同型半胱氨酸通过DNA甲基化途径对神经干细胞增殖分化作用机制的研究

The aim of this study is to explore the epigenetic effects of homocysteine on neural stem cell proliferation and differentiation by DNA methylation. The relationship between homocysteine, DNA methylation and neural stem cell proliferation and differentiation will be studied by primary cell model and rat model with middle cerebral artery occlusion. Global methylation level of genome, DNMTs(DNA methyltransferases) activities, and gene expression level of three DNMTs after RNAi will be detected to elucidate which DNMT is important for DNA methylation status altered by homocysteine. The concentration of several metabolites related to the methyl cycle and gene expression level of DNMT will be measured to find why homocysteine can alter DNMT activities. The methylation level of some genes associated with notch signaling pathway and some markers related to neural stem cell proliferation and differentiation will by analyzed by DNA methylation PCR array and MeDIP-qPCR, and it will be helpful for us to find which genes by methylated modification play an important role during the proliferation and differentiation inhibited by homocysteine. Furthermore, the molecular mechanisms about neural stem cell proliferation and differentiation inhibited by homocysteine will be elucidated based on DNA methylation pathway. This study will provide fundamental basis for clarifying pathophysiological process of the nervous system diseases related to hyperhomocysteinaemia.

本研究将在同型半胱氨酸(Hcy)抑制体外培养神经干细胞(NSC)增殖分化前期工作的基础上，采用原代培养神经干细胞模型和大鼠局灶性脑梗塞模型，开展Hcy-DNA甲基化-NSC增殖分化三者之间的关联研究。通过测定基因组总甲基化水平、甲基转移酶(DNMT)总活性及RNA干扰三种DNMT基因表达，明确Hcy可改变哪种DNMT活性进而影响基因组甲基化状态；通过测定甲基循环相关代谢物的浓度及DNMT基因表达水平，找出Hcy改变DNMT活性的原因；运用Notch信号通路DNA甲基化芯片、甲基化DNA免疫共沉淀PCR法检测Notch通路相关基因及NSC增殖分化中特异标志物基因的甲基化水平，全面分析哪些基因甲基化的改变在Hcy抑制NSC增殖分化中扮演着重要角色；提出基于DNA甲基化途径Hcy抑制NSC增殖分化的分子机制。为从分子水平认识与高Hcy血症有关的神经系统疾病发生发展的病理生理过程奠定基础。

在许多神经疾病中，Hcy在脑中积聚，是潜在的神经毒素，但高Hcy增加疾病发病率并加重病情进展的具体机制尚不清楚。本研究采用原代培养神经干细胞NSCs模型和大鼠局灶性脑梗塞MCAO模型进行Hcy干预，结果显示：Hcy可抑制体外培养的原代NSCs增殖，并减少其向神经元方向分化。Hcy可增大大鼠脑梗死区的面积，损伤MCAO大鼠的学习记忆能力；促进神经细胞的损伤、凋亡，并可抑制体内NSCs增殖，抑制干细胞向神经元方向分化。同时体内外实验均发现Hcy能够降低DNA总甲基化水平，改变DNMT3a、DNMT3b和DNMT1蛋白表达及DNMTs酶总活性，降低甲基循环关键产物 SAM/SAH比值。双免疫荧光标记结果显示：Hcy干预后，在梗死区和海马区神经干细胞和神经元内均发现甲基化水平的改变，暗示DNA甲基化水平变化可能为Hcy抑制脑梗死后神经再生的重要机制。甲基化芯片及MeDIP-qPCR技术分析进一步发现，Hcy主要通过调控Notch1、Notch3、Nestin和GFAP的甲基化状态影响NSC增殖和分化。研究建立了Hcy-DNA甲基化-NSC增殖分化的三者之间直接关联，并从分子水平上阐明Hcy抑制NSC增殖分化的机制，为Hcy神经毒性作用的研究提供新的思路。