人类卟啉症斑马鱼模型的建立、病理新机制和药物筛选

Heme as a structural and signaling molecule plays critical roles in numerous biological processes. Deficiencies in heme-biosynthetic enzymes result in a group of rare and clinically complex syndrome called porphyria. We previously established the first animal model for human hepatoerythropoietic porphyria (HEP), i.e., HEP zebrafish. Supported by a previous NSFC general grant, we used this HEP zebrafish to elucidate a new mechanism underlying porphyria pathogenesis: heme regulation of exocrine pancreatic zymogen genes via Bach1b/Nrf2a-MafK, and also observed possible effects of heme deficiency on the nervous system and circadian regulation. On the basis of our long-term study of porphyria pathogenesis, this research project continues to investigate novel aspects of porphyria pathogenesis, and also expands to generate new zebrafish models for human porphyrias and drug screens. Specific aims of this research project are as follows:1) we will employ new versatile gene-editing tools TALEN and CRISPR-Cas9 to generate 5 new zebrafish models for human porphyrias; 2) we will use new-generation high-throughput sequencing technology to conduct transcriptome and miRNA analyses of 7 zebrafish porphyria models, in order to obtain a deep understanding of porphyria pathogenesis and particularly its epigenetic regulation; and 3) we will take advantage of unique attributes of autofluorescent and pale blood cells of porphyric zebbrafish to conduct high-throughput drug screens in whole animal, in order to develop potential drugs for porphyria patients. This study will provide new targets and drugs for diagnosis, prevention and treatment of porphyria patients.

血红素生物合成缺陷引起一组称为卟啉症的复杂综合症。我们先期率先建立了第一个人类肝红细胞生成性卟啉症的HEP斑马鱼模型。在上一个面上项目资助下，我们利用HEP斑马鱼阐明了血红素通过Bach1b/Nrf2a-MafK通路调控外胰腺消化酶基因的新机制并发现血红素缺失可能危害神经系统和生物钟调控系统。在大量而且成效显著的前期工作基础之上，本项研究将继续深入研究卟啉症病理新机制，并扩展到建立人类卟啉症新的斑马鱼模型和药物筛选。具体研究目标包括采用TALEN和CRISPR-Cas9建立人类卟啉症尚未报道的5种斑马鱼模型；利用新一代高通量测序技术对7种卟啉症斑马鱼模型进行转录组和小RNA分析，以阐明卟啉症病理新机制特别是表观遗传学机制；利用卟啉症斑马鱼模型独特的自发荧光等特性，开展高通量活体药物筛选，以期获得治疗或缓解卟啉症症状的新药物。本项研究将为卟啉症的诊断、预防和治疗提供新的靶点和药物。

血红素生物合成酶缺陷导致一组称为卟啉症的复杂综合症。我们利用TALEN 或CRISPR-Cas9基因修饰技术成功建立了5种尚未有报道的血红素合成缺陷的斑马鱼突变体模型，分别为Alad（Aminolevulinate dehydratase,氨基乙酰丙酸脱水酶）缺陷的突变体,对应相应的人ALA-脱水酶缺陷型卟啉症（ADP）；Hmbsa和Hmbsb（Hydroxymethylbilane synthase a, 羟甲基胆色烷合酶, a Hydroxymethylbilane synthase b,和羟甲基胆色烷合酶 b）缺陷的突变体，对应人急性间歇性卟啉症（AIP）；Uros（Uroporphyrinogen III synthase,尿卟啉原 III 合酶） 缺陷的突变体，对应人先天性红细胞生成性卟啉症（CEP）；Cpox（Coproporphyrinogen oxidase，粪卟啉原氧化酶)）缺陷的突变体，对应人遗传性粪卟啉症（HCP）；Alas1（Delta-aminolevulinate synthase ，delta-氨基酮戊酸合成酶1）突变体。分别对5种血红素合成缺陷斑马鱼模型进行了表型分析以及高通量深度测和miRNA测序分析，发现了一系例与血红素合成、心脏发育以及肝脂代谢的新基因和新通路。我们的研究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。