帕金森氏病成纤维细胞直接转化为多巴胺神经元的分子机理与移植治疗研究

Parkinson’s disease (PD) is one of the most common neurodegenerative disease caused by the selective loss of dopamine neurons in substantia nigra of the middle brain. Since no treatment could stop the disease progression, cell thrasplantation would have the great therapeutic potential for PD. The current cell sources of human neural progenitor cells (hNPCs) of fetal brain and human embryonic stem cells (hESCs) have the immuno-rejection and ethical issues. Even though the recently developed human induced pluripotent stem cells (iPS cells) by somatic cell reprogramming overcome the immune-rejection problem, the differentiated neural stem cells and dopamine neurons of iPS cells might contain some undifferentiated cells to produce the teratoma and carcinogenicity. Recent studies indicated that the human somatic cells could be directly reprogrammed to the neurons and dopamine neurons by overexpression of specific neural transcription factors. But the lentiviral vectors to express the neural transcription factors could be integrated into the genome of the transformed neurons and induce the carcinogenicity. In this study we are going to construct episomal plasmids to overexpress the dopamine neuron-specific transcription factors of Ascl1, Nurr1,Pitx3, Lmx1a, Ngn2 and Fox2a to directly reprogram the PD patients-derived fibroblasts to the dopamine neurns and attain the following aims: To characterize the regrogrammed dopamine neurons by RT-PCR, immuno-fluerescence, the high performance liquid chromatography (HPLC) and whole-cell patch clamp; To use the cDNA microarray, epigenetic modifications, whole-genome miRNA array, Real-time RT-PCR,immuno-precipatation (IP) and chromatin immunoprecipitation (ChIP) to explore the epigenetic molecular mechanisms of the directly regprogramed dopamine neurons; To tranplant the directly reprogrammed dopamine neurons to the 6-OHDA-induced rat PD model and study the therapeutic effects and mechanisms of dopamine cell transplantation by immuno-histochemistry, confocal microscope andbrain-slice patch-clamp to provide the theoretical and experimental evidence for cell based therapy of PD.

帕金森氏病（PD）是中脑多巴胺神经元变性凋亡引起的神经退行性疾病。目前细胞移植治疗PD应用的胎脑神经干细胞，胚胎干细胞和诱导多能干细胞具有免疫排斥，伦理学障碍和可能的致瘤性。 最近研究表明体细胞直接重编程可以转化为神经细胞和多巴胺神经元，但重编程所用的病毒载体整合到基因组可能产生致突变性。本研究构建非染色体整合载体表达Ascl1, Nurr1,Pitx3, Lmx1a, Ngn2 和Fox2a基因，将PD病人成纤维细胞重编程直接转化为多巴胺神经元并取得以下目的：通过RT-PCR, 免疫荧光抗体，多巴胺释放和单细胞膜片钳鉴定直接转化的多巴胺神经元；用cDNA基因芯片，表观遗传修饰，全基因组miRNA等探讨多巴胺神经元直接转化的分子机理；用共聚焦显微镜及脑片膜片钳等研究直接转化的多巴胺神经元移植到PD大鼠模型的治疗作用及作用机理，为PD病人的细胞移植治疗提供理论与实验依据。

帕金森氏病（PD）是中脑多巴胺神经元变性凋亡引起的神经退行性疾病。目前细胞移植治疗PD应用的胎脑神经干细胞，胚胎干细胞和诱导多能干细胞具有免疫排斥，伦理学障碍和可能的致瘤性。最近研究表明体细胞直接重编程可以转化为神经细胞和多巴胺神经元，但重编程所用的病毒载体整合到基因组可能产生致突变性。.我们筛选多种相关转录因子Ascl1, Nurr1, Pitx3, Lmxla, Ngn2 和 Fox2a基因，并筛选小分子化合物组合替代相应转录因子功能，减少外源转录因子数目进行诱导重编程，从而降低了致突变风险。PD病人皮肤成纤维细胞经过6-8周诱导直接重编程转化为神经元和多巴胺神经元，通过RT-PCR，免疫荧光抗体和单细胞膜片钳鉴定证实直接转化的神经元和多巴胺神经元；并用转录组学和甲基化分析等探讨神经元和多巴胺神经元直接转化的分子机理。.我们将成纤维细胞直接转化的神经元移植到6-OHDA诱导的PD大鼠模型，行为学分析显示大鼠运动障碍得到明显改善，移植细胞能与PD大鼠脑内神经细胞形成功能连接，为PD病人的细胞移植治疗提供理论与实验依据。.本研究共发表论文6篇，其中SCI收录4篇，发表论著1部，在国内外学术会议特邀发言3次，获得山东省科技进步奖1项，另有2篇论文正在准备中。