lián)蛋白/碘化丙啶,結(jié)合流式細(xì)胞儀檢測(cè)苦龍膽酯苷成分在作用這三種細(xì)胞48小時(shí)后的凋亡率;利用Transwell法檢測(cè)HepG2和SMMC~7721人肝癌細(xì)胞的遷移情況。在細(xì)胞水平和裸鼠肝癌模型中同時(shí)觀察苦龍膽酯苷對(duì)p53相關(guān)細(xì)胞凋亡信號(hào)通路的影響,利用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(realtime PCR)和蛋白質(zhì)印記法(western blot)分別檢測(cè)人肝癌細(xì)胞p53、AKt、NF~κB、hTERT和p38的mRNA和蛋白表達(dá)水平。結(jié)果:我們用改良過(guò)的提取方法,從獐芽菜提取物中獲得苦龍膽酯苷粉末,其苦龍膽酯苷的獲得率為18.33±1.20%,用高壓液相儀檢測(cè)最終純化的苦龍膽酯苷,其純度大于99.21%。通過(guò)體外細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn)苦龍膽酯苷能夠有效地抑制HepG2和SMMC~7721的增殖,并存在時(shí)間和劑量依賴性。當(dāng)苦龍膽酯苷濃度達(dá)到110μg/mL,作用于肝癌細(xì)胞48小時(shí)后,不僅能夠抑制人肝癌細(xì)胞的遷移,而且對(duì)人肝癌細(xì)胞的凋亡有促進(jìn)作用。其中,p53的mRNA和蛋白表達(dá)明顯增高,同時(shí)Akt、NF~κB和hTERT的mRNA和蛋白表達(dá)明顯被抑制,和裸鼠肝癌模型組織中觀察到的結(jié)果一致,而p38的mRNA和蛋白水平均無(wú)明顯變化(P0.05),差異具有統(tǒng)計(jì)學(xué)意義。說(shuō)明苦龍膽酯苷通過(guò)p53凋亡相關(guān)信號(hào)通路促進(jìn)肝癌細(xì)胞的凋亡,且與p38途徑無(wú)關(guān)。結(jié)論:苦龍膽酯苷上調(diào)了肝癌細(xì)胞p53的mRNA和蛋白質(zhì)表達(dá),下調(diào)了hTERT、Akt、NF~κB亞基RelA(P65)的mRNA和蛋白質(zhì)表達(dá)水平,促進(jìn)了肝癌細(xì)胞的凋亡,對(duì)肝癌模型的治療有一定的效果,為臨床輔助治療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 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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本文編號(hào)：1873259