基因治疗人类CRB1突变引起的视网膜色素变性的方案探索

发布时间：2018-01-20 03:04

本文关键词： 视网膜色素变性利伯氏先天性黑蒙 Crb蛋白蛋白结构域斑马鱼基因重组基因治疗　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：感光细胞退行性病变是人类的一种常见致盲性疾病,严重危害人类健康,但目前针对这类疾病仍没有良好的医疗手段。现今,基因治疗和干细胞治疗是两类具有较好前景的治疗方案。在人类诸多与视网膜退行性病变相关的基因当中,CRB1基因不同位点的突变可以引起视网膜色素变性(Retinitis pigmentosa, RP)和利伯氏先天性黑蒙(Leber congenital amaurosis, LCA),但目前尚未有关于此基因突变进行基因治疗的报道,主要原因之一是CRB1基因比较大,不符合基因治疗的载体腺相关病毒(Adeno-associated virus, AAV)对基因片段大小的要求,且其编码的蛋白质结构复杂,各结构域的功能尚不十分明确。鉴于此,本论文旨在以斑马鱼为模型,分析其Crb2a蛋白各结构域的功能,在此基础上探索对人类因CRB1功能缺陷引起的RP和LCA的基因治疗方案。方法：首先,通过生物信息学比较斑马鱼不同Crb异构体与哺乳动物CRB1之间的同源性,确定crb2a为斑马鱼中与哺乳动物CRB1同源的基因。然后,对斑马鱼Crb2a蛋白的各结构域分区进行预测,结合对比分析自然界原本存在的不同异构体,分别克隆各单个结构域或结构域组合,在斑马鱼和细胞中分别检测其对crb2a突变鱼系表型的修复情况,介导蛋白——蛋白自身粘连的能力等,以此研究Crb2a各结构域的功能。在此基础上,选择基因大小适宜且保留Crb2a功能的重组形式,通过转基因技术在斑马鱼中验证对CRB1突变引起的视网膜疾病进行基因治疗的可行性。结果：生物信息学分析表明, crb2a具有3个Laminin G结构域和16个EGF结构域。同时,尽管自然界中获得广泛认可的具有功能的Crb蛋白通常由以上结构域组成,但cDNA克隆表明crb2a具有多种不同的剪接转录形式。在此基础上,我们通过基因工程技术,获得了8种不同结构域组合的重组crb2a。经体外转录成mRNA后,显微注射到斑马鱼胚胎中,观察是否能修复crb2a突变鱼系表型。结果证明了Crb2a的各个结构域具有相对独立功能,不同重组形式的Crb2a在不同的脏器展现出不同的修复效果。结论：本研究初步证明crb2a的多个结构域具有相对独立功能,通过重组crb2a基因,有望获得基因大小合适AAV载体,且能保留全长crb2a基本功能的修改形式,为基因治疗人类与CRB1相关的疾病提供新的条件。
[Abstract]:Objective: photoreceptor cell degenerative disease is a common blind disease in human, which seriously endangers human health, but there is still no good medical treatment for this kind of disease. Gene therapy and stem cell therapy are two promising therapies. Mutations at different sites of the CRB1 gene can cause retinitis pigmentosa. RPand Leber congenital amaurosis, LCAA, but there are no reports of gene therapy for this mutation. One of the main reasons is that the CRB1 gene is relatively large and does not accord with the gene therapy vector adeno-associated virus (Adeno-associated virus). AAVs require the size of gene fragments, and the structure of proteins encoded by AAVs is complex, and the functions of each domain are not very clear. In view of this, the purpose of this paper is to use zebrafish as a model. On the basis of analyzing the function of Crb2a protein domains, the gene therapy of RP and LCA caused by CRB1 functional defect was explored. Methods: first of all. By comparing the homology between different Crb isomers of zebrafish and mammalian CRB1, crb2a was identified as a gene homologous to mammalian CRB1 in zebrafish. The domain zoning of zebrafish Crb2a protein was predicted and the different isomers existed in nature were compared and cloned into a single domain or domain combination. The phenotypic repair of crb2a mutant fish and its ability to mediate protein-protein self-adhesion were detected in zebrafish and cells respectively. The function of each domain of Crb2a was studied. On the basis of this, the recombination forms of gene size and Crb2a function were selected. The feasibility of gene therapy for retinal diseases caused by CRB1 mutation was verified in zebrafish by transgenic technology. Results: bioinformatics analysis showed. Crb2a has three Laminin G domains and 16 EGF domains. Meanwhile, although widely recognized in nature, functional Crb proteins are usually composed of these domains. However, cDNA cloning shows that crb2a has many different splicing transcriptional forms. On this basis, we use genetic engineering technology. Eight recombinant crb2as with different domain combinations were obtained. After transcribed into mRNA in vitro, they were microinjected into zebrafish embryos. It was observed whether the phenotype of crb2a mutant fish could be repaired. The results showed that each domain of Crb2a had relatively independent function. Different recombination forms of Crb2a showed different repair effects in different organs. Conclusion: this study preliminarily proves that multiple domains of crb2a have relatively independent functions. By recombination of crb2a gene, it is expected that the gene is suitable for AAV vector and can retain the modified form of the basic function of full-length crb2a. It provides new conditions for gene therapy of human CRB1 related diseases.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R774.1

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