中枢神经系统细胞间经胞外体传递的信息交流失调对脑卒中所致血脑屏障通透性增加影响的研究

Cellular components of central nervous system (CNS) exchange signals distantly through secreting and receiving exosomes that contain regulatory proteins and microRNAs. This mechanism plays a pivotal role in maintaining homeostasis of blood-brain barrier (BBB)　and CNS normal functions. Exosomes exist in intercellular fluid and cerebrospinal fluid (CSF). However, due to technical and ethical limitations, serial sampling of CSF is rarely done. Using a new method developed in our lab, we are capable of sampling CSF from monkey cisterna magna in a serial pattern. Based on this new technique, we are able to analyze changing patterns of assorted proteins and miRNAs in CSF of monkeys insulted by ischemic stroke and reperfusion. Moreover, we can determine BBB permeability change in manner of time by calculating the albumin ratio between CSF and blood. Based on our preliminary data, here we propose to study the change of exosome miRNAs in CSF and its relationship with BBB permeability change. This study would help to elucidate how the pathophysiological disruption of exosome-dependent intercellular signal exchanges within CNS may alter the BBB permeability and corresponding stroke injuries. Moreover, this study would lay a foundation for developing new stroke therapies using engineered exosomes containing miRNA mimic or inhibitors of those key miRNAs determined in this proposed study.

中枢神经系统各细胞组份之间通过分泌和接收胞外体（内含微小RNA等调控性分子）实现远距离信息传递，协同维持血脑屏障稳态和其它中枢神经系统正常功能。胞外体存在于细胞组织间液和脑脊液中，由于研究工具和伦理的限制，对于胞外体本身及其所传递成分在脑卒中进程中的动态变化以及对血脑屏障调控影响的研究还严重缺乏。利用能在清醒状态下对恒河猴枕大池脑脊液长期采集的自创方法，我们实现了对脑缺血再灌注后恒河猴脑脊液中关键成分的动态检测，并且通过测定不同时间脑脊液和血液白蛋白比值实现了对血脑屏障通透性的动态检测。根据前期数据，我们拟通过对脑脊液中经胞外体传递的微小RNA进行动态分析，筛查与血脑屏障通透性相关的微小RNA分子，并通过体外氧糖剥夺试验探索因果关系。本研究将有助于阐明脑卒中后细胞间信息交流失调对血脑屏障通透性和脑卒中病生理进程调控的机制，并对今后开发利用人工胞外体传递特定miRNA的新治疗方法打下了基础。

该课题主要目标是通过检测恒河猴脑缺血再灌注后脑脊液中胞外体miRNA以及BBB通透性的变化，并探索其相互之间调控关系。在该课题的执行过程中，我们完善了基于微创介入手术的恒河猴缺血再灌注模型，血脑屏障通透性的时间和空间动态检测，脑脊液胞外体采集和富集等创新方法，完成了申报书中计划的绝大部分关键实验。我们的实验结果最后并未发现恒河猴脑脊液胞外体miRNA与BBB之间的任何相关关系，主要假说证伪。但是，于此同时，我们第一次在单只实验动物上，实现了BBB通透性变化的动态测定，同时证实了BBB通透性的动态变化及其与脑损伤体积的相关关系，并且再灌注后24小时BBB的通透性变化可以预测亚急性期（7天）的脑卒中预后。基于该项目所完善的BBB动态检测技术创新，我们拓展了BBB调控研究，并报导了抗生素导致的肠道菌群紊乱可以导致恒河猴BBB通透性的升高。在项目执行过程中所创新或者技术改进的新模型新方法，在产学研创新合作方面做出了很好成绩，进行了大量临床前新药评价服务，在市场中极具竞争力，为很多新药项目提供了坚实的技术支撑。