SHH缓释信号对Oct-4诱导星形胶质细胞向神经干细胞去分化的作用及分子调控机制

Neural stem cells-transplantation is hitherto regarded as a most perspective strategy for clinical therapies for multilevel neural injury and neurodegenerative disorders. 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 alternative cell pool to overcome the issue. Fascinatingly, Our recent studies evidenced that astrocytes (ASTs) can acquire self-renewal capacility and multipotential, and further express nestin. Concomitantly, our study also revealed that the occurrence of this event is due to the de-differentiation of ASTs to give rise to NSCs. More importantly, some key molecules such as sonic hedgehog (SHH) and FGF4released from the mechanically-injured ASTs actively contribute to the de-differentiation process. The SHH expression therein was significantly up-regulated in the ASTs after injury, and functioned as a key molecule to regulate the AST reprogramming. On the basis of this result, The aims of our present project are to systematically investigate the de-differentiation of rat ASTs transduced with single transcription factor Oct-4 into NSCs in combination of induction microencapsulated SHH. Meanwhile, The characteristics of NSCs derived ASTs were identified by using the morphology, cellular and biochemical assays, electrophysiology technology. Moreover, the molecular mechanism underlying the reprogramming of AST was revealed to stablish an efficient technology platform for the generation NSCs from ASTs. We will eventually develop an effective strategy for de-differentiation of ASTs to NSCs, which could contribute immensely to clinical applications of ASTs as a novel alternative to treat the central nervous system injury and the neural degenerative diseases. Up to date, an effort still remains in understanding of the molecular mechanisms underlying de-differentiation of ASTs. Thus, the present study will be undertaken to further elucidate the molecular mechanism by which ASTs directly de-differentiate into NSCs through signaling pathways. It may provide noval theoretical basis for elucidation of the molecular mechanism of ASTs dedifferentiation.

神经干细胞(NSC)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来源严重匮乏等问题使其成为再生医学应用的瓶颈。因此寻求理想的替代细胞尤为重要。我们前期研究发现：体外培养的星形胶质细胞（AST）损伤后能表达nestin并可获得NSC特征，同时实验证实AST发生重编程为NSC与其受损后释放的SHH和FGF4等分子诱导密切相关。其中SHH在AST损伤后表达显著上调，是AST重编程的关键调控分子。本项目以AST为切入点，利用细胞和分子生物学及电生理学等技术探讨AST在转录因子Oct4修饰后SHH缓释信号对其重编程为NSC的作用和调控机制，拟建立高效诱导AST去分化为NSC的新技术平台, 为临床采用自体AST源性的NSC治疗神经损伤及退变性疾病提供新细胞来源和治疗手段。目前AST去分化机制尚不清楚，本项目将探明AST逆分化为NSC的分子调控机制，为阐释AST重编程提供新的理论依据。

中枢神经系统(CNS)损伤以及神经退变性疾病治疗一直是当今国际神经医学领域最为棘手的科学难题。由于CNS结构组成的复杂性，在CNS损伤或退变发生后，进行性的动态病理变化所引起神经元变性、坏死是最终导致不可逆系统功能丧失及机体功能障碍的主要原因，迄今尚无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)被认为是当今治疗神经损伤和神经退变性疾病最具有应用前景的细胞，然而由于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。我们先前研究发现：当CNS受损后，在远离损伤区有许多星形胶质细胞（ASTs）可呈现nestin 阳性反应，经过培养可以获得具有自我更新和有一定分化潜能的 NSCs。进一步体外损伤实验证实ASTs能够发生重编程产生NSCs，而且ASTs重编产生 NSCs与SHH分子及转录因子调控密切相关。因此本项目拟以ASTs为切入点，利用细胞分子生物学、形态学及电生理学等技术探讨ASTs在pHIV-HB2病毒载体介导的转录因子Oct4修饰后SHH缓释信号对其重编程为NSCs的作用，进一步阐明SHH诱导Oct4介导ASTs重编程为NSCs分子调控机制，最终拟建立高效诱导ASTs重编程为NSCs新的技术平台。我们目前研究结果显示：单转录因子Oct4能有效诱导ASTs产生具有野生型NSCs形态特征、生化表型以及自我更新能力和多分化潜能的诱导型NSCs。进一步当诱导体系加入SHH，这一重编程效率和进程被显著提高，表现为产生NSCs的数量显著增加、生化表型、自我更新能力和分化潜能更接近野生型NSCs。最重要的是体外诱导后，这些ASTs源性的NSCs能够分化产生神经元和胶质细胞，而且分化产生的神经元具有野生型神经元的典型特征如突触形成、神经元的电生理活动及神经元特异性的钙成像。这种诱导ASTs发生细胞重编过程可能与Sox2/Shh靶向下游分子调控以及PI3K/Cdk2/Smurf2信号通路的共同调控密切相关。因此本项目研究有望为临床上细胞治疗采用自体细胞重编程产生NSCs提供重要的细胞来源，也为CNS损伤及神经退变性疾病治疗提供了新的治疗策略。