本文關(guān)鍵詞： SWI/SNF Brm 人端粒酶逆轉(zhuǎn)錄酶 選擇性剪接 腫瘤　出處：《天津醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的： SWI/SNF (switch/sucrose non-fermenting)是位于基因編碼區(qū)的染色質(zhì)重塑復(fù)合物,具有調(diào)控選擇性剪接體的功能。Brm (Brahma)是染色質(zhì)重塑復(fù)合物SWI/SNF的催化亞單位,具有DNA依賴的ATP酶活性,通過調(diào)節(jié)染色質(zhì)結(jié)構(gòu)影響基因表達(dá)；同時(shí)Brm也參與多種基因前體mRNA的剪接調(diào)控,可以誘導(dǎo)E-鈣粘著蛋白,BIM,細(xì)胞周期蛋白D1,CD44等多種基因mRNA發(fā)生外顯子融合作用。選擇性剪接是真核生物基因中普遍存在的一種現(xiàn)象,它在真核基因表達(dá)調(diào)控中起著十分重要的作用。選擇性剪接是人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptase, hTERT)基因表達(dá)調(diào)控的機(jī)制之一。本研究利用Brm-siRNA轉(zhuǎn)染永生化人胃黏膜上皮細(xì)胞,觀察RNA干擾前后Brm基因?qū)TERT選擇性剪接調(diào)控的影響,進(jìn)一步闡明端粒酶逆轉(zhuǎn)錄酶選擇性剪接的分子機(jī)制,為腫瘤的臨床診斷和治療提供了新的實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 研究方法： 實(shí)驗(yàn)用永生化人胃黏膜上皮細(xì)胞系GES-1購自北京腫瘤醫(yī)院遺傳室。傳代培養(yǎng)GES-1細(xì)胞4-5代后,隨機(jī)選取對(duì)數(shù)生長期細(xì)胞并轉(zhuǎn)染入Brm-siRNA.提取RNA干擾前后GES-1細(xì)胞總RNA,以β-actin為內(nèi)參,利用RT-PCR分別檢測(cè)目的基因Brm的表達(dá)情況；然后分別提取RNA干擾前后GES-1細(xì)胞總蛋白,以β-actin為內(nèi)參,經(jīng)Western blot檢測(cè)BRM蛋白的表達(dá)情況,確定Brm是否已被干擾。最后用RNA干擾前后提取的GES-1細(xì)胞總RNA進(jìn)行逆轉(zhuǎn)錄,以逆轉(zhuǎn)后的cDNA為模板,檢測(cè)hTERT選擇性剪接變異體的類型及表達(dá)情況,進(jìn)一步觀察Brm基因?qū)θ硕肆Ｃ改孓D(zhuǎn)錄酶選擇性剪接的調(diào)控作用。數(shù)據(jù)分析采用SPSS17.0統(tǒng)計(jì)軟件進(jìn)行處理,采用單因素方差分析,P≤0.05作為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1. RT-PCR法：對(duì)于空白對(duì)照組和陰性對(duì)照組,提取細(xì)胞總RNA逆轉(zhuǎn)錄后可以擴(kuò)增出Brm目的基因,并且Brm mRNA的表達(dá)量無明顯差異(P0.05)；對(duì)于轉(zhuǎn)染入Brm-siRNA的實(shí)驗(yàn)組,提取細(xì)胞總RNA逆轉(zhuǎn)錄后Brm mRNA表達(dá)量明顯低于空白對(duì)照組和陰性對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P≤0.01)。 2. Western blot法：對(duì)于空白對(duì)照組和陰性對(duì)照組,提取細(xì)胞總蛋白進(jìn)行Western blot實(shí)驗(yàn)后可以檢測(cè)到BRM蛋白的表達(dá),并且BRM蛋白的表達(dá)量無明顯差異(P＞0.05)；對(duì)于轉(zhuǎn)染入Brm.siRNA的實(shí)驗(yàn)組,提取總蛋白進(jìn)行Westernblot實(shí)驗(yàn)檢測(cè)到BRM蛋白的表達(dá)量明顯低于空白對(duì)照組和陰性對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。 3.RT.PCR擴(kuò)增hTERT選擇性剪接轉(zhuǎn)錄本的表達(dá)：將空白對(duì)照組,陰性對(duì)照組以及轉(zhuǎn)染Brm.siRNA的實(shí)驗(yàn)組中提取得到的總RNA進(jìn)行逆轉(zhuǎn)錄,以逆轉(zhuǎn)后的cDNA為模板PCR擴(kuò)增后可檢測(cè)到三種hTERT選擇性剪接變異體(hTERTASV),分別為的α+β+hTERT ASV,α-β+hTERT ASV和α+β-hTERT ASV.并且在轉(zhuǎn)染入Brm-siRNA的實(shí)驗(yàn)組中可檢測(cè)到α+β+hTERT ASV表達(dá)量較空白對(duì)照組和陰性對(duì)照組明顯下降,α+β-hTERT ASV的表達(dá)量較空白對(duì)照組和陰性對(duì)照組明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 轉(zhuǎn)染Brm-siRNA后,細(xì)胞中Brm mRNA表達(dá)量以及BRM蛋白的表達(dá)量均明顯低于空白對(duì)照組和陰性對(duì)照組,提示Brm-siRNA可以有效的沉默目的基因和蛋白的表達(dá)。永生化胃黏膜上皮細(xì)胞GES-1 hTERT mRNA可產(chǎn)生三種選擇性剪接轉(zhuǎn)錄產(chǎn)物,分別為α+β+hTERT ASV,α+β-hTERT ASV和α-β+hTERT ASV,且沉默Brm基因后,α+β+hTERT ASV表達(dá)明顯下降,α+β-hTERT ASV表達(dá)明顯升高。Brm可以調(diào)控永生化人胃黏膜上皮細(xì)胞GES-1 hTERT選擇性剪接變異體的表達(dá),從而引起端粒酶活性的改變,為腫瘤的臨床治療提供新的思路和方法。
[Abstract]:The purpose of the study is:
SWI/SNF (switch/sucrose non-fermenting) is a chromatin remodeling complex located in the gene encoding function,.Brm can regulate alternative splicing (Brahma) catalyzes the chromatin remodeling complex SWI/SNF subunit, with ATP enzyme activity dependent DNA, by regulating chromatin structure influence gene expression; at the same time also participate in a variety of Brm splicing the precursor of mRNA gene can induce E-, E-cadherin, BIM, cyclin D1, mRNA gene CD44 exon fusion. Alternative splicing is a common phenomenon in eukaryotic gene expression, it plays an important role in regulation of eukaryotic genes selectively. Splicing is the human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) of the regulation of gene expression. This study used Brm-siRNA transfected immortalized human gastric mucosal epithelial cells. We observed the influence of Brm gene on hTERT alternative splicing regulation before and after RNA interference, and further elucidated the molecular mechanism of the alternative splicing of hTERT, which provided a new experimental basis and theoretical basis for the clinical diagnosis and treatment of tumors.
Research methods:
The experiment used immortalized human gastric epithelial cell line GES-1 was purchased from Beijing Cancer Hospital. Genetic laboratory cultured GES-1 cells were randomly selected after 4-5 generations, the logarithmic growth phase were extracted and transfected into Brm-siRNA. RNA interference in GES-1 cells before and after total RNA with beta -actin expression using RT-PCR as reference, respectively detection of Brm gene and then extract; RNA interference in GES-1 cells before and after total protein respectively, with beta -actin for reference, the expression of BRM protein was detected by Western blot, to determine whether Brm has been interference. Finally RNA interference before and after extraction of GES-1 cell RNA by reverse transcriptase, to reverse after cDNA as template to detect the expression and type hTERT splicing variants. Further, observe the regulation effect of Brm gene on human telomerase reverse transcriptase gene splicing. Data were analyzed by SPSS17.0 statistical software for processing, using single factor variance Analysis of P is less than or equal to 0.05 as the difference was statistically significant.
Result錛，

本文編號(hào)：1533282