小窝蛋白-1介导裸鼹鼠海马神经元低氧耐受的作用及机制研究

Hypoxia and ischemia are always life-threatening among central nervous system diseases (CNS) and associated with poor clinical and functional outcomes. The responses of Naked mole-rat(NMR) CNS to hypoxia is extremely complex and is not fully understood. Thus, a thorough understanding of the molecular mechanisms that control the CNS hypoxia tolerance would be of great significance to develop hypoxia enhancement therapies for hypoxic-ischemic encephalopathy. In this study, cav-1 was screened by the transcriptome sequencing within the NMR hippocampus after the treatment in hypoxia conditions (O2, 3%) for 12hrs. Immunocytochemistry showed that cav-1 protein levels in NMR hippocampal neurons were significantly decreased during hypoxia conditions. Down-regulation of cav-1 by siRNA resulted in the significantly increasement of hippocampal neuron apoptosis during hypoxia, which were detected by Flow cytometry analysis and Western blotting. These primary results indicated that cav-1 may play critical roles in regulating NMR hippocampal neuron hypoxia tolerance. In this proposal, we aim 1) to further confirm cav-1 protein levles in regulating NMR hippocampal neurons in culterd cell hypoxia model and NMR hypoxia model. And investigate roles of cav-1 in regulating NMR hippocampal neuron hypoxia tolerance.2) to explore the function and mechanisms of cav-1 in NMR hippocampal neuron hypoxia tolerance. 3）to study the expression of cav-1 protein levels in ischemia lesions.Various techniques including immunostaining, lentivirus mediated gene over-expression and down-regulation, nuclear-cytoplasmic fraction extraction, flow cytometry, electrophysiological observation will be used to examine the hypoxia tolerance and mechamisms of NMR hippocampal neurons. This study will reveal the role and mechanisms of cav-1 in regulating NMR hippocampal neurons, which will provide novel therapeutic targets and strategies for the treatment of CNS hypoxia and ischemia diseases.

中枢神经系统缺血缺氧性损伤广泛存在且缺乏有效的防治手段。裸鼹鼠作为一种天然适应低氧的啮齿类动物，其独特的中枢耐低氧特性在生物医学领域有着重要的应用前景，但是其中的生物学机制尚不明确。本课题组利用转录组测序获得了低氧情况下仅在裸鼹鼠海马中表达显著上调而在小鼠海马中无显著差异的分子cav-1。流式细胞仪检测发现，敲减cav-1导致低氧情况下裸鼹鼠海马神经元凋亡水平显著升高，据此提出cav-1介导裸鼹鼠海马神经元低氧耐受的研究假设。本研究将利用裸鼹鼠及小鼠海马神经元、海马组织脑片、低氧刺激的整体模型等，结合人类缺血缺氧性脑病样本，采用慢病毒介导的基因过表达或干扰等分子生物学方法，采用细胞形态学、生化分析、流式细胞术、激光共聚焦成像及钙成像、电生理等检测手段，详细证明上述科学假设并阐明内在的分子机制，有望为中枢神经系统缺血缺氧性疾病防治提供新靶点和思路。

中枢神经系统缺血缺氧性损伤广泛存在且缺乏有效的防治手段。裸鼹鼠作为一种天然适应低氧及高二氧化碳环境的啮齿类动物，但是其中的生物学机制尚不明确，其独特的中枢耐低氧特性在生物医学领域有着重要的应用前景。本项研究期望揭示裸鼹鼠适应低氧环境的生理机制，为解决缺血缺氧性疾病的防治及低氧环境下的生物防御措施提供重要线索。. 利用转录组测序筛选到低氧刺激时裸鼹鼠脑内特异性高表达的分子——Caveolin-1(Cav-1)，这是一个在中枢神经系统具有促存活作用的跨膜支架蛋白。为了阐述Cav-1在裸鼹鼠低氧耐受中的生物学作用，首先利用免疫光染色及Western blotting检测发现，Cav-1在裸鼹鼠海马神经元经历低氧刺激时表达显著上调。利用免疫荧光染色、Western blotting及流式细胞术检测发现，在裸鼹鼠神经元中过表达Cav-1能显著促进存其在低氧情况下的耐受能力，而敲除Cav-1则导致低氧情况下裸鼹鼠海马神经元凋亡率显著升高。此外，我们将裸鼹鼠来源LV-Cav-1转染至小鼠海马神经元中进行低氧刺激发现，裸鼹鼠Cav-1可以显著降低低氧情况下小鼠海马神经元的凋亡水平。此外，在裸鼹鼠海马内注射Cav-1过表达或shRNA，然后再进行整体动物低氧刺激，结果发现，Cav-1敲除导致低氧情况下裸鼹鼠海马神经元凋亡水平显著升高，而在小鼠海马中过表达裸鼹鼠来源Cav-1则显著降低了小鼠海马神经元的凋亡水平。进一步利用行为学检测发现，低氧刺激后，过表达Cav-1的裸鼹鼠空间学习记忆能力不受影响，而敲除Cav-1的裸鼹鼠空间学习记忆能力显著降低。低氧刺激后，与对照组相比，在小鼠中过表达Cav-1之后则能提高小鼠的空间学习记忆能力。本研究还初步揭示了HIF-1α调控Cav-1来介导裸鼹鼠海马神经元低氧耐受，为今后进一步探索裸鼹鼠Cav-1介导其海马神经元低氧耐受的机制指明了方向，也为寻找人类缺氧性疾病的基础医学研究提供了潜在的重要的线索。