本文選題：表沒(méi)食子兒茶素沒(méi)食子酸酯 + 放射敏感性　； 參考：《江蘇大學(xué)》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(diǎn)。研究顯示表沒(méi)食子兒茶素沒(méi)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(duì)人食管鱗癌TE-1細(xì)胞放射敏感性的影響,進(jìn)而探究凋亡和自噬在其中發(fā)揮的作用,尋找促進(jìn)食管鱗癌放射敏感性的方法�！痉椒ā�(1)四甲基偶氮唑藍(lán)(MTT)法檢測(cè)不同濃度(20、40、60、80μg/ml)的EGCG分別作用24、48、72小時(shí)對(duì)食管鱗癌TE-1細(xì)胞的生長(zhǎng)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(lián)合應(yīng)用對(duì)TE-1細(xì)胞的抑制效應(yīng),確定最適工作劑量。自噬抑制劑3-MA預(yù)處理食管鱗癌細(xì)胞TE-1后,檢測(cè)EGCG和X射線聯(lián)合處理對(duì)細(xì)胞增殖能力的影響。(2)將實(shí)驗(yàn)分為空白對(duì)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(shí)驗(yàn)記錄四組含大于50個(gè)細(xì)胞的克隆個(gè)數(shù),流式細(xì)胞法檢測(cè)四組細(xì)胞的凋亡率。X射線單獨(dú)作用或與聯(lián)合EGCG作用于TE-1細(xì)胞,通過(guò)克隆形成實(shí)驗(yàn)檢測(cè)單純放射組和放射聯(lián)合EGCG組兩組TE-1細(xì)胞放射敏感性的變化。(3)免疫印跡法(Western blot法)檢測(cè)不同處理組TE-1細(xì)胞中Cleaved caspase-3、Bcl-2、LC3-II、Belin1蛋白的相對(duì)表達(dá)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(xì)胞TE-1后,采用MTT法和Western blot法檢測(cè)EGCG和X射線聯(lián)合處理后TE-1細(xì)胞的增殖能力及LC3-II表達(dá)水平的變化�！窘Y(jié)果】(1)EGCG抑制食管鱗癌TE-1細(xì)胞的增殖,呈時(shí)間和劑量依賴效應(yīng),選擇80μg/ml作用48小時(shí)作為最適工作濃度。EGCG可以使X射線生長(zhǎng)抑制作用加強(qiáng),選用4Gy X射線作為聯(lián)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(yù)處理食管鱗癌細(xì)胞TE-1后減弱了EGCG對(duì)TE-1細(xì)胞的生長(zhǎng)抑制。(2)與空白對(duì)照組、EGCG組、X射線組相比,EGCG+X射線組造成TE-1細(xì)胞克隆個(gè)數(shù)顯著減少,細(xì)胞凋亡率顯著降低。細(xì)胞存活曲線顯示與單純放射組相比,放射聯(lián)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(xì)胞48小時(shí)后,LC3-II、Beclin1表達(dá)量與劑量呈正相關(guān)。EGCG+X射線組,與空白對(duì)照組、EGCG組、X射線組相比,Bcl-2相對(duì)表達(dá)量顯著降低,Cleaved caspase-3、LC3-II、Beclin1表達(dá)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(xì)胞TE-1后,EGCG聯(lián)合X射線對(duì)其LC3-II的上調(diào)作用明顯減弱,對(duì)TE-1細(xì)胞生長(zhǎng)的抑制作用也明顯減弱�！窘Y(jié)論】(1)EGCG與X射線均對(duì)人食管鱗癌TE-1細(xì)胞有細(xì)胞毒性作用。(2)EGCG能增加人食管鱗癌TE-1細(xì)胞的放療敏感性。(3)EGCG增加人食管鱗癌TE-1細(xì)胞的放療敏感性的機(jī)制可能是下調(diào)Bcl-2蛋白和上調(diào)Beclin1蛋白的表達(dá),誘導(dǎo)食管鱗癌TE-1細(xì)胞發(fā)生凋亡和自噬,最終導(dǎo)致細(xì)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(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.1

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