GDNF修饰的嗅鞘细胞联合小分子诱导大鼠SSCs转分化为功能性脊髓神经元的作用及分子调控机制

Neural stem cells (NSCs) are hitherto regard as powerful perspective candidates for cell transplantation in clinical therapies for multilevel spinal cord injury and fucntion restoration. 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 reservoir as an alternative to further overcome the above-mentioned issue. Fascinatingly, Our recent studies evidenced that spermatogonial stem cells(SSCs) and C18-4 cells （SSC cell line） could transdifferentiate into spinal cord (SC) neural cells under a special condition, namely a combination of GDNF -modifed olfactory enseathing cells(OECs) and defined bio-active molecules such as RA, IBMX and TGFβ3, etc, 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 rat SSCs to SC neurons, to further screen most optimal approach for the generation of SC from SSCs to establish an efficient technology platform. Meanwhile, SSCs derived SC neurons were identified by means of the morphological characteristics and biochemical phenotypies. Concomitantly, the assessement of SC neurons in vitro and in vivo physiological functions was also conducted. We will eventually develop an effective strategy for transdifferentiation of rat SSCs to SC neurons, which could contribute immensely to clinical applications of SSCs as a novel alternative to current NSC source in treating SCI, and also provide an alternative therapeutic solution for neural degenerative diseases. Furthermore, up till now 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 possible molecular mechanism by which SSCs directly transdifferentiate into functional SC neurons through PI3K-Wnt/Catenin and Smad1/5/8 signaling pathways.This present project may provide noval theoretical basis for clarification of the molecular mechanism of SSCs transdifferentiation.

神经干细胞（NSC）目前是治疗脊髓损伤实现功能恢复最具有应用前景的细胞，然而NSC来源匮乏及伦理等诸多问题使其成为再生医学应用的瓶颈。因此找寻最佳的替代细胞尤为重要。基于我们前期研究，GDNF修饰的嗅鞘细胞(OEC)和RA、IBMX等小分子能有效地诱导大鼠精原干细胞(SSC)及C18-4细胞系转分化为脊髓神经元。本项目拟以大鼠SSC为切入点，采用GDNF修饰的OEC联合限定性小分子诱导其转分化为脊髓神经元。从细胞形态特征和生化表型对产生的脊髓神经元进行鉴定,进一步对其体外和在体生物功能进行检测和评估，拟建立高效诱导SSC转分化为功能性脊髓神经元的技术平台，为临床上应用细胞治疗脊髓损伤和组织工程提供重要的细胞来源及新思路。迄今SSC转分化为神经元的分子机制尚不清楚。本项目将探究SSC直接分化为功能性脊髓神经元的PI3K-Wnt/Smad信号转导通路，为阐明SSC跨谱系分化提供新的分子机制。

脊髓损伤（spinal cord injury，SCI）是目前一种致残率极高的中枢神经系统创伤性疾病，也是困扰临床工作者的难题，迄今仍然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。尽管目前一些治疗手段可以改善、纠正神经损伤退变的微环境进而实现神经元再生和系统修复及功能恢复，但这些手段无法恢复由于神经元退变缺失而导致的系统功能障碍。目前采用植入干细胞、刺激神经组织中潜在的干细胞分化替代受损的、变性的、退变的神经元形成局部环路来修复由于各种的损伤造成不可逆的神经功能障碍成为研究热点。相比之下，精原干细胞(SSCs)独具的功能特性——干细胞特性、无可比拟的遗传放大效应以及本身的优点（来源容易，转分化潜能强，无致瘤性、免疫原性低等特点）使其有望成为最具有临床应用前景的治疗细胞。我们前期研究发现，GDNF修饰的嗅鞘细胞(OECs)条件培养基和RA、IBMX等小分子能有效地诱导大鼠 SSCs及其干细胞系C18-4细胞可转分化为脊髓神经元。因此本项目拟以大鼠SSCs为切入点，采用GDNF修饰的OECs联合限定性小分子RA、Shh、IBMX、FGF4、TGFβ3等可将其诱导转分化为脊髓神经元。随后对分化产生的神经元进行细胞形态特征和生化表型鉴定，并结合体外和在体生物功能进行检测和评估，拟建立高效诱导SSCs转分化为功能性脊髓神经元的技术平台。最后进一步探讨SSCs转分化脊髓神经元在过程中相关分子调控机制及下游分子靶位的调控作用。我们目前研究结果显示：应用GDNF修饰的嗅鞘细胞的条件培养基可将大鼠SSCs转分化为功能性脊髓神经元以及胶质细胞，而且这些SSCs转分化产生的神经元已经具有野生型CHAT、CGRP、AchE神经元生化表型和细胞功能特性，将预诱导的SSCs到移植SCI动物后可改善其功能缺陷，表现为BBB评分、爬坡实验、SEP以及MEP相关参数都有所改变。这一转分化过程可能与PI3K/AKT/Wnt以及Smad1Smad1/5/8激活及下游通路调控因子Cip1及细胞周期退出的靶分子Nolz1和Smurf2 的变化变化密切相关。本研究为临床上应用SSCs治疗脊髓损伤和组织工程提供重要的细胞来源及新思路。