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。

远程低氧预适应是组织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，保护远隔的异位组织器官，但其机制尚不明确。外泌体携带各种生物信息，远程传递信号，可能参与了远程低氧预适应的保护机制。本课题成功构建UPR荧光报告系HSPA4-EGFP稳定表达的SH-SY5Y细胞系，并证明HSPA4-EGFP可直观实时反映UPR的变化情况。利用UPR荧光报告系HSPA4-EGFP，我们发现在低氧和低氧预适应中UPR反应的变化趋势不同，提示UPR反应可能参与低氧预适应的低氧保护作用。提取低氧以及低氧预适应后外泌体，进行外泌体的测序，测序结果显示低氧预适应后血管内皮的外泌体高表达信息物质miRNA-126。进一步研究证实miRNA-126能够靶向神经细胞中的DNMT3B，通过降低DNMT3B和整体甲基化水平起到低氧保护的作用。采用斑马鱼低氧模型，结合抑制剂和Cas9敲除技术，抑制和敲除外泌体释放关键酶nSMase2；注射外源低氧预适应后外泌体，初步证实外泌体参与低氧预适应远程保护效应，可能通过携带信息物质促进神经细胞抵御低氧刺激。本研究探索了UPR反应和外泌体在低氧预适应远程保护效应中的作用和可能机制，为低氧预适应在预防和治疗缺血缺氧疾病提供理论基础。