本文選題：BTG2　切入點(diǎn)：BRCA1　出處：《蘇州大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：BTG2基因是BTG/TOB抗增殖基因家族重要成員之一，作為抗細(xì)胞增殖和促進(jìn)細(xì)胞凋亡的分子可能是腫瘤治療中一重要的靶向治療基因。目前，國(guó)內(nèi)外對(duì)BTG2在腫瘤放射治療中的影響尚無(wú)任何研究報(bào)道。本課題旨在了解電離輻射對(duì)BTG2表達(dá)水平的影響，，以及BTG2基因在腫瘤細(xì)胞放射治療中的作用和可能的相關(guān)調(diào)節(jié)機(jī)制，為BTG2作為一新的腫瘤放射治療靶基因的深入基礎(chǔ)研究和臨床研究提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 方法：采用乳腺癌細(xì)胞MCF-7和MDA-MB-231，前列腺癌細(xì)胞C33A,子宮頸癌細(xì)胞HeLa，肺癌細(xì)胞A549等腫瘤細(xì)胞作為研究對(duì)象。 (1)采用Western blot法檢測(cè)電離輻射對(duì)腫瘤細(xì)胞中BTG2蛋白表達(dá)水平的影響。 (2)采用pcDNA3-BTG2脂質(zhì)體轉(zhuǎn)染的方法獲得BTG2高表達(dá)的乳腺癌細(xì)胞MCF-7/BTG2和MDA-MB-231/BTG2穩(wěn)定轉(zhuǎn)染細(xì)胞株，利用四甲基偶氮唑鹽比色法（MTT）以及細(xì)胞克隆集落形成法了解BTG2表達(dá)水平的提高對(duì)細(xì)胞放射敏感性的影響。 (3)采用免疫共沉淀分析方法檢測(cè)BTG2與BRCA1蛋白的相互結(jié)合和作用。 (4)采用BRCA1基因真核表達(dá)質(zhì)粒以提高BTG2高表達(dá)的乳腺癌細(xì)胞BRCA1的表達(dá)水平了解人為提高BRCA1對(duì)BTG2的腫瘤放射增敏作用的影響。采用siRNA-BRCA1以沉默BTG2高表達(dá)的乳腺癌細(xì)胞中BRCA1的表達(dá)水平了解人為降低BRCA1對(duì)BTG2的腫瘤放射增敏作用的影響。 (5) Western blot檢測(cè)姜黃素處理后腫瘤細(xì)胞中BTG2的表達(dá)水平。 結(jié)果： (1)137Cs γ射線照射導(dǎo)致不同腫瘤細(xì)胞包括乳腺癌細(xì)胞MCF-7和MDA-MB-231，前列腺癌細(xì)胞C33A,子宮頸癌細(xì)胞HeLa，肺癌細(xì)胞A549腫瘤細(xì)胞中的BTG2蛋白和BTG2mRNA的表達(dá)水平明顯提高，而且輻射誘導(dǎo)的BTG2水平增加與所受到的照射劑量成正相關(guān)。 (2)人為地采用pcDNA3-BTG2基因真核表達(dá)質(zhì)粒轉(zhuǎn)染可以明顯提高腫瘤細(xì)胞中BTG2的表達(dá)水平，而且這種BTG2高表達(dá)的細(xì)胞的放射敏感性明顯比未轉(zhuǎn)染BTG2質(zhì)粒的腫瘤母細(xì)胞和轉(zhuǎn)染了pcDNA3空質(zhì)粒的對(duì)照腫瘤細(xì)胞的放射敏感性提高。 (3)采用免疫共沉淀分析方法發(fā)現(xiàn)BTG2蛋白與BRCA1蛋白直接結(jié)合形成復(fù)合體。BRCA1明顯地下調(diào)了BTG2的轉(zhuǎn)錄活性，而B(niǎo)TG2則輕微地增加了BRCA1的轉(zhuǎn)錄活性。 (4)高表達(dá)BRCA1的表達(dá)水平明顯地抑制了BTG2高表達(dá)對(duì)乳腺癌細(xì)胞的放射敏感性的調(diào)節(jié)作用，而降低BRCA1的表達(dá)水平則提高了BTG2對(duì)乳腺癌細(xì)胞的放射敏感性的調(diào)節(jié)作用。 (5)姜黃素處理可以增加腫瘤細(xì)胞中BTG2的表達(dá)水平。 結(jié)論： (1)電離輻射照射可以誘導(dǎo)腫瘤細(xì)胞BTG2表達(dá)水平的提高，并且呈現(xiàn)劑量依賴性。 (2) BTG2的表達(dá)水平與腫瘤細(xì)胞的放射敏感性成正相關(guān)，提高BTG2的表達(dá)可以明顯增加腫瘤細(xì)胞的放射敏感性。 (3) BTG2蛋白與DNA損傷修復(fù)蛋白BRCA1在細(xì)胞中形成蛋白-蛋白復(fù)合物，BRCA1蛋白水平的改變可以調(diào)節(jié)BTG2對(duì)腫瘤細(xì)胞放射敏感性的影響。 (4)姜黃素的腫瘤放療增敏作用可能與其對(duì)BTG2表達(dá)水平的調(diào)節(jié)相關(guān)，更進(jìn)一步提示BTG2可以在放射敏感性調(diào)節(jié)中起到重要作用。 總之，BTG2是一種新的電離輻射損傷誘導(dǎo)基因，其表達(dá)水平改變可以明顯影響腫瘤細(xì)胞的放射敏感性，為腫瘤放射治療和放射治療敏感性提供了新的預(yù)測(cè)基因標(biāo)志物。
[Abstract]:Objective: BTG2 gene is one of the important members of BTG/TOB gene family as anti proliferation, anti molecular cell proliferation and promote cell apoptosis may be an important target in tumor therapy to gene therapy. At present, the domestic and foreign influence on BTG2 in tumor radiotherapy there is no research reports. This paper aims to study the expression levels of ionizing radiation effect of BTG2, and the role of BTG2 gene in tumor cells in radiation therapy and possible related regulatory mechanisms for BTG2 as a new tumor radiotherapy target gene into the basic research and clinical studies provide experimental and theoretical basis.
Methods: MCF-7 and MDA-MB-231 of breast cancer cells, C33A of prostate cancer cell, HeLa of cervical cancer cell and A549 of lung cancer cells were used as the research object.
(1) the effect of ionizing radiation on the expression of BTG2 protein in tumor cells was detected by Western blot.
(2) pcDNA3-BTG2 by liposome transfection to obtain the high expression of BTG2 in breast cancer cell MCF-7/BTG2 and stably transfected MDA-MB-231/BTG2 cell line, using four methyl thiazolyl tetrazolium colorimetric assay (MTT) and cell colony formation method to understand the influence of BTG2 expression level on cell radiosensitivity.
(3) immunoprecipitation assay was used to detect the interaction and interaction of BTG2 and BRCA1 protein.
(4) the eukaryotic expression plasmid of BRCA1 gene to improve the expression level of BRCA1 breast cancer cells with high expression of BTG2 is known about the effects of BRCA1 on BTG2 tumor artificially enhance the radiosensitizing effect of BRCA1. Using siRNA-BRCA1 to silence BTG2 expression in breast cancer cells the expression level of human understanding to reduce the impact of sensitization of BRCA1 on BTG2 radiation oncology.
(5) Western blot was used to detect the expression level of BTG2 in the tumor cells after curcumin treatment.
Result錛

本文編號(hào)：1558468