人类卟啉症及贫血的斑马鱼疾病模型的建立与解析

发布时间：2017-12-28 07:26

本文关键词：人类卟啉症及贫血的斑马鱼疾病模型的建立与解析　出处：《苏州大学》2015年博士论文　论文类型：学位论文

【摘要】：血红素(heme)是生物体内一种重要的分子,参与组成血红蛋白、肌红蛋白、细胞色素P450、维生素B1等。血红素的合成由8步酶催化的级联反应组成,相应的催化酶分别由对应的基因编码,包括ALAS1和ALAS2、ALAD、HMBS、UROS、UROD、CPOX、PPOX、FECH。血红素合成过程中的酶的功能的缺陷导致一系列被称为卟啉症(porphyria)的综合症。卟啉症的临床症状复杂,目前的诊断和治疗手段相对有限。斑马鱼作为一种模式生物,在人类疾病的研究中有着广泛的应用,其相应基因缺陷的突变体能够作为人类卟啉症的疾病模型。已有报道斑马鱼alas2,urod,ppox,fech基因缺陷的突变体能够分别作为人类先天性铁粒幼细胞性贫血症(CSA)、肝性红细胞生成性卟啉症(HEP)、杂色卟啉症(VP)、细胞生成性原卟啉症(EPP)的疾病模型,但其他几种卟啉症还没有可稳定遗传的斑马鱼疾病模型。本文运用TALEN技术建立了斑马鱼alad基因缺陷的突变体,作为人类5-氨基酮戊酸脱水酶缺乏性卟啉症(ALA dehydratase deficient porphyria,ADP)的疾病模型。还运用CRISPR-Cas9技术建立了斑马鱼cpox基因缺陷的突变体,作为人类遗传性粪卟啉症(Hereditary coproporphyria,HCP)的疾病模型。对两种突变体的表型分析显示,与野生型相比,alad-/-和cpox-/-突变体的血红素合成受阻,体内血红素含量显著减少,血细胞发育异常,血红蛋白含量显著下降,中间产物异常累积,同时血红素合成通路中的相关基因表达水平异常。此外,人ALAD m RNA和CPOX m RNA分别能够解救斑马鱼alad-/-和cpox-/-的表型,说明alad和cpox的功能在人和斑马鱼中具有保守性。斑马鱼中存在两个编码血红素合成第三步反应酶的基因,分别为hmbsa和hmbsb。系统树分析表明这两个基因为复制基因,是人HMBS基因的共同直系同源基因(co-orthologs)。本文运用CRISPR-Cas9和TALEN技术建立了斑马鱼hmbsa和hmbsb基因缺陷的突变体,作为人类急性间歇性卟啉症(Acute intermittent porphyria,AIP)的疾病模型,并对其表型进行了分析。两种纯合突变体均显示血红素缺失的贫血症状,并且突变体表型的显著程度随等位基因的缺失程度而增加。同时与野生型相比,两种突变体幼鱼和成鱼的行为均显示异常。为了对卟啉症的病理机制做出更详细的解析,本文还对两种已有报道的斑马鱼卟啉症及贫血模型urod-/-和alas2-/-进行了高通量深度测序转录组分析,结果显示血红素的缺失导致大量基因的表达发生了改变。不同发育阶段和不同突变体中的差异表达基因也不尽相同,显示了卟啉症的复杂性。血红素合成的限速酶基因alas1的表达受生物钟系统调控,生物钟系统通过血红素偶联能量代谢途径。为了深入探究生物钟系统与血红素合成的相互作用关系,本文还运用CRISPR-Cas9技术建立了斑马鱼per1a基因的突变体以及per基因全部敲除的突变体。并证实了alas1的表达节律在per1a突变体中发生了改变。本文首次运用反向遗传学技术建立了四种人类卟啉症的斑马鱼疾病模型,分别模拟人ADP,HCP和AIP,为相应疾病的病理机制研究提供了重要的动物模型,并为治疗卟啉症的药物筛选工作提供了有力的工具。本文还建立了生物钟突变体,为研究斑马鱼生物钟系统与血红素代谢间的相互作用关系提供了独特的方向。
[Abstract]:Heme (heme) is an important molecule in the organism, which is involved in the formation of hemoglobin, myoglobin, cytochrome P450, vitamin B1 and so on. The synthesis of heme is composed of 8 steps enzyme catalyzed cascade reaction. The corresponding catalytic enzymes are encoded by corresponding genes, including ALAS1 and ALAS2, ALAD, HMBS, UROS, UROD, CPOX, PPOX and FECH. The process of synthesis of defects in heme enzyme function leads to a series called porphyria (porphyria) syndrome. The clinical symptoms of porphyria complex, diagnosis and treatment method at present is relatively limited. Zebrafish as a model organism, is widely used in the study of human disease, the corresponding gene deficient mutants can be used as a model of human disease porphyria. There have been reports of zebrafish ALAS2, UROD, PPOX, fech deficient mutants can respectively as human congenital sideroblastic anemia (CSA), hepatoerythropoietic porphyria variegate porphyria (HEP), (VP), cell generative protoporphyrin (EPP) disease model, zebrafish model but several other diseases there is no stable genetic porphyria. This paper uses TALEN technology to establish the zebrafish ALAD gene deficient mutant, as human 5- aminolevulinic acid dehydratase deficiency porphyria (ALA dehydratase deficient porphyria, ADP) disease model. Using CRISPR-Cas9 technology to establish the zebrafish CPOX gene deficient mutant, as human hereditary coproporphyria (Hereditary coproporphyria HCP) disease model. Two kinds of mutant phenotype analysis showed that compared with wild-type alad-/- and cpox-/- mutant heme synthesis is blocked, the body heme content decreased significantly, the abnormal development of blood cells, hemoglobin content decreased significantly, the intermediate product at the same time as abnormal accumulation of heme related gene pathway in abnormal expression. In addition, human ALAD m RNA and CPOX m RNA can save the phenotype of zebrafish alad-/- and cpox-/-, indicating that the functions of ALAD and CPOX are conserved in human and zebrafish. In zebrafish, there are two genes that encode heme to synthesize third - step reactivity enzymes, hmbsa and hmbsb, respectively. Phylogenetic analysis shows that these two genes are replicating genes and are common direct homologous genes (co-orthologs) of the human HMBS gene. This paper uses CRISPR-Cas9 and TALEN technology to establish the zebrafish hmbsa and hmbsb gene deficient mutant, as human acute intermittent porphyria (Acute intermittent, porphyria, AIP) of the disease model, and analyzed its phenotype. The two homozygous mutants all showed heme deficiency anemia, and the phenotype of the mutant was significantly increased with the degree of loss of allele. At the same time compared with the wild type, two mutants of juvenile and adult fish showed abnormal behavior. In order to make the analysis more detailed pathological mechanism of porphyria, the two kinds of reported porphyria and anemia model urod-/- and zebrafish alas2-/- high-throughput deep sequencing transcriptome analysis, results show that heme deficiency results in a large number of gene expression changes. The difference in different developmental stages and different mutants of gene expression are not the same, showing the complexity of porphyria. The expression of the speed limiting enzyme gene ALAS1 in heme synthesis is regulated by the biological clock system, and the biological clock system is mediated by the heme coupling energy metabolism pathway. In order to further explore the interaction relationship between the biological clock system and heme synthesis, we also used CRISPR-Cas9 technology to establish the mutant of per1a gene in zebrafish and the knockout mutant of per gene. It was also confirmed that the expression rhythm of ALAS1 changed in the per1a mutant. In this paper, using reverse genetics technology established four models of zebrafish human disease porphyria were simulated for the first time, ADP, HCP and AIP, provides an important animal model for the study of pathological mechanism of the disease, provide a powerful tool for drug treatment and screening of porphyria. The biological clock mutants have been established in this paper, which provides a unique direction for the study of the interaction between the biological clock system of zebrafish and the heme metabolite.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R-332;R55

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