C16肽联合Reg-2修饰的骨髓间充质干细胞移植在脑缺血后的神经修复机制

It is a pivotal task to explore more effective ischemic neuronal protective mechanism and new therapeutic method for treating cerebral ischemia. The transplantation of bone marrow mesenchymal stem cells (BMSCs) is one of the most potent measures to repair ischemic brain injury. However, the lack of nutritious support, inflammatory reactions and immune responses to the transplanted cells will lead to the implanted cells death. Previous research showed that BMSCs have protective effects on cerebral ischemic injury. In this study, we combine established Reg-2 lentivirus with C16 peptide, which owns anti-inflammatory property to solve the crucial aporia to increase the survival rate of implanted cells for cell replacement therapy. We are planning to do in vitro experiments with C16 peptide, which may conduct its protective role on cerebral ischemia by influencing vascular and inflammatory mediators. And cell culture, immunofluorescence, RT-PCR, confocal, magnetic resonance imaging methods are employed in this study to observe the expression of Reg-2 BMSCs growth, proliferation, differentiation and apoptosis, autophagy to explore the neuroprotective effects of Reg-2 BMSCs. Therefore, we illuminate the ischemia repairing function and mechanism of Reg-2 modified neural BMSCs transplantation after focal cerebral. Moreover, we aim to investigate the regulatory mechanism of C16 and BMSCs transplantation via PI3K/Akt and TLR4 signal transduction pathway on ischemia reperfusion injury. Moreover, this study can provide a theoretical basis for searching innovative therapies and new drug targets of cerebral ischemia injury.

探索更有效的脑缺血治疗新方法是目前缺血性卒中研究的迫切任务，其中骨髓间充质干细胞移植（BMSCs）技术最具潜能，但营养缺乏、炎症反应以及宿主的免疫排斥等导致BMSCs植入后大量死亡。为此，在前期已构建Reg-2慢病毒基础上，本研究将通过构建Reg-2修饰的BMSCs，探索解决BMSCs在脑缺血治疗中增加存活和增殖等难题。本项目首先通过体外实验，联合具有抗炎作用的C16肽，采用细胞培养、免疫荧光、RT-PCR和激光共聚焦成像等方法，研究过表达Reg-2的BMSCs增殖、分化、自噬和凋亡等情况，探索该细胞的神经保护作用；其次阐明Reg-2修饰的BMSCs移植在脑缺血小鼠神经修复的细胞和分子机制，并通过磁共振成像和行为学评估，研究其疗效；同时探讨C16和BMSCs移植对脑缺血损伤过程中PI3K/Akt和TLR4信号转导通路的调控机制。项目成果同时为脑缺血损伤的机制研究新药开发提供依据。

缺血性脑卒中目前缺乏有效治疗方法。脑内骨髓间充质干细胞移植（BMSCs）后小胶质细胞活化和炎症反应等影响植入细胞存活。本研究聚焦脑缺血治疗中增加移植细胞存活难点，探索脑内定位注射过表达Reg-2的BMSCs联合C16肽等抗炎症、抑制脑内自噬活动和小胶质细胞活化的神经修复机制。利用自噬缺陷和小胶质细胞敲除转基因小鼠，建立离体和在体缺血模型，采用细胞培养、免疫荧光、RT-PCR、磁共振成像、行为学评估和双光子活体成像等方法，开展BMSCs移植联合C16肽，雷公藤甲素（TP）和小胶质细胞特异性抑制剂Ki20227预处理，分析BMSCs移植后脑内自噬状态和炎症相关信号通路变化，阐述了BMSCs移植和TP干预调控脑缺血后小胶质细胞自噬和活化、神经元突触可塑性的神经保护机制。C16和BMSCs移植对脑缺血损伤过程中炎症信号转导通路的调控机制以及TP调控小胶质细胞的具体机制为脑卒中的进展研究提供非常直观的依据。项目成果同时为脑缺血损伤的机制研究新药开发提供依据。