基底节特异性GCH1基因敲除小鼠模型的构建及多巴反应性肌张力障碍基底核通路异常的研究

Dopa-Responsive Dystonia (DRD) is a neurogenetic disease of children which presents the symptom of fluctuating dystonia. GCH1 gene is the primary disease-causing gene of DRD. GCH1 gene mutation leads to a decrease in the reduction or enzyme activity and the protein expression, thereby causing the basal ganglia dopamine synthesis of obstacles. Clear the mechanism of basal ganglia nucleus in DRD is the core problem of the exploration of the mechanism of dysfunction of DRD, especially the role of GCH1 gene mutation. The research urgently needs some kind of appropriate transgenic animal. The DRD transgenic animal model we have commonly used is deficient .The application of CRISPR/Cas9 transgenic technology is able to knock out target genes specifically, which is expected to build DRD mouse model successfully. Our team has brought in CRISPR/Cas9 transgenic mice in preliminary work and carried out the research which focuses on the influence of GCH1 gene mutation on basal ganglia neural pathway. we have knocked out GCH1 gene of mice, analyzed the number of dopamine neurons and nerve fibers in basal ganglia, the variation of neurotransmitter and neuroreceptor, as well as the change of nerve electrophysiology.

多巴反应性肌张力障碍（DRD）是好发于儿童期的的神经系统遗传性疾病，GTP环化水解酶Ⅰ（GCH1）是DRD的主要致病基因，GCH1基因突变导致其蛋白表达减少或酶活性下降，从而引起基底节区多巴胺、5-羟色胺等多种神经递质的合成障碍。DRD机制探索的核心问题在于明确基底节区各核团功能障碍的机制，特别是探明GCH1基因突变在其中的作用。DRD的基底核功能障碍及通路异常的机制研究需要动物模型来实现，但目前的模型仍不理想。本课题组近期引进了Cre依赖的Cas9 转基因小鼠，拟在此基础上构建基底节区特异性GCH1基因敲除小鼠模型，进一步围绕GCH1基因突变对基底节神经通路的影响这一核心问题展开研究，分析GCH1基因突变后，基底节区神经元、纤维束、神经递质、受体及电生理的改变，以明确GCH1基因突变的具体发病机制。

多巴反应性肌张力障碍（DRD）是好发于儿童期的神经系统遗传性疾病，GTP环化水解酶Ⅰ（GCH1）是DRD的主要致病基因，GCH1基因突变导致其蛋白表达减少或酶活性下降，从而引起基底节区多巴胺、5-羟色胺等多种神经递质的合成障碍。DRD的基底核功能障碍及通路异常的机制研究需要动物模型来实现，但目前的动物模型仍不理想。本项目先后探索了定点注射sgRNA靶向敲除Cre依赖的Cas9转基因小鼠模型及Cre-Lox条件敲除GCH1基因小鼠模型，分析基因敲除效率、病理变化及动物行为学改变，发现定点注射sgRNA小鼠基因敲除后GCH1基因及蛋白水平变化不明显，而Cre-Lox条件敲除GCH1小鼠脑组织RNA、蛋白含量明显降低，酶活性下降，引起多巴胺、去甲肾上腺素和5-羟色胺水平下降，旷场实验和步态分析发现小鼠存在运动能力下降。该研究构建的条件敲除GCH1基因小鼠模型，为进一步开展该病的发病机制及药物治疗研究提供了可靠的动物模型。