本文關(guān)鍵詞： BAT1 Hsf4b 白內(nèi)障 αB hsp25　出處：《河南大學》2014年碩士論文　論文類型：學位論文

【摘要】：背景 先天性白內(nèi)障是導(dǎo)致兒童失明的主要原因，大約有1/3先天性白內(nèi)障是遺傳性的，其中常染色體顯性遺傳所占比列較高，，遺傳性先天性白內(nèi)障多數(shù)是由于基因突變引起的。先天性白內(nèi)障主要的病理改變?yōu)榫铙w的發(fā)育障礙、晶狀體內(nèi)纖維組織瘢塊形成，最終導(dǎo)致晶狀體渾濁。研究表明先天性白內(nèi)障的發(fā)生與多種基因的突變有相關(guān)性，如晶狀體細胞結(jié)構(gòu)相關(guān)基因的突變（alpha-,bete-和gamma-crystallins and GJA3/A8，AQPO,BFSP2）和調(diào)控晶狀體發(fā)育相關(guān)的轉(zhuǎn)錄因子基因突變（PITX3,PAX6，F(xiàn)OXE3,EYA1,MAF和Hsf4）。 熱休克轉(zhuǎn)錄因子4（Hsf4）在新生兒晶狀體發(fā)育的過程中起關(guān)鍵作用。Hsf4由于剪切方式不同形成不同的轉(zhuǎn)錄產(chǎn)物：Hsf4a和Hsf4b。Hsf4a為轉(zhuǎn)錄抑制因子，而Hsf4b則為轉(zhuǎn)錄激活因子。晶狀體中只有Hsf4b這一種活性形式，它主要參與調(diào)控小分子熱休克蛋白（Hsp25、γ-crystallin、α-crystallin）和纖維骨架蛋白（imentin、filesin）等蛋白的表達。研究發(fā)現(xiàn)Hsf4b基因的缺失可導(dǎo)致下游蛋白表達的缺失，如Hsp25、γ-晶狀體蛋白中的γ-F，γ-S晶體蛋白等。這些發(fā)現(xiàn)都說明Hsf4b是調(diào)控晶狀體早期發(fā)育的一個重要轉(zhuǎn)錄因子。Hsf4b受磷酸化、乙酰化、類泛素化、sumo化等的調(diào)控。但是，調(diào)控Hsf4b轉(zhuǎn)錄活性的信號通路以及Hsf4b調(diào)控晶狀體早期發(fā)育的分子機制仍不清楚。 我們通過酵母雙雜交實驗,發(fā)現(xiàn)了一個新的與Hsf4相互作用的蛋白BAT1（又名UAP56，56KD,U2AF56-associated protein）。BAT1基因最早從豬克隆出來，它含有9個高度保守的結(jié)構(gòu)域。BAT1蛋白屬于DAED-box家族蛋白成員之一，DAED-box家族廣泛存在于從細菌到哺乳動物的許多物種中，是一個ATP依賴的RNA解螺旋酶家族，既具有RNA激活的ATP結(jié)合和水解活性，又具有ATP依賴的RNA解螺旋活性。BAT1參與RNA的各種代謝過程如:RNA轉(zhuǎn)錄、mRNA前體剪接、核糖體和剪接體裝配、核質(zhì)運輸、蛋白翻譯、mRNA降解及維持mRNA的穩(wěn)定性等重要生命活動。本研究在小鼠晶狀上皮細胞中分析BAT1與Hsf4b的相互作用及細胞內(nèi)定位，探討B(tài)AT1對Hsf4b調(diào)控蛋白的影響，豐富先天性白內(nèi)障的分子機制，為先天性白內(nèi)障的早期診斷和治療提供理論基礎(chǔ)。 目的 探討B(tài)AT1在小鼠晶狀上皮細胞中與Hsf4b的相互作用及對Hsf4b下游蛋白表達的調(diào)控機理。 方法 (1)用pwzl-HA、pwzl-HA-Hsf4b、pbabe-HA-BAT1、pwzl-HA-Hsf4b+pbabe-HA-BAT1質(zhì)粒的逆轉(zhuǎn)錄病毒感染hsf4-/-小鼠晶狀上皮細胞（mLEC/hsf4-/-）,用blasticidin和puromycin篩選，構(gòu)建細胞穩(wěn)定株。突變體BAT1K95E、Hsf4b+BAT1K95E質(zhì)粒的逆轉(zhuǎn)錄病毒感染hsf4-/-小鼠晶狀上皮細胞（mLEC/hsf4-/-）,用puromycin篩選,構(gòu)建細胞穩(wěn)定株。 (2)應(yīng)用細胞免疫熒光技術(shù)，分析Hsf4b與BAT1亞細胞定位。 (3)應(yīng)用Realtime-PCR方法，分析BAT1對Hsf4b下游基因在mRNA水平的影響，應(yīng)用免疫印跡（western blotting）實驗，分析BAT1對Hsf4b調(diào)控蛋白αB晶狀體蛋白、hsp25蛋白的表達。 (4)應(yīng)用Luciferase assay實驗，檢測BAT1對Hsf4b下游基因aB-晶狀體蛋白基因啟動子的調(diào)控。 (5)應(yīng)用細胞核質(zhì)分離，Realtime-PCR方法，分析BAT1對Hsf4b下游蛋白的核質(zhì)輸出的影響，利用Pull Down實驗，探討Hsf4b是否可以促進BAT1的泛素化。 (6)利用BAT1在mRNA水平具有核輸出功能，而95位點是其核輸出功能的關(guān)鍵位點。構(gòu)建pbabe-HA-BAT1K95E突變質(zhì)粒，酶切測序及鑒定突變位點。 (7)應(yīng)用Realtime-PCR方法，分析BAT1基因核輸出位點突變對Hsf4b下游基因αΒ在mRNA水平的影響。 結(jié)果 (1)在小鼠晶狀上皮細胞mLEC中，BAT1與Hsf4b在細胞核共定位，同時抑制hsf4b的下游蛋白αB基因啟動子的轉(zhuǎn)錄活性。BAT1對Hsf4b下游蛋白αB、hsp25的表達起下調(diào)的作用。 (2)根據(jù)BAT1的生物學功能進一步分析，BAT1對Hsf4b下游基因aB在核質(zhì)輸出方面無調(diào)控作用。同時BAT1對Hsf4b下游蛋白αB的抑制作用不是由于BAT1 的核輸出起作用，BAT1核輸出功能突變后對Hsf4b的下游基因αB在mRNA水平 有所下調(diào)。同時，Hsf4b不能促進BAT1的泛素化。結(jié)論 BAT1與Hsf4b在體外相結(jié)合，同時在小鼠晶狀上皮細胞內(nèi)共定位于細胞核。BAT1降低Hsf4b下游基因αB、hsp25mRNA水平、減少αB、hsp25蛋白表達，且此作用與BAT1的ATP結(jié)合能力無明顯關(guān)系。
[Abstract]:Background Congenital cataract is the main cause of blindness in children . Approximately 1 / 3 of congenital cataract is hereditary , in which autosomal dominant inheritance is high , hereditary congenital cataract is mostly caused by gene mutation . The main pathological changes of congenital cataract are caused by gene mutation . The main pathological changes of congenital cataract are caused by mutation of lens . The research shows that the occurrence of congenital cataract is associated with mutation of various genes ( alpha - , bete - and gamma - crystallins and GJA3 / A8 , AQPO , BFSP2 ) and transcription factor gene mutation related to regulation of lens development ( PITX3 , PAX6 , FoxE3 , eyA1 , MAF and Hsf4 ) . Hsf4 plays a key role in the process of neonatal lens development . Hsf4 forms different transcription products due to shear . Hsf4 is an active form of transcription - inhibiting factor . It is mainly involved in the regulation of the expression of downstream protein , such as Hsp25 , 緯 - crystallin , 偽 - crystallin , and so on . We found a new protein BAT1 ( also named UAP56 , 56KD , U2AF56 - associated protein ) interacting with Hsf4 through yeast two - hybrid experiment . BAT1 protein belongs to one of DAED - box family proteins . The DAED - box family is widely present in many species of DAED - box family . The DAED - box family is a ATP - dependent RNA helicase family , which has both RNA - activated ATP binding and hydrolytic activity and ATP - dependent RNA helicase activity . Purpose Objective To investigate the interaction between BAT1 and Hs4b and the mechanism of protein expression in the downstream protein of BAT1 . method ( 1 ) Retroviral infection of hsf4 - / - mouse crystalline epithelial cells ( mLEC / hsf4 - / - ) with the recombinant retroviral infection hsf4 - / - mouse crystalline epithelial cells ( mLEC / hsf4 - / - ) , which were transfected with the plasmid ptimiticidin and puromycin , by using the retroviral infection hsf4 - / - mouse crystalline epithelial cells ( mLEC / hsf4 - / - ) of the plasmid of pw1 - HA , pw2 - HA - HSV4b , pounce - HA - BAT1 , pw1 - HA - HSV4b + p. - HA - BAT1 , and then screened with puromycin to construct a cell - stable strain . ( 2 ) Using cellular immunofluorescence technique , we analyzed the localization of Hs4b and BAT1 sub - cells . ( 3 ) Using the method of Realtime PCR , the effect of BAT1 on mRNA level of downstream gene of HSV4b was analyzed . Western blotting was used to analyze the expression of BAT1 on the expression of 偽 - B lens protein and Hsp25 protein . ( 4 ) The regulation of the promoter of aB - lens protein in the downstream gene of BAT1 was detected by Lucifer assay . ( 5 ) To investigate the effect of BAT1 on the nuclear output of downstream protein by using nuclear separation and Realtime - PCR . Using Pull Down experiment , it was discussed whether HSF4b could promote the ubiquitination of BAT1 . ( 6 ) The expression of BAT1 has nuclear output function at mRNA level , and the 95 site is the key point of its nuclear output function . Construction of ptase - HA - BAT1K95E mutant plasmid , enzyme digestion and sequencing and identification of mutation site . ( 7 ) Using the method of Realtime PCR , the effect of the mutation of BAT1 gene nuclear output site on the mRNA level of the gene 偽尾 in the downstream gene was analyzed . Results ( 1 ) In the mouse crystalline epithelial cell mLEC , BAT1 and HSV4b were co - located in the nucleus , while inhibiting the transcription activity of the downstream protein 偽B gene promoter of hsf4b . The BAT1 could regulate the expression of downstream protein 偽B and Hsp25 . ( 2 ) According to the biological function of BAT1 , BAT1 has no regulatory effect on the nuclear output of downstream gene aB . The inhibitory effect of BAT1 on downstream protein 偽B is not due to BAT1 . The downstream gene 偽B downstream of the BAT1 nuclear output function after the mutation of the BAT1 nuclear output function is at the mRNA level There was a downregulation . At the same time , Hs4b could not promote the ubiquitination of BAT1 . Conclusion At the same time , BAT1 and HSV4b were combined in vitro . At the same time , the cells of BAT1 were located in the nucleus . BAT1 reduced the levels of the downstream gene 偽B , Hsp25 mRNA , and decreased the expression of 偽B and Hsp25 , and this effect was not significantly associated with the ATP binding ability of BAT1 .

【學位授予單位】：河南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R776.1

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