本文選題：表沒食子兒茶素沒食子酸酯 + 放射敏感性　； 參考：《江蘇大學》2016年碩士論文

【摘要】：【目的】放療作為食管癌患者最有效的治療手段之一,如何提高放療的效果成為如今的研究熱點。研究顯示表沒食子兒茶素沒食子酸酯(epigallocathechin-3-gallate,EGCG)可以增加鼻咽癌的放療敏感性,但EGCG是否可以增加食管癌的放射敏感性尚不清楚。本課題旨在研究EGCG對人食管鱗癌TE-1細胞放射敏感性的影響,進而探究凋亡和自噬在其中發(fā)揮的作用,尋找促進食管鱗癌放射敏感性的方法�！痉椒ā�(1)四甲基偶氮唑藍(MTT)法檢測不同濃度(20、40、60、80μg/ml)的EGCG分別作用24、48、72小時對食管鱗癌TE-1細胞的生長抑制作用,確定EGCG最適工作濃度。80μg/ml EGCG與不同劑量的X射線(2、4、8Gy)聯合應用對TE-1細胞的抑制效應,確定最適工作劑量。自噬抑制劑3-MA預處理食管鱗癌細胞TE-1后,檢測EGCG和X射線聯合處理對細胞增殖能力的影響。(2)將實驗分為空白對照組、EGCG組(培養(yǎng)液中EGCG終濃度為80μg/ml)、X射線組(放射劑量為4Gy)、EGCG+X射線組(培養(yǎng)液中EGCG終濃度80μg/ml+放射劑量4Gy)四組,克隆形成實驗記錄四組含大于50個細胞的克隆個數,流式細胞法檢測四組細胞的凋亡率。X射線單獨作用或與聯合EGCG作用于TE-1細胞,通過克隆形成實驗檢測單純放射組和放射聯合EGCG組兩組TE-1細胞放射敏感性的變化。(3)免疫印跡法(Western blot法)檢測不同處理組TE-1細胞中Cleaved caspase-3、Bcl-2、LC3-II、Belin1蛋白的相對表達情況,熒光倒置顯微鏡觀察自噬小體熒光量的變化。(4)自噬基因Beclin1小干擾RNA(siRNA-Beclin1)處理食管鱗癌細胞TE-1后,采用MTT法和Western blot法檢測EGCG和X射線聯合處理后TE-1細胞的增殖能力及LC3-II表達水平的變化�！窘Y果】(1)EGCG抑制食管鱗癌TE-1細胞的增殖,呈時間和劑量依賴效應,選擇80μg/ml作用48小時作為最適工作濃度。EGCG可以使X射線生長抑制作用加強,選用4Gy X射線作為聯合放射組和單純放射組的最適工作劑量。自噬抑制劑3-MA預處理食管鱗癌細胞TE-1后減弱了EGCG對TE-1細胞的生長抑制。(2)與空白對照組、EGCG組、X射線組相比,EGCG+X射線組造成TE-1細胞克隆個數顯著減少,細胞凋亡率顯著降低。細胞存活曲線顯示與單純放射組相比,放射聯合EGCG組,D0、Dq值均變小,放射增敏比為1.19。(3)不同濃度EGCG處理TE-1細胞48小時后,LC3-II、Beclin1表達量與劑量呈正相關。EGCG+X射線組,與空白對照組、EGCG組、X射線組相比,Bcl-2相對表達量顯著降低,Cleaved caspase-3、LC3-II、Beclin1表達量明顯升高且自噬體熒光量明顯增多。(4)用siRNA-Beclin1處理食管鱗癌細胞TE-1后,EGCG聯合X射線對其LC3-II的上調作用明顯減弱,對TE-1細胞生長的抑制作用也明顯減弱�！窘Y論】(1)EGCG與X射線均對人食管鱗癌TE-1細胞有細胞毒性作用。(2)EGCG能增加人食管鱗癌TE-1細胞的放療敏感性。(3)EGCG增加人食管鱗癌TE-1細胞的放療敏感性的機制可能是下調Bcl-2蛋白和上調Beclin1蛋白的表達,誘導食管鱗癌TE-1細胞發(fā)生凋亡和自噬,最終導致細胞死亡。
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.1

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