错构素及其DNA甲基化在低氧耐受中作用的研究

Brain Hypoxia/Ischemia preconditioning was a kind of endogenous neurve adaptation which induced by sublethal hypoxia/ischemia and its mechanism is still unclear. A new study reported that hamartin was up-regulated by ischemic preconditioning and was unaffected by ischemia in hippocampal CA1 neuron. Hamartin worked as endogenous neuroprotective molecule in brain. The mechanism of its expression in ischemic preconditioning was unknown. DNA methylation play an improtant role in regulation the genes expression in nerve system. There was a potential CpG island in the promoter of TSC1(hamartin) and which may affect the expression of hamartin. It is unknow whether the expression of hamartin induced by ischemic preconditioning via the change of DNA methylation in its promoter.So it is.essential to reveal the the change of DNA methylation in promoter of TSC1 during the process of ischemic preconditioning.Using hypoxic preconditioning mouse model which was treated with 5-Aza,we want to investigate the role of DNA methylaton in the expression on hamartin induced by hypoxic preconditioning. In order to further clarify the mechanism of DNA methylation in promoter of hamartin induced by hypoxia preconditioning，its effect was also supported by cell culture in which three kinds of DNA methyltransferase and hamartin were over-expressed or knocked-out. This study is important for understanding the mechanisms of neuroprotection of hypoxia preconditioning.

脑缺血/低氧预适应(I/HPC)是神经细胞对缺血/低氧刺激产生的一种内源性保护措施，到目前为止其机制尚未明确。研究显示错构素（hamartin）在海马CA1区神经细胞IPC时表达上调而在单纯缺血时不变，是神经保护的内源性机制之一，但其表达改变的原因尚未清楚。DNA甲基化在调节基因表达中有重要作用，错构素的启动子区有潜在的DNA甲基化位点并可以调节其表达。I/HPC是否是通过DNA甲基化来调节错构素的表达来参与I/HPC的形成，目前尚未见报道。因而有必要对错构素及其DNA甲基化在I/HPC过程中的变化进行深入研究。拟利用已建立的HPC动物（分离其CA1和CA3组织）和细胞模型，应用基因过表达技术及Crispr/cas9基因编辑技术等技术，并辅以DNA甲基转移酶抑制剂5-Aza，明确错构素表达变化及其启动子区的DNA甲基化在表达改变的作用，本研究将进一步丰富对脑缺血/低氧内源性机制的认识。

低氧/缺血是临床和高原高空等极端环境作业常见的生理/病理过程，严重威胁人类健康。缺血/低氧预处理（Ischemic/Hypoxia preconditioning, I/HPC）是指通过亚致死性的缺血/低氧刺激，可以增加组织或器官对随后致死性刺激的耐受，是一种由内源性细胞保护机制。大脑是对低氧/缺血最为敏感的器官，其中海马是中枢神经系统中一个重要的脑区，其中海马CA1区和CA3脑区对低氧/缺血耐受的差异，报道显示缺血预适应可以诱导CA1区hamartin表达参与神经保护，但其分子机制到目前仍未十分清楚。.本研究利用低氧预适应小鼠和神经细胞模型，将动物和细胞分为对照组、低氧对照组和低氧预适应组，通过real time-PCR、Western Blot和免疫荧光等方法探究低氧预适应中TSC1（hamartin）在小鼠海马CA1和CA3区的表达及对与TSC2和其下游mTOR/自噬通路的影响。为了证明TSC1的作用，在体外过表达或敲除小鼠海马HT22细胞中的TSC1基因后，给予低氧处理，通过对细胞活性和细胞凋亡的检测证明TSC1的神经保护作用。由于在低氧条件下，DNA甲基化对基因表达有重要的调节作用，且TSC1启动子区符合CpG岛特征。本研究通过重亚硫酸盐修饰后测序PCR（Bisulfite sequencing PCR，BSP）对小鼠海马CA1和CA3区TSC1基因启动子（-360 bp～-720 bp）区域进行CpG岛甲基化测序，观察低氧预适应能否通过影响TSC1基因启动子区CpG岛甲基化的改变进而影响TSC1的表达；为了进一步证明DNA甲基化对TSC1的影响，通过体内外给予DNA甲基化抑制剂5-Aza-CdR，观察5-Aza-CdR对TSC1及mTOR/自噬的影响，进一步证明DNA甲基化对TSC1表达的意义。.本研究得出以下结论：1. 低氧预适应可上调小鼠海马CA1区TSC1表达，并通过抑制通过mTOR/ p70S6K活性、增加自噬活性，进而增加CA1区神经细胞的对低氧的耐受； 2. TSC1在小鼠海马CA1和CA3区表达量的不同及对低氧的反应不同提示：TSC1的差异表达可能是CA1和CA3区神经细胞低氧耐受差异的机制之一； 3. 低氧预适应条件下，TSC1的表达量可能与TSC1启动子（-360 bp～-720 bp）区域的10, 21, 22号CpG位点呈现甲基化改