本文關(guān)鍵詞： ataxin-3 基因表達芯片 逆轉(zhuǎn)錄實時定量PCR 小鼠胚胎成纖維細胞 EphA3基因 啟動子 熒光素酶報告基因載體 轉(zhuǎn)錄活性 Ataxin-3 EphA3 組蛋白乙�；� 組蛋白去乙�；�3 核受體共抑制因子 組蛋白去乙�；敢种苿�　出處：《華中科技大學》2012年博士論文　論文類型：學位論文

【摘要】：第一部分：Ataxin-3敲除導致基因表達譜改變 目的：采用基因表達芯片技術(shù)(Microarray)觀察ataxin-3敲除對基因表達變化的影響。 方法：選取ataxin-3敲除的小鼠胚胎成纖維細胞(ataxin-3 KO MEF)和正常小鼠胚胎成纖維細胞(ataxin-3 WT MEF)為研究對象,提取RNA并用基因表達譜芯片技術(shù)觀察ataxin-3敲除對基因表達的影響,所得實驗結(jié)果數(shù)據(jù)經(jīng)ThomsonReuters/GeneGo公司的生物信息學軟件進行分析,并采用逆轉(zhuǎn)錄實時定量PCR技術(shù)證實基因表達芯片結(jié)果的可靠性。 結(jié)果：與正常細胞相比,ataxin-3敲除導致410個基因表達明顯異常(基因表達信號上調(diào)或下調(diào)超過2倍及以上),其中260個基因表達上調(diào),而150個基因表達下調(diào)。GeneGo軟件分析這410個基因的生物學功能,并對其功能進行分類。發(fā)現(xiàn)ataxin-3敲除導致多個細胞通路轉(zhuǎn)錄異常,尤其是炎癥免疫和細胞粘附相關(guān)基因。 結(jié)論：Ataxin-3分子丟失導致多種細胞通路相關(guān)基因轉(zhuǎn)錄異常。 第二部分：EphA3基因啟動子區(qū)域定位表達及活性分析 目的：構(gòu)建含有不同長度EphA3基因啟動子片段的報告基因載體,研究其在293T細胞和MEF細胞中的轉(zhuǎn)錄活性。 方法：以Balb/C小鼠基因組DNA為模板,擴增不同長度的EphA3基因啟動子片段,并克隆進入熒光素酶報告基因質(zhì)粒pGL3-Basic真核表達載體內(nèi)。酶切鑒定及基因測序無誤后,將重組質(zhì)粒和pRL-CMV內(nèi)對照質(zhì)粒共轉(zhuǎn)染293T或MEF細胞,分析不同長度的EphA3基因啟動子片段的轉(zhuǎn)錄活性。 結(jié)果：酶切鑒定和基因測序結(jié)果提示表達載體構(gòu)建成功,EphA3基因的核心啟動子區(qū)域位于-279～+110bp之間,在293T細胞和MEF細胞中啟動子片段的轉(zhuǎn)錄活性相似。 結(jié)論：成功構(gòu)建了熒光素酶報告基因重組質(zhì)粒,并確定了EphA3基因的核心啟動子區(qū)域。 第三部分：HDAC3和NCoR介導了ataxin-3對EphA3基因的轉(zhuǎn)錄調(diào)控 脊髓小腦共濟失調(diào)3型(SCA3)是多聚谷氨酸誘導神經(jīng)退行性疾病的一種,其疾病基因編碼一個小分子蛋白ataxin-3。Ataxin-3蛋白外顯子區(qū)域CAG異�？截悓е录膊〉陌l(fā)生。研究發(fā)現(xiàn)Ataxin-3分子是一個去泛素化酶,調(diào)節(jié)某些蛋白的活性和穩(wěn)定性,是泛素-蛋白酶體的重要成分。同時,ataxin-3分子也參與細胞轉(zhuǎn)錄調(diào)控,病理性ataxin-3導致多種細胞通路基因轉(zhuǎn)錄異常,加重疾病進程。為探討正常Ataxin-3分子轉(zhuǎn)錄調(diào)控的機制,我們選用ataxin-3 KO MEF和ataxin-3 WT MEF細胞為研究對象�；虮磉_芯片、逆轉(zhuǎn)錄實時定量PCR和免疫印跡結(jié)果均顯示ataxin-3敲除導致EphA3基因表達明顯上調(diào)。因此我們構(gòu)建了EphA3基因的啟動子片段,雙熒光素酶活性測定結(jié)果發(fā)現(xiàn)ataxin-3缺失導致EphA3基因啟動子轉(zhuǎn)錄活性增強,而瞬時表達人源ataxin-3分子進入ataxin-3 KO MEF細胞內(nèi)可抑制EphA3基因啟動子的轉(zhuǎn)錄活性。為闡明ataxin-3分子對EphA3基因啟動子調(diào)控的機制,我們采用染色體免疫共沉淀技術(shù),結(jié)果發(fā)現(xiàn)雖然ataxin-3分子不能直接結(jié)合到EphA3啟動子區(qū)域,但可以調(diào)節(jié)EphA3啟動子區(qū)域組蛋白H3和H4乙�；乃�。進一步免疫印跡結(jié)果證實Ataxin-3丟失導致總的組蛋白H3和H4乙�；皆黾�,而調(diào)節(jié)組蛋白H3和H4乙�；拿附M蛋白去乙�；�3(HDAC3)和核受體共抑制因子(NCoR)的水平減少。在正常MEF細胞內(nèi)給予HDAC3特異的阻斷劑可以模擬ataxin-3丟失的情況。綜上所述,我們的研究首次發(fā)現(xiàn),ataxin-3敲除可導致多種基因轉(zhuǎn)錄異常。Ataxin-3通過調(diào)節(jié)組蛋白去乙�；窰DAC3和NCoR的水平來調(diào)控基因啟動子區(qū)域組蛋白的乙酰化,進而調(diào)控基因的轉(zhuǎn)錄。Ataxin-3功能缺失導致的轉(zhuǎn)錄異�？赡芤藏暙I于SCA3疾病病理,探索Ataxin-3分子的轉(zhuǎn)錄調(diào)控機制,有助于尋找合適的治療靶點。
[Abstract]:Part I : Ataxin - 3 Knockout Causes Gene Expression Profile Changes Objective : To observe the effect of attemperance - 3 knock on gene expression by gene expression chip technique . Methods : The experimental results were analyzed by the bioinformatics software of Thomson Reuters / GeneGo , and the reliability of gene expression chip was confirmed by reverse transcription - real - time quantitative PCR . Results : Compared with normal cells , at least one hundred and forty - four genes were differentially expressed ( up - regulated or down - regulated by more than 2 - fold or more ) , and the expression of 260 genes was up - regulated and 150 genes were down - regulated . GeneGo software was used to analyze the biological functions of the 410 genes and classify their functions . Conclusion : The loss of Ataxin - 3 molecules leads to abnormal gene transcription in various cell pathways . Second part : Expression and activity analysis of EphA3 gene promoter region Objective : To construct a reporter gene vector containing EphA3 gene promoter fragment with different length , and to study the transcriptional activity of EphA3 gene promoter . Methods : Using Balb / C mouse genomic DNA as a template , the EphA3 gene promoter fragment of different length was amplified and cloned into the luciferase reporter plasmid pGL3 - Basic eukaryotic expression vector . After the enzyme digestion and the gene sequencing were correct , the recombinant plasmid and the control plasmid in pRL - CMV were co - transfected into 293 cells , and the transcription activity of EphA3 gene promoter fragment of different length was analyzed . Results : The results of enzyme digestion and gene sequencing showed that the expression vector was constructed successfully . The core promoter region of EphA3 gene was located between -279 and + 110bp , and the transcriptional activity of the promoter fragment was similar to that of the promoter fragment . Conclusion : The recombinant plasmid of luciferase reporter gene was successfully constructed and the core promoter region of EphA3 gene was determined . The third part : HDAC3 and NCoR mediate the transcriptional regulation of EphA3 gene . In this paper , we have constructed the promoter fragment of EphA3 gene . The results showed that the expression of Ataxin - 3 could not bind directly to the promoter region of EphA3 , but it could regulate the transcription of histone H3 and H4 .

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R346

【共引文獻】

相關(guān)期刊論文 前1條

1 姜淼,金春蓮,林長坤,邱廣榮,柳宗蘭,王朝祥,孫開來;東北地區(qū)正常漢族人群SCA1及SCA3/MJD基因內(nèi)CAG重復變異研究[J];中華醫(yī)學遺傳學雜志;2004年01期

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