体内重编程的神经干细胞对脑出血模型大鼠的脑保护作用及其机制研究

Intracerebral hemorrhage(ICH), which is lacking of effective treatments, has higher lethality, disability rate and recurrence rate. Recently, we have found that transplantation of induced pluripotent stem cells (iPSCs) can enhance the functional recovery in rats with experimental ICH(81301007). Induced neural stem cells (iNSCs) that directly reprogrammed from various adult somatic cells possess more potentials than iPSCs for autologous cell transplantation in treating ICH, because they have shorter induction time and less tumor risk than that of iPSCs. In this proposal, firstly, rat astrocytes would be directly reprogrammed into iNSCs in vitro by definited transcription factors and exogenous genes with an episomal HSV-1-derived amplicon vector which we have successfully constructed before. Subsequently, these iNSCs would be implanted into the rat ICH models to assess their neuroprotective effects on brain damage after ICH, and comprehensively analyze the corresponding molecular and cellular mechanisms from multiple perspectives such as neural cell replacement, neurotrophy, neuroprotection and anti-inflammation. Eventually, iNSCs would be induced directly in the brain of ICH rats and followed with assessment of their possible therapeutic effects. The aims of this study are to explore the therapeutic effects and mechanisms of iNSCs that are reprogrammed both in vivo and in vitro on experimental ICH. These new insights into the therapeutic effects and corresponding mechanisms of iNSCs may further facilitate their autologous transplantation becoming a valuable strategy for treating hemorrhagic stroke.

脑出血（ICH）的致死率、致残率和复发率均较高，并缺乏有效治疗手段。我们在发现诱导多能干细胞（iPSCs）移植能改善ICH模型大鼠神经功能缺陷的基础上（81301007），本项目拟探讨在个体化治疗方面具有更大应用潜力的、组织类型特异干细胞直接重编程技术诱导的神经干细胞（iNSCs）对实验性ICH的脑保护作用。我们在前期构建的非整合单纯疱疹病毒（HSV）扩增子载体的基础上，拟首先利用该载体携带特定外源基因体外将大鼠星形胶质细胞直接重编程为iNSCs；随后评估iNSCs移植对ICH模型大鼠脑损伤的保护作用，并从神经细胞替代、神经营养、神经保护和免疫调节等角度综合分析其分子与细胞机制；最后在实验性ICH大鼠脑内直接重编程iNSCs并评估其可能疗效。本项目通过探讨直接重编程的iNSCs对实验性ICH的神经保护作用及其可能机制，为利用ICH患者自体iNSCs进行个体化细胞治疗奠定相关理论及技术基础。

脑出血（intracerebral hemorrhage，ICH）具有较高发病率、致死率、致残率，且缺乏有效特异性的治疗措施。目前影响ICH内科治疗的关键因素是如何改善由氧化应激和血液毒性产物等造成的级联神经损伤，以及受损的功能性神经元的再生与替代。在本课题研究中我们以腺相关病毒（adeno-associated virus，AAV）为载体，在体内外星形胶质细胞中特异性过表达NeuroD1基因，完成星形胶质细胞向诱导型神经细胞（induced neural cells, iNCs）原位转化这一过程，并利用单细胞测序技术对不同时间点ICH纹状体组织进行分析，我们观察到：①ICH模型中星形胶质细胞过表达NeuroD1后可逐渐出现神经元标志物NeuN的表达；②单细胞测序分析提示ICH后小胶质细胞可分化为不同功能的细胞亚型，在抗炎、促炎、细胞死亡方式、神经营养、轴突导向等方面发挥不同作用，并影响星形胶质细胞发育、活化与分化；③ICH后星形胶质细胞呈现不同功能亚型，参与ICH后炎症反应、吞噬作用、神经细胞营养、突触导向等发挥重要作用。④本课题其他相关研究发现急性ICH患者外周血中性粒细胞与淋巴细胞比值、淋巴细胞与单核细胞比值与ICH患者3月预后明显相关。本课题发现在急性ICH模型中星形胶质细胞中过表达NeuroD1可诱导其向神经元转化，后续我们将继续探索iNCs转化对ICH后的免疫调节及神经保护的影响，对转化而来的神经元功能及替代作用进行评价。本研究还对小鼠ICH模型进行了单细胞转录组分析，对ICH后免疫应答过程、免疫细胞与神经细胞的交互作用以及星形胶质细胞在ICH后发挥的作用有了进一步认识，为后续探索iNCs转化过程、提高iNCs转化率等提供了理论依据，将可能促进个体化iNCs在急性ICH中的临床应用。