G6PD转基因斑马鱼模型的建立及其G6PD缺陷突变型酶与红细胞发育的分子研究

Glucose - 6 - phosphate dehydrogenase (G6PD) deficiency is the worldwide hereditary enzyme deficiency in the clinical investigation .The deficiency is due to G6PD gene mutantions induced decrease in enzyme activity. As a result, red blood cells can not be destroyed by the anti-oxidative damage caused hemolytic anemia. It is high incidence, about 100 million patients in China. Guizhou province is one of the Chinese high incidence area.G6PD enzymology and the relationship of structure-function studies have gradually been concerned about. To study G6PD structure-function relationships for the interpretation of the molecular mechanisms of disease plays an important role in the future. However, the lack of ideal experimental model lead to the constraint of further research of the disease. The advantages of zebrafish embryos are development in vitro, transparent,achieve the direct dynamic monitoring of hematopoietic development process, hematopoietic regulatory network is highly conserved in human.therefore,zebrafish is a suitable model to study the disease. The research group previously established two mutant g6pd transgenic lines by zebrafish model with containing highly conserved in two Chinese people common G6PD variant sites and human G6PD enzyme NADP+ bind sites. On that basis, the project intends to establish mutant G6PD zebrafish model, Using the model to detect the function of g6pd gene in erythroid development and to clarify the possible pathogenic mechanisms of G6PD deficiency . Furthur to provide a basis for deeply research and to discover of new intervention strategies.

葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是临床最常见的红细胞遗传性酶缺陷病，因基因突变致酶活性降低，进而使红细胞不能抗氧化而损伤，引起溶血性贫血，对人类危害较大。该病的临床表现多样，疾病严重程度与G6PD的酶活性密切相关。G6PD酶学、结构与功能关系研究渐被关注，探讨G6PD结构与功能的关系对于诠释发病的分子机制有着重要的作用。但目前缺乏有效的研究模型制约了对该病系统和深入的研究。斑马鱼因胚胎体外发育、透明，可直接动态监测造血发育过程、造血调控网络与人类高度保守等优势，是研究该病的合适模型。课题组前期已建立斑马鱼转基因模型平台，制备了含两种中国人常见G6PD变异型位点的斑马鱼突变缺陷型基因，本课题拟建立可示踪的突变缺陷型G6PD转基因斑马鱼模型，并基于该模型深入探讨突变型G6PD基因在红系发育和红细胞中的功能，系统全面地阐明G6PD缺乏症发病机理,为进一步研发新的干预策略提供理论依据。

根据我们对斑马鱼G6PD基因的生物信息学分析，以及前期对斑马鱼不同组织G6PD酶活性的测定结果，实验室构建了两个G6PD基因常见突变位点的质粒：zgata1:g6pdM118-144-egfp-pBSK-IsceⅠ质粒；gata1-g6pdM1315-1443-EGFP-PBSK-IsceI质粒，通过显微注射的方式将质粒与IsceI 酶共注射入tüebingen 野生型品系斑马鱼单细胞期胚胎中，筛选后代可以表达绿色荧光蛋白的斑马鱼作为F0代，通过原位杂交，Western-blot技术对F1代进行了鉴定，证明G6PD基因突变斑马鱼模型建立成功。采用氧化性药物α-萘酚对F1代胚胎进行处理，发现118-144位点突变的斑马鱼药物处理后反应不明显，1315-1443位点突变的斑马鱼药物处理后斑马鱼胚胎死亡率增加，心包肿大，心率变慢。流式细胞术分析表明，随着药物浓度的增加，表达绿色荧光的斑马鱼红细胞百分比减少，提示斑马鱼有溶血症状发生。全胚胎G6PD酶活性，还原型谷胱甘肽表达量，膜结合巯基表达水平，均有所降低；膜脂质体氧化水平，全胚胎ROS水平升高。提示G6PD缺乏可能通过降低红细胞膜抵抗氧自由基攻击，进而造成细胞变脆，易发溶血导致发病。通过本研究，我们首次通过基因显性负的技术和crispr/cas9基因编辑技术建立了中国人常见G6PD突变基因类型的斑马鱼模型，并应用该模型研究了氧化物质对红细胞发育影响的分子机制，为进一步开展G6PD缺陷的研究和寻找保护性抗氧化药物提供了一个重要的实验模型和研究手段。本课题培养研究生6名，发表学术论文11篇，其中SCI 3篇，北图核心期刊3篇，统计源核心期刊3篇，会议论文2篇，在投SCI论文1篇；申报发明型专利5项，目前已进入实质审查期；获得贵州医学科技奖一等奖1项。