基因靶向修饰AMSCs结合可控因子缓释三维支架移植复合体对PD模型功能性DA能神经网络重建的研究

The key gene targeted modification of DA neurons can boost the differentiation of AMSCs into DA neurons, which is considered as ideal seed cells for treatment. However, whether functional DA neural network could be reconstructed in brains of PD becomes a restrained factor for the further research. In this study, the key role of AMSCs gene in differentiation of transfected DA neural network was first investigated by perfusion-3D-parallel microreactor. To assure the most effective program of gene transfection, the efficient and rapid screen of DA neurons from AMSCs with high differentiation rate is developed through parallel screening system. With stratified perfusion 3D microreactor culture system, AMSCs with high differentiation rate and neural network constructing capacity were secondary screened by dynamically comparing the formation of synapses between high differentiation rate AMSCs and DA neurons in SNc. As the seed cells of gene targeted modification, the transplantation of AMSCs into CNS in PD models for DA neural network construction ensured the seed cells transplanting in animal models with high efficiency and functionality. The technology of 3D scaffold with stained releasing controllable factors as well as targeting gene modified AMSCs graft complexes with high differentiation rate and neural network constructing capacity were first introduced to achieve the controlling of differentiation in a certain value and direction during the transplantation of AMSCs in PD models, which is contributed to valuing the targeting gene modification. With the evaluations of DA neurons differentiation and treatment in vivo and in vitro from different angles, this project provided a variety of critical evidence of targeting gene modified AMSCs differentiating into functional DA neurons and reconstructing functional DA neural network in CNS of PD hosts. Further, it also established new ideas and possible technical supports to solve bottlenecks restricting the applications of clinical stem cells transplantation in treatment of PD, with crucial scientific and social values.

多巴胺（DA）能神经元关键基因修饰能提高脂肪间充质干细胞（AMSCs）向成熟DA能神经元分化率，被认为是治疗帕金森病（PD）的潜在理想种子细胞，其能否在PD模型脑内重建功能性DA能神经网络成为深入研究的制约因素。本项目首次通过平行筛选及复层培养三维灌注微生物反应器筛选高分化率/高神经网络构建能力的基因靶向修饰AMSCs；结合可控性外源因子缓释支架构建移植复合体，切实解决干细胞移植中定植及定向诱导分化难题；应用灵长类PD模型研究基因靶向修饰AMSCs在PD模型脑内的分化、转归，以及对PD模型功能性DA能神经网络重建的作用及机制。研究将为基因靶向修饰AMSCs分化为成熟的功能性DA能神经元、在PD宿主中枢神经系统重建功能性DA能神经网络提供关键证据与实验数据，为临床开展干细胞移植治疗PD提供新思路与可行技术支撑，解决制约干细胞临床治疗PD的瓶颈问题，具有重要的科学与应用价值

项目顺利完成了构建稳定表达外源基因的AMSCs细胞系及分化比率筛选工作，将Nurr1、Pitx3、Mash1、Lmx1a四种组合定量克隆至AMSCs中。结果显示Pitx3与Lmx1a共同转染组具有较好的分化潜能。并比较不同关键基因转染AMSCs体外构建功能性神经网络的能力。通过微流控芯片技术建立了体外NSCs/神经元网络模型；并对NSCs来源的神经元功能进行动态监测及评价，比较表达不同外源基因组合的AMSCs的神经网络形成能力。针对磁性氧化铁Ferumoxytol纳米粒子标记AMSCs的体外安全性，我们对细胞表面抗原、增殖、凋亡/死亡、损伤及三系分化作以全面评估，尤其对标记后淋巴细胞增殖情况做了评价，为体内移植hADSCs后的安全示踪提供前期实验依据。基本完成项目计划书内的研究内容。通过脑立体定位注射标记细胞的大鼠脑内MRI，采用脑立体定位注射法将HPF-hADSCs移植入SD大鼠脑内，分别对大鼠在移植标记细胞1 h，7 d，14 d，28 d后的MRI进行分析，结果显示，在移植标记细胞28 d的过程中始终能够检测到大鼠脑内移植细胞部位T2信号的降低；脑立体定位注射标记细胞的大鼠各组织切片普鲁士蓝染色，结果显示，脾脏中出现蓝染，而肝脏和肾脏均无明显的染色阳性现象；HPF标记细胞注射后淋巴细胞增殖变化，结果显示注射HPF标记的脂肪干细胞后淋巴细胞增殖能力较未标记组明显升高，具有统计学差异。