帕金森氏病GBA基因突变诱导多能干细胞模型建立及致病机理研究

Parkonson' disease (PD) is the neurodegenerative disease caused by the selective loss of dopamine neurons in the substantia nigra of the middle brain. Recent genetic studies have identified genetic mutations in several genes for Parkinson's disease and suggested that the mitochondria dysfunction is one of the important mechanisms in the pathogenesis of PD. However the molecular mechanisms underlining the pathogenesis of PD are largely unknown because the dopamine neuron loss was rarely shown in the transgenic and knockout animal models. Previously we identified mutations in the Nurr1, PARL and GBA genes for patients with PD. In this project we will collect skin fibroblasts from PD patients with mutations in the GBA gene，use the somatic reprogramming of the episomal plasmids to generate induced pluripotent stem cells (iPS cells) and differentiate the iPS cells to neural precursor cells and dopamine neurons. Then we will use the cell culture, cDNA Microarray，RT-PCR, Western blot, immuno-fluorescence and cell patch-clamp techniques to study the functions of iPS cell-derived dopamine neurons and the molecular mechanism of the mutations in the GBA gene on the mitochondria dynamic pathways as well as to provide the human PD model for screening the therapeutic drugs . In addition we will use the gene targeting technique of the Zinc Finger Nucleases to correct the mutation in the GBA gene and explore the iPS cell-based transplantation therapy for PD.

帕金森氏病（PD）是由于中脑多巴胺神经元凋亡引起的神经退行性疾病。近年来的分子遗传学研究鉴定出多个基因与PD发病有关并且提示线粒体功能障碍是PD基因突变的主要致病机理之一。但由于转基因动物和基因敲除动物模型极少能呈现PD的特异性多巴胺神经元凋亡，无法阐明PD基因突变的致病机理。我们以前的研究鉴定出PD病人的Nurr1，PARL和GBA基因突变。本研究采用体细胞重编程技术建立GBA基因突变的PD病人诱导多能干细胞模型。 通过细胞培养，全基因组cDNA芯片分析,RT-PCR, Western blot,免疫荧光及膜片钳技术，从细胞和分子水平研究GBA基因突变对多巴胺神经元发育及线粒体信号通路影响的机理。同时我们将用锌指核酸酶基因打靶技术修复诱导多能干细胞的GBA 基因突变，探讨诱导多能干细胞对PD动物的移植治疗作用，为筛选PD药物和自体移植治疗提供理论和实验依据.

帕金森氏病（Parkinson's disease，PD）是中脑多巴胺神经元凋亡引起的神经退行性疾病。转基因和基因敲除动物模型极少能呈现 PD 特异性多巴胺神经元凋亡，无法阐明 PD 基因突变的致病机理。本项目主要内容包括：1） 筛选PD病人GBA基因突变位点；2）利用非整合质粒载体技术，将PD病人(GBA突变)和正常人的皮肤成纤维细胞重编程为诱导多能干细胞（iPS）；3）iPS细胞的定向神经分化及神经元功能分析；4）利用CRISPR Cas9基因编辑技术对iPS细胞GBA基因进行校正，分析多巴胺神经元损伤机理；5）探讨iPS细胞来源NSCs对 PD 大鼠的移植治疗作用及机理。本项目通过体细胞重编程，cDNA 芯片，RT-PCR，Western blot，膜片钳，细胞移植，CRISPR Cas9校正等技术，成功建立PD病人和正常人诱导多能干细胞库，分析GBA 基因突变对PD多巴胺神经元发育的影响，为筛选 PD 药物和自体移植治疗提供理论和实验依据。