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X连锁特发性眼球震颤相关FRMD7基因新剪切变体的克隆及其在神经发育过程中相关功能的研究

发布时间:2018-01-17 16:29

  本文关键词:X连锁特发性眼球震颤相关FRMD7基因新剪切变体的克隆及其在神经发育过程中相关功能的研究 出处:《浙江大学》2011年博士论文 论文类型:学位论文


  更多相关文章: 选择性剪切 先天性特发性眼球震颤 FRMD7 神经突触生长


【摘要】:研究背景与目的: 先天性特发性眼球震颤(Idiopathic congenital nystagmus, ICN)是一种出生后数月内发病、主要临床表现为非自主性节律性眼球颤动的疾病,其震颤程度一般不随患者年龄的增长而加重。ICN与其他后天获得性眼球震颤(如白内障,青光眼,白化病等)有明显区别,该病罕有伴随眼球、脑或其他系统的器质性病变。ICN的发病机制目前尚不明确,主要推测可能与脑部控制眼球运动及凝视功能的区域发育异常有关。约7%-30%的ICN患者有家族史,其中X连锁遗传方式最为常见。FERM domain-containing 7 (FRMD7)基因(NM_194277)为X连锁ICN的一个致病基因,迄今为止已报道40余个不同的突变位点及突变类型,但目前对FRMD7基因的相关功能了解甚少。选择性剪切(Alternative splicing,AS)是真核生物体内广泛存在的现象,单个基因通过选择性剪切可以产生多个不同结构的蛋白产物,这对真核基因的功能及调节机制产生了重要影响。大规模基因组测序发现选择性剪切可能与组织多样性相关,超过90%的基因存在不同数目的剪切变体。值得注意的是,相对其他组织来说选择性剪切现象在脑组织中出现机率更高。近年来,越来越多的研究发现,选择性剪切现象在多种疾病包括肿瘤、遗传性疾病及神经系统疾病等的发生发展进程中扮演了重要的角色。本课题克隆及鉴定了FRMD7基因的两个新剪切变体,这也是首次对FRMD7剪切变体的报道;随后对全长FRMD7及两个新剪切变体在神经系统发育过程中的作用进行了研究。 研究方法: 使用RT-PCR分别从人NT2细胞及小鼠胚脑中克隆得到FRMD7全长及两个新的剪切变体并检测了它们在人胚胎组织中的表达分布。通过实时荧光定量PCR方法,检测了分别用维甲酸及BMP-2诱导NT2细胞分化后3个转录本的相对表达量变化情况。分别使用绿色荧光蛋白及红色荧光蛋白融合全长hFRMD7_FL及剪切本hFRMD7_SV1的重组蛋白进行共转染NT2细胞,观察亚细胞共定位。HA或Myc标签蛋白融合重组质粒瞬时共转染NT2细胞后进行免疫共沉淀实验。瞬时转染hFRMD7_FL或hFRMD7_SV1,荧光定量PCR技术检测另一方的表达量变化情况。构建稳定过表达hFRMD7_FL或hFRMD7_SV1的NT2细胞系,间接免疫荧光技术观察与classⅢβ-tubulin的共定位,及维甲酸诱导后NT2细胞突触生长速度。 结果: (1)本课题首次克隆并报道了X连锁ICN的致病基因FRMD7的两个新剪切变体。分别命名为FRMD7_SV1(包含一个N端缺失45个碱基的截短的4号外显子)及FRMD7_SV2(缺失整个2、3、4号外显子共227个碱基)。(2)人FRMD7全长基因及hFRMD7_SV1在人胚胎小脑高表达,hFRMD7_SV2随着胚脑发育逐渐限制性表达在小脑内。(3)人FRMD7全长基因及两个剪切变体的表达水平在维甲酸诱导的NT2分化过程早期出现一个显著升高,而在BMP-2诱导的分化过程早期受到明显抑制。(4)细胞内共定位及免疫共沉淀实验结果揭示了hFRMD7_FL与hFRMD7_SV1在NT2细胞内存在共定位及相互作用。(5) hFRMD7_FL及hFRMD7_SV1与神经元特异性骨架蛋白classⅢβ-tubulin在维甲酸诱导5天的NT2细胞内共定位。(6)过表达hFRMD7_FL会引起hFRMD7_SV1转录水平显著升高并可以促进维甲酸诱导的NT2细胞突触发育的生长速度。 结论: (1) FRMD7基因至少存在两个剪切变体,命名为FRMD7_SV1的剪切本包含了缺少45个碱基的截短的4号外显子,命名为FRMD7_SV2的剪切本缺失了整个2、3、4号外显子并可能编码了一个严重截短的蛋白。(2)组织表达分布的研究提示了FRMD7全长及两个剪切本可能与小脑的发育及相关区域的功能有关。(3维甲酸诱导NT2细胞分化实验证实了人FRMD7全长及两个剪切本均在神经元分化早期显著升高,提示了其特异性的参与了神经元早期的分化和发育过程。4)hFRMD7_FL与hFRMD7_SV1在NT2细胞内存在共定位及相互作用,提示了两者很可能在细胞内形成蛋白复合物而行使功能。(5)hFRMD7_FL及hFRMD7_SV1与classⅢβ-tubulin在细胞内共定位,提示了其作用可能与微管及骨架相关蛋白有关或参与了相关的分子通路。(6)过表达hFRMD7_FL引起hFRMD7_SV1转录水平升高,并可以促进维甲酸诱导的NT2细胞神经突触的生长速度,进一步证实了全长FRMD7在神经元的突触发生及发育过程中发挥了重要的作用,同时也提示了hFRMD7_SV1剪切本可能在此过程中起辅助的作用。
[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锛,

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