帕金森小型猪疾病模型的制备和应用

Parkinson’s disease (PD) is a common neurodegenerative movement disorder disease. It is estimated that around 15-328 in 100 thousand people in the world suffer from PD. Mutations in Parkin, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of PD. However, single knockout mice lacking Parkin, DJ-1, or PINK1 or triple knockout mice lacking all three genes exhibited no nigral degeneration and did not mimic the major clinical features of PD. Pigs are more similar to humans than mice in.anatomy, physiology, and genome. Thus, we will generate DJ-1, Parkin and PINK1 triple knockout miniature pigs to model human PD in this proposal using CRISPR/Cas9-mediated genome engineering, which has been commonly adapted for pig gene modifications in our group. Combined with MPTP or rotenone injection, the gene knockout pigs will be phenotyped by the pathological feature of substantia nigra and striatum, dopaminergic (DA) neurons number and apoptosis, neuroglia cell proliferation, inflammatory factor secretion and the expression level of tyrosine hydroxylase (HT) and dopamine transporter (DAT). Lastly, the behavioral phenotypes analysis of gene knockout pigs will be investigated by field test and gait analysis. We hope this study will provide pig models that are able to recapitulate specific aspects of human PD, and promote the study of PD physiology and disease biology, therefore show huge promise for identifying and validating new drug targets.

帕金森病（Parkinson’s disease, PD）是一种常见的神经退行性疾病，患病率为15～328/10万人口。Parkin、DJ-1和PINK1是被发现与常染色体隐性遗传性PD相关的3个基因，但上述基因的单个或者联合敲除小鼠模型并不能很好的模拟帕金森病的主要病理特征。与小鼠相比，猪在解剖结构、生理特征和基因组上都与人更接近，因此，我们计划利用CRISPR/Cas9高效基因打靶工具，构建DJ-1、Parkin、PINK1三基因敲除小型猪，结合MPTP或鱼藤酮注射诱导，检测基因敲除猪中脑黑质和纹状体的病理学特征、多巴胺神经元的数量及凋亡、胶质细胞的增生及炎症因子分泌以及纹状体中酪氨酸羟化酶（TH）和多巴胺转运体（DAT）的表达水平等分子信号通路；通过矿场实验和步态分析基因敲除猪的帕金森疾病表型，为研究人类帕金森病的发病机理、药物开发和治疗提供合适的工具。

猪在解剖结构、生理特性等方面与人类非常接近，是研究人类疾病的理想模型动物。帕金森病（PD）是一种常见的神经退行性疾病，其确切病因还未得到完全阐释，推测与遗传因素、环境因素、年龄老化、氧化应激等相关。对家族性PD的研究结果发现许多基因的突变与PD相关，包括DJ-1，Parkin,PINK1等基因。虽然有多种基因修饰小鼠模型的报道，但均未能很好的重现PD病人的病理特征，这可能与小鼠的生理特性相关。本研究在我们前期研究工作的基础上，优化了CRISPR/Cas9介导的高效基因打靶平台，获得了DJ-1，Parkin，PINK1三基因均为双等位基因打靶的小型猪，并对基因敲除猪进行了扩繁；建立了检测猪中脑黑质和纹状体病理特征的技术方法及大动物（猪）行为分析系统；测定了基因敲除猪血清中的微量元素含量，为铁、铜等的沉积在帕金森病的发生发展提供参考；制备了NCSTN（γ外泌酶的一个亚基）点突变猪，分析了其疾病表型并解析了其相应的信号通路；创制了包含Mondini内耳畸形、瓦登伯革氏综合症2A型、小丑鱼鳞癣等在内的多种疾病模型猪；发现小胶质细胞自噬缺陷通过PDE10-cAMP信号激活NLRP3炎症小体而增加神经炎症，从而引起帕金森病样症状，并进一步探究了炎症小体激活的机制，发现造血细胞激酶（HCK）在NLRP3炎性小体激活中起关键作用。本项目将为人类帕金森病的发病机理研究和治疗策略开发提供良好的疾病模型。