iNPC定向分化为胆碱能神经元的实验研究

Recent reports have demonstrated that sets of transcription factors are sufficient to convert mouse fibroblasts directly into other differentiated cell types, such as neural progenitor cells (iNPCs). We hypothesized that iNPCs may efficiently differentiate into cholinergic neuron by a novel combination of several transcription factors and small molecules, and have the function of cell therapy in the treatment of Alzheimer's disease. Toward this goal, we will optimize mouse embryonic fibroblasts derived from Nestin-EGFP mice with a set of several transcription factors, which highly expressed both in forbrain NPCs and during the development of cholinergic neuron. Then, we will detect whether the iNPCs have qualities of primary brain-derived NPCs (WT-NPCs), including the expression of NPC-specific marker genes, tripotent differentiation potential, mature neuron differentiation capability and physiological properties. Moreover, the function of small molecules would be investigated in the iNPCs-differentiation. These Qtracker565 labeled iNPCs will further transplanted into hippocampus of three-month-old C57BL/6 mice to test their survival ability, differentiation potential and behavioral changes during 4 weeks. Our project might suggest an alternative strategy for direct lineage reprogramming using target cell-type-specific transcription factors to induce NPC-like cells，which potentially could be used for autologous cell transplantation-based therapies in the brain.

针对当前人们关注的诱导性神经干细胞（iNPCs）治疗阿尔茨海默病（AD）的科学问题，本项目拟通过优化转录因子组合，将皮肤成纤维细胞转分化为iNPCs。进一步探讨在小分子化合物的作用下， iNPCs在体内、外分化为胆碱能神经元的效率。本研究针对iNPCs分化胆碱能神经元的影响因素，通过不同转录因子组合（决定细胞命运转录因子和胆碱能神经元特异性转录因子组合）转分化Nestin-EGFP小鼠皮肤成纤维细胞为iNPCs，进而筛选最佳转录因子组合。利用分子生物学、电生理技术等验证iNPCs的功能特点和与原代神经干细胞的相似性。进一步筛选小分子化合物，以提高iNPCs分化为胆碱能神经元的效率。将Qtracker565标记iNPCs，定点注射在C57BL/6小鼠海马区，明确iNPCs在体内定向分化为胆碱能神经元的影响因素。该研究为iNPCs治疗阿尔茨海默病提供了科学基础，也具有重要的研究意义和临床价值。

针对当前人们关注的诱导性神经干细胞治疗阿尔茨海默病的科学问题，本项目拟通过转录因子优化组合诱导皮肤成纤维细胞转分化为神经前体细胞，进一步探讨其分化为胆碱能神经元的效率和体内功能。本研究建立在转分化的基础上，通过转录因子优化组合（Sox2、Brn2、FoxG1），转分化Nestin-EGFP小鼠皮肤成纤维细胞，获得具有前脑特征的神经前体细胞。进一步，iNPC中过表达胆碱能神经元分化相关基因lhx8（l3）基因，能够显著提高iNPC向胆碱能神经元的分化效率，降低GABA神经元的分化效率。我们继续通过优化转录因子组合，将l3与其它转录因子Sox2、Brn2不同的组合感染入小鼠皮肤成纤维细胞中，已成功诱导不同转录因子组合的神经前体细胞。扩大培养细胞后，比较不同细胞系的增殖能力、NPC特异基因表达水平，表明诱导性神经前体细胞具有NPC相关特性。然而，iNPCs分化为星形胶质细胞、少突胶质细胞的分化特异性基因表达水平，较野生型神经前体细胞显著降低。目前，正在进行iNPCs的神经元分化实验，需进一步检测其分化为胆碱能神经元的效率。该研究为诱导性神经前体细胞治疗阿尔茨海默病提供了科学基础，也具有重要的研究意义和临床价值。