EGCG对人食管鳞癌TE-1细胞放疗增敏的作用及其机制研究

发布时间：2018-05-14 19:48

本文选题：表没食子儿茶素没食子酸酯 + 放射敏感性　；参考：《江苏大学》2016年硕士论文

【摘要】：【目的】放疗作为食管癌患者最有效的治疗手段之一,如何提高放疗的效果成为如今的研究热点。研究显示表没食子儿茶素没食子酸酯(epigallocathechin-3-gallate,EGCG)可以增加鼻咽癌的放疗敏感性,但EGCG是否可以增加食管癌的放射敏感性尚不清楚。本课题旨在研究EGCG对人食管鳞癌TE-1细胞放射敏感性的影响,进而探究凋亡和自噬在其中发挥的作用,寻找促进食管鳞癌放射敏感性的方法。【方法】(1)四甲基偶氮唑蓝(MTT)法检测不同浓度(20、40、60、80μg/ml)的EGCG分别作用24、48、72小时对食管鳞癌TE-1细胞的生长抑制作用,确定EGCG最适工作浓度。80μg/ml EGCG与不同剂量的X射线(2、4、8Gy)联合应用对TE-1细胞的抑制效应,确定最适工作剂量。自噬抑制剂3-MA预处理食管鳞癌细胞TE-1后,检测EGCG和X射线联合处理对细胞增殖能力的影响。(2)将实验分为空白对照组、EGCG组(培养液中EGCG终浓度为80μg/ml)、X射线组(放射剂量为4Gy)、EGCG+X射线组(培养液中EGCG终浓度80μg/ml+放射剂量4Gy)四组,克隆形成实验记录四组含大于50个细胞的克隆个数,流式细胞法检测四组细胞的凋亡率。X射线单独作用或与联合EGCG作用于TE-1细胞,通过克隆形成实验检测单纯放射组和放射联合EGCG组两组TE-1细胞放射敏感性的变化。(3)免疫印迹法(Western blot法)检测不同处理组TE-1细胞中Cleaved caspase-3、Bcl-2、LC3-II、Belin1蛋白的相对表达情况,荧光倒置显微镜观察自噬小体荧光量的变化。(4)自噬基因Beclin1小干扰RNA(siRNA-Beclin1)处理食管鳞癌细胞TE-1后,采用MTT法和Western blot法检测EGCG和X射线联合处理后TE-1细胞的增殖能力及LC3-II表达水平的变化。【结果】(1)EGCG抑制食管鳞癌TE-1细胞的增殖,呈时间和剂量依赖效应,选择80μg/ml作用48小时作为最适工作浓度。EGCG可以使X射线生长抑制作用加强,选用4Gy X射线作为联合放射组和单纯放射组的最适工作剂量。自噬抑制剂3-MA预处理食管鳞癌细胞TE-1后减弱了EGCG对TE-1细胞的生长抑制。(2)与空白对照组、EGCG组、X射线组相比,EGCG+X射线组造成TE-1细胞克隆个数显著减少,细胞凋亡率显著降低。细胞存活曲线显示与单纯放射组相比,放射联合EGCG组,D0、Dq值均变小,放射增敏比为1.19。(3)不同浓度EGCG处理TE-1细胞48小时后,LC3-II、Beclin1表达量与剂量呈正相关。EGCG+X射线组,与空白对照组、EGCG组、X射线组相比,Bcl-2相对表达量显著降低,Cleaved caspase-3、LC3-II、Beclin1表达量明显升高且自噬体荧光量明显增多。(4)用siRNA-Beclin1处理食管鳞癌细胞TE-1后,EGCG联合X射线对其LC3-II的上调作用明显减弱,对TE-1细胞生长的抑制作用也明显减弱。【结论】(1)EGCG与X射线均对人食管鳞癌TE-1细胞有细胞毒性作用。(2)EGCG能增加人食管鳞癌TE-1细胞的放疗敏感性。(3)EGCG增加人食管鳞癌TE-1细胞的放疗敏感性的机制可能是下调Bcl-2蛋白和上调Beclin1蛋白的表达,诱导食管鳞癌TE-1细胞发生凋亡和自噬,最终导致细胞死亡。
[Abstract]:Objective: as one of the most effective treatment methods for esophageal cancer patients, how to improve the effect of radiotherapy has become a hot topic. It has been shown that epigallocathechin-3-gallate EGCG can increase the radiosensitivity of nasopharyngeal carcinoma, but it is not clear whether EGCG can increase the radiosensitivity of esophageal carcinoma. The aim of this study was to investigate the effects of EGCG on radiosensitivity of human esophageal squamous cell carcinoma (TE-1) cells, and to explore the role of apoptosis and autophagy. To find a method to promote the radiosensitivity of esophageal squamous cell carcinoma. [methods] the growth inhibition of esophageal squamous cell carcinoma (TE-1) cells was detected by tetramethylazolium methylene tetrozolium (MTT-1) method. The growth inhibition of TE-1 cells was detected by EGCG at different concentrations of 2040 ~ 6080 渭 g / ml for 24 ~ 4872 hours, respectively. To determine the inhibitory effect of the optimal working concentration of EGCG (.80 渭 g/ml EGCG) combined with different doses of X-rays (4Gy) on TE-1 cells, and to determine the optimal working dose. Autophagy inhibitor 3-MA pretreated esophageal squamous cell TE-1. To detect the effect of combined treatment of EGCG and X-ray on cell proliferation, the experiment was divided into four groups: blank control group (the final concentration of EGCG in culture medium was 80 渭 g 路ml ~ (-1) X ray group (radiation dose was 4 渭 g 路ml ~ (-1) and the final concentration of EGCG in culture medium was 80 渭 g/ml radiation dose (4 Gy). Clone formation assay recorded the number of clones containing more than 50 cells in four groups. Flow cytometry was used to detect the apoptosis rate of four groups of cells. X ray alone or in combination with EGCG was used to detect the apoptosis rate of TE-1 cells. The changes of radiosensitivity of TE-1 cells were detected by clone formation assay. The relative expression of Cleaved caspase-3 Bcl-2LC3-IIP Belin1 protein in TE-1 cells of different treatment groups was detected by Western blot. Fluorescence inversion microscope was used to observe the fluorescence changes of autophagy bodies. (4) Beclin1 siRNA-Beclin1), a autophagy gene, was used to treat TE-1 of esophageal squamous carcinoma cells. The proliferative ability and LC3-II expression of TE-1 cells treated with EGCG and X ray were detected by MTT and Western blot methods. [results] the proliferation of TE-1 cells of esophageal squamous cell carcinoma was inhibited in a time and dose-dependent manner. The optimal working concentration of 80 渭 g/ml was 48 hours. EGCG could enhance the growth inhibition of X-ray, and 4Gy was used as the optimal working dose of combined radiation group and simple radiation group. The growth inhibition of EGCG on TE-1 cells was attenuated after TE-1 pretreated with autophagy inhibitor 3-MA.) compared with the control group, the number of TE-1 cell clones was significantly decreased and the apoptosis rate was significantly decreased in the TE-1 X ray group compared with the control group. The cell survival curve showed that the D0 DQ value of TE-1 cells in the radiation combined with EGCG group was smaller than that in the control group, and the radiosensitization ratio was 1.19 ~ (3) after 48 hours of EGCG treatment with different concentrations of EGCG, there was a positive correlation between the expression of LC3-IIP Beclin1 and the dose of TE-1 cells. Compared with the blank control group, the relative expression of Bcl-2 was significantly decreased. The expression of Cleaved caspase-3LC3-IIP Beclin1 was significantly increased, and the fluorescence of autophagy was significantly increased. 4) the up-regulation of LC3-II in esophageal squamous carcinoma cells treated with siRNA-Beclin1 was obviously weakened after treated with siRNA-Beclin1 combined with X-ray. [conclusion] both TE-1 and X-ray have cytotoxic effect on human esophageal squamous cell carcinoma (TE-1) cells. [conclusion] the chemotoxicity of TE-1 can increase the radiosensitivity of human esophageal squamous cell carcinoma (TE-1) cells to radiotherapy. [conclusion] it can increase TE-1 of human esophageal squamous cell carcinoma. The mechanism of cell radiosensitivity may be down-regulation of Bcl-2 protein and up-regulation of Beclin1 protein expression. Apoptosis and autophagy of esophageal squamous cell carcinoma (TE-1) cells were induced and eventually resulted in cell death.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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