嗅鞘细胞协同限定性生长因子诱导人精原干细胞转分化为多巴胺神经元的作用及分子机制

Neural stem cells (NSCs) are hitherto regard as powerful perspective candidates for cell transplantation in clinical therapies for multilevel neural injury and degenerative diseases. However,the extreme shortage of NSCs available for transplantation still makes its therapeutic application a significant bottleneck in neural regenerative medicine.Therefore it is pivotal solution to seek an ideal cellular pool, which offers an alternative to further overcome the issue. Fascinatingly, Our recent studies evidenced that spermatogonial stem cell(SSC) line could transdifferentiate into DAergic(DA) neuronal cells under a special condition, namely a combination of olfactory enseathing cells(OECs) and defined factors such as GDNF, Shh, TGFβ3 and FGF8a, suggesting that SSCs could potentially become an acceptable alternative cellular resource. On the basis of this result, The aims of our present project are to systematically investigate transdifferentiation of human spermatogonial stem cells to DA neurons, to further optimize these protocols for the generation of DA neurons from SSCs to establish an efficient technology platform. Meanwhile, SSCs derived DA neurons were identified by means of the morphological characteristics and biochemical phenotypies. Concomitantly, the assessement of DA neurons in vitro and in vivo physiological functions was also conducted. We will eventually develop an effective strategy for transdifferentiation of human SSCs to DA neurons, which could contribute immensely to clinical applications of SSCs as a novel alternative to current neural stem cell source in treating PD, and also provide an alternative therapeutic solution for neural degenerative diseases. Furthermore, up to date an effort still remains in understanding of the molecular mechanisms underlying neural transdifferentiation of SSCs. Thus, the present study will be undertaken to further elucidate the molecular mechanism by which SSCs directly transdifferentiate into functional DA neurons through PI3K/AKT and Smad1/5/8 signaling pathways.It may provide noval theoretical basis for clarification of the molecular mechanism of SSCs transdifferentiation.

神经干细胞(NSC)迄今是治疗神经损伤和神经退变性疾病最具有应用前景的细胞，然而NSC来源严重匮乏等诸多问题使其成为神经再生医学应用的瓶颈。因此寻求理想的替代细胞显得尤为重要。基于我们前期研究，嗅鞘细胞(OEC)和Shh、FGF8a、TGFβ3 等因子能协同诱导小鼠精原干细胞(SSC)系转分化为功能性多巴胺(DA）神经元。本项目以人的SSC为切入点，采用OEC合并限定性生长因子诱导使其转化为DA神经元。从形态特征和生化表型对产生的DA神经元进行鉴定，进一步检测并评估它的体外和在体功能，拟建立高效诱导人SSC转分化为功能性DA神经元的技术平台，为临床上帕金森等神经退变性疾病的细胞治疗和组织工程提供重要的细胞来源和新的思路。目前SSC转分化的分子机制尚不清楚。本项目将探明SSC直接分化为功能性DA神经元的PI3K/AKT和Smad1/5/8信号转导通路，为阐释SSC跨谱系分化提供新的分子机理。

帕金森氏病PD引起的机体功能障碍是困扰临床工作者的难题，这种神经退变性疾病病理机制主要归咎于黑质纹状体系多巴胺(DA）能神经元不断退变缺失最终引起不可逆系统功能丧失，迄今为止没有切实有效的治疗手段，因此寻求有效的治疗手段迫在眉睫。神经干细胞(NSCs)被当今认为是治疗神经损伤和神经退变性疾病最具有应用前景的细胞，然而NSCs来源严重匮乏以及伦理等诸多问题使其成为神经再生医学应用的瓶颈。因此寻求理想的替代细胞显得尤为重要。我们先前的研究发现，SSCs具有其它干细胞没有的独特优势（来源容易，转分化潜能强，无致瘤性及免疫原性低等特点），在嗅鞘细胞(OECs)联同限定性因子Shh、FGF8a、TGFβ3 等能诱导下可使小鼠精原干细胞(SSCs)系转分化为功能性DA神经元。因此本项目拟以人的SSCs为切入点，采用OECs合并优化的限定性生长因子诱导使其转化为DA神经元，并从细胞形态特征和生化表型对产生的DA神经元进行鉴定，进一步检测并评估它的体外和在体的细胞学功能，最终拟建立高效诱导人SSCs转分化为功能性DA神经元的技术平台。与此同时探明SSCs直接分化为功能性DA神经元的PI3K/AKT和Smad1/5/8信号转导通路的分子调控机制。我们目前研究结果显示：采用OECs条件培养基联合DA神经元发生发育的过程中关键特化因子可诱导人的SSCs转分化为具有DA神经元形态特征和生化表型的细胞，并且产生多巴胺，表达突触素，获得神经元特有Ca+成像以及神经电生理活动。这一转分化过程可能与PI3K/AKT以及Smad信号通路的调控密切相关。最重要的是将它们移植到MTPT作用后PD模型动物的纹状体内，这些SSCs可以迁移并能分化为DA神经元细胞，而且能改善动物的功能缺陷如：爬竿犯错次数减少，前后肢动作协调性改善以及动物行动迟缓得等到纠正。因此本项目的研究为阐释SSCs跨谱系分化提供新的分子机理,同时也为为临床上帕金森等神经退变性疾病的细胞治疗和组织工程提供重要的细胞来源和新的思路。