组合诱导CiPSCs-NSC与DA神经元前体移植治疗帕金森病模型猪体内功能建立及调控机制研究

Parkinson's disease (PD) is a serious human central nervous system degenerative disease. In recent years, transplant of iPSCs to replace the loss of dopamine neurons, which has become the most suitable for PD treatment. In this study, we chose the wuzhishan miniature pig as experimental animals, and reprogramed the fetal pig fibroblasts to CiPSC derived NSCs-EGFP and CiPSC derived DA neural progenitor using a combination of pluripotent transcription factors and genes alternative small molecule compounds (such as: VC6TFZ/VC6T) under low oxygen conditions. Meanwhile, the epigenetic molecular mechanism of small molecule compounds and their combination control the reprogrammed cells would be evaluated; Through co-culture with PD cell model in vitro, the repair function and interaction mechanism of CiPSC derived NSCs-EGFP and CiPSC derived DA progenitors were analysized; The acute injury and chronic damage of PD wuzhishan miniature pig model were prepared by MPTP and 6-OHDA induction respectively. The CiPSC-derived NSCs-EGFP and CiPSC derived DA progenitors were transplanted into PD model nigra striatum, and normal NSC was served as control at the same time. Through exploring the DA differentiation, survival, migration, integration efficiency into the pig brain and synaptic connection between the host and the grafted cells, to evaluate the transplanting efficacy and choose the best transplanting donor and pathological animal model; And, to discuss the feasibility of the clinical application of CiPSC-derived NSCs-EGFP and CiPSC derived DA progenitors transplantation in the treatment of Parkinson's disease, and provide theoretical and practical basis, which could significantly promote the researches in this area.

帕金森病（PD）是一种严重影响人类健康的中枢神经系统退行性疾病。近年来，利用iPS细胞移植替代损失的多巴胺神经元，已成为医学研究的重要热点。本研究选用我国特有五指山小型猪作为实验动物，利用猪自身4种转录因子，同时组合使用基因替代性小分子化合物（如：VC6TFZ/VC6T）诱导猪胚胎成纤维细胞去分化为CiPSC-NSCs与CiPSC-DA 神经元前体，探析组合调控细胞重编程的表观遗传分子机理；通过与PD体外细胞模型共培养，分析其对PD细胞修复作用及互作机制；并尝试制备五指山猪急性损伤PD动物模型与慢性损伤模型，以CiPSC-NSCs-GFP+与CiPSC-DA前体-GFP+为移植供体，分别移植PD五指山小型猪黑质-纹状体，术后观察宿主运动能力恢复状况，对比观察供体细胞在宿主脑内的存活、分化多巴胺神经元及迁移情况，为组合诱导CiPSC-DA神经元前体移植治疗帕金森病和再生医学提供依据。

利用五指山猪自身4种再程序化因子，组合使用基因替代性小分子化合物（Repsox与CHIR99021），诱导五指山小型猪胚胎成纤维细胞重编程为CiPSCs，对小分子化合物Melatonin、Reversine、Repsox与VPA调控成纤维细胞重编程表观遗传学及分子机制进行系统研究，研究发现Reversine通过激活多能性干细胞因子OCT4与间充质干细胞因子CD29、CD44与CD73的表达，诱发成纤维细胞重编程。而RepSox 与VPA 通过激活BMP信号通路，抑制TGF-β信号通路，诱发细胞重编程。通过6-羟基多巴胺（6-OHDA）右侧内侧双点黑质前脑束毁损法制备帕金森病SD大鼠模型方法体系，初步进行帕金森猪PD动物模型研究。并建立体外定向分化为GABA能神经元前体（CiPSCs-iGABA）与原始神经上皮细胞（CiPSCs-NECs）的方法体系。分别定点移植MiPSCs-iGABA前体、CiPSCs-NECs至PD大鼠黑质-纹状体区，PD大鼠运动障碍明显改善，在大鼠脑内微环境中存活并分化为TH+、GABA+、PSD95+与GFAP+的细胞，MiPSCs-GABA可在宿主脑内存活至少32周，CiPSCs-NECs可存活至少13周，并与宿主脑形成突触联系，能够定向分化为多巴胺能、GABA能、Glu神经元与胶质细胞。这为MiPSCs-iGABA前体与CiPSCs-NECs作为治疗帕金森病的供体细胞提供可能，为帕金森病细胞移植临床应用奠定基础。项目负责人刘长青获评安徽省教学名师，郭俣获评安徽省线上教学名师，发表科技论文9 篇，其中SCI 收录6 篇，medline收录2篇，另投稿论文4篇。培养硕士研究生12人，指导大学生获9项国家级大学生创新训练计划项目，国家级生命科学竞赛一等奖2项，二等奖1项，三等奖2项，省级一等奖5项，省挑战杯二等奖2项，全国基础医学竞赛三等奖1项。