利用斑马鱼低氧模型研究外泌体在低氧预适应中的作用及其机制

Hypoxic preconditioning is a kind of endogenous protective mechanism induced by sublethal hypoxia. The effects of hypoxic preconditioning are broadened remotely to protect different organ and tissues in other regions, which is called as remote hypoxic preconditioning. However, the mechanism of the remote hypoxic preconditioning is still unexplored. HPC can increase the number of misfolded proteins, and these misfolded proteins can trigger neuroprotection. Exosomes can carry biological informations and transmit signals remotely. Thus we speculate that exosomes may carry biological informations such as misfolded proteins and initiate the remote hypoxic preconditioning. This study will inhibit and knock out the key enzyme nSMase2 using inhibitors and Cas9 technology, and inject exogenous HPC exosomes in zebrafish hypoxia model, in order to explore the role of exosomes in HPC remote protection. Secondly, the UPR fluorescence reporter system are constructed and the exosomes after HPC will be extracted and injected into hypoxic zebrafish to explain whether the exosomes carrying misfolded proteins to initiate remote hypoxic preconditioning. Lastly, exogenous and endogenous exosomes labeled by MemBright might be tracked to provide a high resolution description of their transfer and uptake physiological processes in hypoxic and hypoxic preconditioning zebrafish. This study will further enrich the role and mechanism of exosomes in the hypoxic preconditioning remote protection effect, and provide a theoretical basis for the prevention and treatment of ischemia and hypoxia diseases.

低氧预适应（HPC）是机体对低氧刺激产生的一种内源性保护机制，可产生多器官、交叉联动的效应，对远隔的异位组织器官产生保护，即远程低氧预适应，但其分子机制尚不明确。HPC可导致错误蛋白增加，这些错误折叠蛋白可以触发神经保护。外泌体可远程传递信号，据此我们推测外泌体携带错误折叠蛋白等生物信息，启动了远程低氧预适应。本课题采用斑马鱼低氧模型，结合抑制剂和Cas9技术，抑制和敲除外泌体释放关键酶nSMase2，注射外源HPC后外泌体，探讨外泌体在HPC远程保护效应中的作用；其次构建UPR荧光报告系并提取HPC后外泌体，注射入低氧斑马鱼，阐述外泌体是否携带错误折叠蛋白启动远程低氧预适应；第三标记外泌体，全时空追踪并解析内源和外源外泌体在斑马鱼体内的运行、摄取、靶细胞和命运等细胞学机制。本研究将进一步丰富外泌体在低氧预适应远程保护效应中的作用和机制，为低氧预适应预防和治疗缺血缺氧疾病提供理论基础。