槲皮素对缺氧诱导的人肝癌细胞上皮间质转化影响的初步研究

发布时间：2018-01-17 01:12

本文关键词：槲皮素对缺氧诱导的人肝癌细胞上皮间质转化影响的初步研究　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:上皮间质转化(epithelial-mesenchymaltransition,EMT)是指在特定的生理和病理状态下上皮细胞向间质细胞转化的现象。肿瘤细胞发生侵袭和迁移的主要机制之一即肿瘤细胞发生了上皮间质转化。诱导细胞发生上皮间质转化的因素有很多,缺氧也被认为是其重要的诱导因素之一。槲皮素(Quercetin,Que)是广泛存在于植物中的天然黄酮类化合物,具有抗炎、抗氧化和抗肿瘤等作用,已被证实可抑制多种肿瘤细胞的上皮间质转化进而抑制肿瘤细胞的侵袭和迁移。但大量的实验研究仅证明了槲皮素在常氧条件下对肿瘤细胞上皮间质转化的抑制效应,目前尚无研究报道槲皮素在缺氧条件下对肿瘤细胞上皮间质转化的影响。本研究通过体外细胞实验探讨槲皮素对缺氧诱导的人肝癌Hep G2细胞上皮间质转化的影响及其可能的机制。方法:体外培养人肝癌Hep G2细胞,以100umol/L的氯化钴(Cobaltous chloride,Co Cl2)作用于细胞建立缺氧模型,将对数生长期的Hep G2细胞分为四组:对照组、Co Cl2组、Que组、Co Cl2+Que组,采用噻唑蓝比色法(MTT)检测不同浓度的槲皮素在常氧和缺氧条件下作用48h、72h后肝癌细胞的活力变化,并计算出相应条件下槲皮素对肝癌细胞的半数抑制浓度;采用细胞划痕实验检测槲皮素作用后四组肝癌细胞迁移的距离并比较其差异;采用q PCR和Western blotting法分别检测槲皮素作用后四组Hep G2细胞中缺氧诱导因子1α(hypoxia inducible factor-1alpha,HIF-1α)和上皮间质转化相关标志因子Snail、E-cadherin和vimentin的m RNA及蛋白的表达水平。结果:MTT结果显示槲皮素可显著抑制Hep G2细胞的增殖,且在一定的浓度范围内其抑制作用存在剂量依赖性。槲皮素在常氧、缺氧条件下作用48h和72h的IC50值分别为69.25±3.02umol/L、56.78±1.99umol/L和48.76±2.63umol/L、41.07±2.51umol/L。细胞划痕实验结果显示槲皮素能明显减缓常氧和缺氧条件下Hep G2细胞的迁移能力。q PCR实验结果显示槲皮素可使Snail、vimentin的m RNA表达降低和E-cadherin的m RNA表达增加,而对HIF-1α的m RNA表达无影响。Western blotting实验结果显示槲皮素可下调HIF-1α、Snail和vimentin的蛋白表达和上调E-cadherin的蛋白表达。结论:槲皮素能显著抑制缺氧诱导的人肝癌Hep G2细胞的上皮间质转化,其机制可能与槲皮素抑制缺氧诱导的人肝癌细胞中HIF-1α蛋白的累积有关。
[Abstract]:Objective: to study the epithelial-mesenchymal transition. EMT). The transformation of epithelial cells into interstitial cells in certain physiological and pathological conditions. One of the main mechanisms of invasion and migration of tumor cells, I. E. epithelial interstitial transformation of tumor cells, induces the occurrence of epithelial cells. There are many factors involved in mesenchymal transformation. Quercetin Quercetinine (Quercetin) is a natural flavonoid widely found in plants and has anti-inflammatory properties. Antioxidation and anti-tumor effects. It has been proved that quercetin can inhibit the epithelial interstitial transformation of many tumor cells and thus inhibit the invasion and migration of tumor cells. However, a large number of experimental studies have only proved that quercetin inhibits the epithelial interstitial transformation of tumor cells under normoxic conditions. Effects. There is no study on the effect of quercetin on epithelial mesenchymal transformation of tumor cells under hypoxia. In this study, we studied the effects of quercetin on hypoxia induced Hep of human hepatocellular carcinoma in vitro. Effects of epithelial mesenchymal transformation in G2 cells and its possible mechanisms methods: human hepatoma Hep G2 cells were cultured in vitro. The model of hypoxia was established by the action of Cobaltous chloride Co Cl2 (100 umol / L) on the cells. The Hep G2 cells in logarithmic growth phase were divided into four groups: control group, Co Cl2 group, que group, Co Cl2 Que group. Thiazolyl blue colorimetric assay (MTT) was used to detect the changes of the viability of hepatoma cells treated with quercetin at different concentrations for 48 h or 72 h under normoxic and hypoxic conditions. The inhibition concentration of quercetin on hepatoma cells was calculated. Cell scratch assay was used to detect the migration distance of liver cancer cells in the four groups after quercetin treatment and the difference was compared. Q PCR and Western blotting methods were used to detect hypoxia inducible factor-1 伪 (HIF-1 伪) in four groups of Hep G2 cells after quercetin treatment. Hypoxia inducible factor-1alpha. HIF-1 伪 and Snail. Results the results showed that quercetin could significantly inhibit the proliferation of Hep G2 cells. The inhibitory effect of quercetin was in a dose-dependent manner in a dose-dependent manner. The IC50 values of quercetin exposed to normoxic oxygen, hypoxia for 48h and 72h were 69.25 卤3.02 umol/ L, respectively. 56.78 卤1.99umoll / L and 48.76 卤2.63umol/ L respectively. 41.07 卤2.51umol / L. Cell scratch test showed that quercetin could significantly decrease the migration ability of Hep G2 cells under normoxic and hypoxic conditions. Q. PCR results showed that quercetin could make Snail. The expression of m RNA in vimentin was decreased and the expression of m RNA in E-cadherin was increased. The results of Western blotting assay showed that quercetin could down-regulate HIF-1 伪. The protein expression of Snail and vimentin and up-regulation of E-cadherin protein were up-regulated. Conclusion: quercetin can significantly inhibit hypoxia induced Hep of human hepatocellular carcinoma. Epithelium mesenchymal transformation of G2 cells. The mechanism may be related to the inhibitory effect of quercetin on the accumulation of HIF-1 伪 protein in human hepatoma cells induced by hypoxia.
【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】：R735.7

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