本文關(guān)鍵詞：X連鎖特發(fā)性眼球震顫相關(guān)FRMD7基因新剪切變體的克隆及其在神經(jīng)發(fā)育過程中相關(guān)功能的研究　出處：《浙江大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 選擇性剪切 先天性特發(fā)性眼球震顫 FRMD7 神經(jīng)突觸生長

【摘要】：研究背景與目的： 先天性特發(fā)性眼球震顫(Idiopathic congenital nystagmus, ICN)是一種出生后數(shù)月內(nèi)發(fā)病、主要臨床表現(xiàn)為非自主性節(jié)律性眼球顫動(dòng)的疾病,其震顫程度一般不隨患者年齡的增長而加重。ICN與其他后天獲得性眼球震顫(如白內(nèi)障,青光眼,白化病等)有明顯區(qū)別,該病罕有伴隨眼球、腦或其他系統(tǒng)的器質(zhì)性病變。ICN的發(fā)病機(jī)制目前尚不明確,主要推測可能與腦部控制眼球運(yùn)動(dòng)及凝視功能的區(qū)域發(fā)育異常有關(guān)。約7%-30%的ICN患者有家族史,其中X連鎖遺傳方式最為常見。FERM domain-containing 7 (FRMD7)基因(NM_194277)為X連鎖ICN的一個(gè)致病基因,迄今為止已報(bào)道40余個(gè)不同的突變位點(diǎn)及突變類型,但目前對FRMD7基因的相關(guān)功能了解甚少。選擇性剪切(Alternative splicing,AS)是真核生物體內(nèi)廣泛存在的現(xiàn)象,單個(gè)基因通過選擇性剪切可以產(chǎn)生多個(gè)不同結(jié)構(gòu)的蛋白產(chǎn)物,這對真核基因的功能及調(diào)節(jié)機(jī)制產(chǎn)生了重要影響。大規(guī)模基因組測序發(fā)現(xiàn)選擇性剪切可能與組織多樣性相關(guān),超過90%的基因存在不同數(shù)目的剪切變體。值得注意的是,相對其他組織來說選擇性剪切現(xiàn)象在腦組織中出現(xiàn)機(jī)率更高。近年來,越來越多的研究發(fā)現(xiàn),選擇性剪切現(xiàn)象在多種疾病包括腫瘤、遺傳性疾病及神經(jīng)系統(tǒng)疾病等的發(fā)生發(fā)展進(jìn)程中扮演了重要的角色。本課題克隆及鑒定了FRMD7基因的兩個(gè)新剪切變體,這也是首次對FRMD7剪切變體的報(bào)道；隨后對全長FRMD7及兩個(gè)新剪切變體在神經(jīng)系統(tǒng)發(fā)育過程中的作用進(jìn)行了研究。 研究方法： 使用RT-PCR分別從人NT2細(xì)胞及小鼠胚腦中克隆得到FRMD7全長及兩個(gè)新的剪切變體并檢測了它們在人胚胎組織中的表達(dá)分布。通過實(shí)時(shí)熒光定量PCR方法,檢測了分別用維甲酸及BMP-2誘導(dǎo)NT2細(xì)胞分化后3個(gè)轉(zhuǎn)錄本的相對表達(dá)量變化情況。分別使用綠色熒光蛋白及紅色熒光蛋白融合全長hFRMD7_FL及剪切本hFRMD7_SV1的重組蛋白進(jìn)行共轉(zhuǎn)染NT2細(xì)胞,觀察亞細(xì)胞共定位。HA或Myc標(biāo)簽蛋白融合重組質(zhì)粒瞬時(shí)共轉(zhuǎn)染NT2細(xì)胞后進(jìn)行免疫共沉淀實(shí)驗(yàn)。瞬時(shí)轉(zhuǎn)染hFRMD7_FL或hFRMD7_SV1,熒光定量PCR技術(shù)檢測另一方的表達(dá)量變化情況。構(gòu)建穩(wěn)定過表達(dá)hFRMD7_FL或hFRMD7_SV1的NT2細(xì)胞系,間接免疫熒光技術(shù)觀察與classⅢβ-tubulin的共定位,及維甲酸誘導(dǎo)后NT2細(xì)胞突觸生長速度。 結(jié)果： (1)本課題首次克隆并報(bào)道了X連鎖ICN的致病基因FRMD7的兩個(gè)新剪切變體。分別命名為FRMD7_SV1(包含一個(gè)N端缺失45個(gè)堿基的截短的4號(hào)外顯子)及FRMD7_SV2(缺失整個(gè)2、3、4號(hào)外顯子共227個(gè)堿基)。(2)人FRMD7全長基因及hFRMD7_SV1在人胚胎小腦高表達(dá),hFRMD7_SV2隨著胚腦發(fā)育逐漸限制性表達(dá)在小腦內(nèi)。(3)人FRMD7全長基因及兩個(gè)剪切變體的表達(dá)水平在維甲酸誘導(dǎo)的NT2分化過程早期出現(xiàn)一個(gè)顯著升高,而在BMP-2誘導(dǎo)的分化過程早期受到明顯抑制。(4)細(xì)胞內(nèi)共定位及免疫共沉淀實(shí)驗(yàn)結(jié)果揭示了hFRMD7_FL與hFRMD7_SV1在NT2細(xì)胞內(nèi)存在共定位及相互作用。(5) hFRMD7_FL及hFRMD7_SV1與神經(jīng)元特異性骨架蛋白classⅢβ-tubulin在維甲酸誘導(dǎo)5天的NT2細(xì)胞內(nèi)共定位。(6)過表達(dá)hFRMD7_FL會(huì)引起hFRMD7_SV1轉(zhuǎn)錄水平顯著升高并可以促進(jìn)維甲酸誘導(dǎo)的NT2細(xì)胞突觸發(fā)育的生長速度。 結(jié)論： (1) FRMD7基因至少存在兩個(gè)剪切變體,命名為FRMD7_SV1的剪切本包含了缺少45個(gè)堿基的截短的4號(hào)外顯子,命名為FRMD7_SV2的剪切本缺失了整個(gè)2、3、4號(hào)外顯子并可能編碼了一個(gè)嚴(yán)重截短的蛋白。(2)組織表達(dá)分布的研究提示了FRMD7全長及兩個(gè)剪切本可能與小腦的發(fā)育及相關(guān)區(qū)域的功能有關(guān)。(3維甲酸誘導(dǎo)NT2細(xì)胞分化實(shí)驗(yàn)證實(shí)了人FRMD7全長及兩個(gè)剪切本均在神經(jīng)元分化早期顯著升高,提示了其特異性的參與了神經(jīng)元早期的分化和發(fā)育過程。4)hFRMD7_FL與hFRMD7_SV1在NT2細(xì)胞內(nèi)存在共定位及相互作用,提示了兩者很可能在細(xì)胞內(nèi)形成蛋白復(fù)合物而行使功能。(5)hFRMD7_FL及hFRMD7_SV1與classⅢβ-tubulin在細(xì)胞內(nèi)共定位,提示了其作用可能與微管及骨架相關(guān)蛋白有關(guān)或參與了相關(guān)的分子通路。(6)過表達(dá)hFRMD7_FL引起hFRMD7_SV1轉(zhuǎn)錄水平升高,并可以促進(jìn)維甲酸誘導(dǎo)的NT2細(xì)胞神經(jīng)突觸的生長速度,進(jìn)一步證實(shí)了全長FRMD7在神經(jīng)元的突觸發(fā)生及發(fā)育過程中發(fā)揮了重要的作用,同時(shí)也提示了hFRMD7_SV1剪切本可能在此過程中起輔助的作用。
[Abstract]:Research background and purpose:
Congenital idiopathic nystagmus (Idiopathic congenital, nystagmus, ICN) is a few months after birth incidence, clinical manifestation of involuntary rhythmic eyeball fibrillation disease, the tremor degree is generally not with age of patients increases.ICN and the other was acquired nystagmus (such as cataract, glaucoma albinism, etc.) have the obvious difference, the disease is accompanied by eye, the pathogenesis of pathological changes of cerebral.ICN or other systems are still not clear, presumably with the main brain eye movement and gaze control function of regional development abnormalities. About 7%-30% of ICN patients have a family history, the X genetic linkage way common.FERM domain-containing 7 (FRMD7) gene (NM_194277) is a pathogenic gene X chain ICN, have been reported so far more than 40 different mutations and mutations of FRMD7 gene, but at present The correlation function is poorly understood. Alternative splicing (Alternative splicing, AS) is widespread in eukaryotic organisms, single gene by alternative splicing can generate a number of different structural protein products, the eukaryotic gene function and regulation had a major impact. The discovery of alternative splicing of large-scale genome sequencing and organization diversity, more than 90% of the gene variants have different number. It is worth noting that, relative to other organizations of alternative splicing phenomenon is likely in the brain. In recent years, more and more research found that alternative splicing phenomena in a variety of diseases including cancer, plays an important role in the occurrence and development of genetic diseases and diseases of the nervous system in this subject. Cloning and two new variants of FRMD7 gene were identified, this is the first time for FRMD7 shear The effect of the full length FRMD7 and two new shear variants on the development of the nervous system was subsequently studied.
Research methods:
The use of RT-PCR from human NT2 cells and mouse embryonic brain cloned FRMD7 gene and two new variants and detect their expression in human embryonic tissues distribution. By real-time fluorescence quantitative PCR method, testing the differentiation of NT2 cells induced by retinoic acid and BMP-2 3 transcripts expression changes. Using green fluorescent protein and red fluorescent protein fusion recombinant protein of the hFRMD7_SV1 full length hFRMD7_FL and shear were co transfected into NT2 cells to observe the subcellular co localization of.HA or Myc tag fusion protein recombinant plasmids were transiently co transfected NT2 cells by CO immunoprecipitation experiments. Transient transfection of hFRMD7_FL or hFRMD7_SV1, the expression changes of fluorescence detection quantitative PCR of the other party. To build a stable NT2 cell line expressing hFRMD7_FL or hFRMD7_SV1, indirect immunofluorescence observation and class III beta -tubul The co localization of in and the growth rate of synapses in NT2 cells after retinoic acid were induced.
Result錛，

本文編號(hào)：1437061