斑马鱼HMDPC疾病模型建立及金属离子代谢分子机制研究

The manganese homeostasis is essential for organic health. Hyper-manganesemia with dystonia, polycythemia, and cirrhosis (HMDPC) is an inherited metabolism disorder induced by human SLC30A10 mutations, in which the element manganese accumulated and pathological characterized by dystonia, polycythemia, and hepatic dysfunction. There is no specific medicine for this early-onset and lifelong disease by now. Furthermore, contrast to metabolism disorders concerned to other trace elements, there is no any animal model for the metabolism disorders concerned to manganese. To further discovering of the physiological process of manganese metabolism and to obtain a good model in early drug screening of HMDPC, we use CRISPR/Cas technology to generate zebrafish slc30a10 mutant lines. Preliminary analysis revealed that the slc30a10 mutants simulate the pathological symptoms of this human disorder, which show a series of phenotypes of manganese accumulation, suggesting zebrafish would be a good model for studying the mechanism of manganese metabolism and the pathogenesis of related diseases. This project plans to comprehensively and systematically analyze phenotypical features and pathological symptoms of this mutant line, research on drug reaction features of the mutants and the molecular interaction networks of slc30a10. It will provide data for the metal metabolic theory and functional mechanisms in organism, suggest new methods for clinical trials of related diseases, and serve as a new animal model for early drug screening and development.

锰元素稳态代谢对机体健康至关重要。由SLC30A10基因突变导致的人类HMDPC遗传病是近年来发现的一种锰离子蓄积型代谢疾病，病理症状主要表现为运动失调、肝功能损伤和红细胞增多。而这种早发性和终身性遗传代谢疾病至今尚无特效药。同时不像其它微量元素代谢疾病，目前锰元素代谢相关疾病尚无任何动物模型。为深入探索锰离子稳态代谢这一生理过程，并提供HMDPC疾病早期药物筛选的良好模型，我们采用CRISPR/Cas技术构建了slc30a10基因突变斑马鱼品系。初步分析表明此突变体能够模拟HMDPC的病理症状，并表现出锰离子蓄积的一系列表型，提示该模型适于研究锰离子代谢及相关疾病的发病机制。本项目拟全面系统分析该突变体的表型特点和病理症状，考察突变体药物反应特性及slc30a10分子互作网络。将为阐明机体金属离子代谢理论及机制提供坚实数据，为相关疾病的防治提供新思路，为早期药物的筛选和开发提供新模型。

锰元素作为人体必需微量元素之一，其稳态代谢对机体健康至关重要。由SLC30A10基因突变导致的人类HMDPC遗传病是近年来发现的一种锰离子蓄积型代谢疾病，病理症状主要表现为运动失调、肝功能损伤和红细胞增多。而这种早发性和终身性遗传代谢疾病至今尚无特效药。同时不像其它微量元素代谢疾病，目前锰元素代谢相关疾病尚无任何动物模型。为深入探索锰离子稳态代谢这一生理过程，并提供HMDPC疾病早期药物筛选的良好模型，我们采用CRISPR/Cas技术构建了slc30a10基因突变斑马鱼品系。通过对斑马鱼与人类锰离子代谢的相似性分析，检测slc30a10 基因突变斑马鱼锰离子代谢缺陷的表型，探究slc30a10 调控锰离子代谢的分子网络与作用机制，我们发现突变体斑马鱼成鱼体内锰离子显著蓄积，尤其是在脑部和肝脏大量蓄积。同时，突变体斑马鱼呈现了运动障碍，肝脏损伤，红细胞增多等一系列与人类疾病类似的表型。这些表型可以通过补充螯合剂EDTA- CaNa2或富马酸亚铁进行拯救。尤其重要的是，我们发现在斑马鱼胚胎表达SLC30A10之前，ATP2C1的表达可以保护胚胎免受锰离子蓄积的毒性效应，提示ATP2C1是在SLC30A10不表达时的一个代偿基因。然而，无论是后期斑马鱼表达正常的SLC30A10还是突变体SLC30A10，ATP2C1的表达和外排锰的能力均会受到抑制。SLC30A10 与ATP2C1的相互作用暗示，ATP2C1是一个潜在的临床治疗靶点，可以通过激活ATP2C1的活性或增强ATP2C1对锰离子的转运来避免机体遭受锰离子蓄积毒性。本项目的研究发现为阐明机体金属离子代谢理论及机制提供了坚实基础，为相关疾病的防治提供了新思路，为早期药物的筛选和开发提供了全新的动物模型。

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