基于Notch 信号通路的脑缺血预处理对脑缺血再灌注保护机制研究

Cerebral ischemic preconditioning （CIP） is the strong endogenous mechanism for brain protection against cerebral ischemiareperfusion(I/R) injury. But up to now, its specific mechanism is not clear. Others' and our experiments indicated that Notch pathway was activated in cerebral I/R to promot nerve regeneration and recovery through increasing the proliferation of nerve stem cell( NSCs) and decreasing its apoptosis. In previous study, we found that NICD, the activated form of Notch, was up-regulated in Cerebral I/R, which was strengthened by CIP. We therefore hypothesized that the activated Notch signaling may be one of the important mechanisms for CIP. CIP may protect the brain from the injury for the decreased apoptosis of nerve cell in ischemic region and the increased proliferation of NSCs by Notch pathway after ischemia. In order to verify the hypothesis, we will detect the expression of the key proteins of Notch signaling by RT-PCT, western-blot and immunohistochemistry, and investigate the apoptosis of nerve cell and the proliferation of NSCs. Notch signaling specific mechanisms in CIP will also be disscussed through over-expression, siRNA technique, and medication block Notch-signaling-pathway in vitro and vivo. The investigation may provide the theoretical support for the study of the molecular mechanism for CIP and the molecular treatment of I/R.

脑缺血预处理（CIP）是脑缺血再灌注（I/R）损伤强有力的内源性保护方法，但其确切机制未明。现有文献和我们前期研究表明： Notch信号通路在脑I/R后被激活，诱导神经干细胞增殖、并抑制其凋亡，促进脑缺血后神经再生、修复。我们还发现：CIP能促进Notch活化分子NICD蛋白在脑I/R后表达增高并起脑保护作用。我们推测：Notch信号激活可能是CIP的重要机制之一；CIP可能通过Notch信号促进神经干细胞增殖分化、减轻缺血区神经细胞凋亡而产生脑保护作用。为验证这一假说，我们将利用RT-PCR、 Western-blot和免疫组化等方法,检测Notch信号通路关键蛋白在大鼠脑缺血预处理后的表达,并观察神经干细胞增殖、分化和神经细胞凋亡；通过Notch1过表达和表达抑制的体外、体内实验，探讨Notch信号在CIP中的重要作用及机制。为深入研究CIP机制及脑I/R的分子靶向治疗提供理论依据。

脑缺血预处理（CIP）是脑缺血再灌注（I/R）损伤强有力的内源性保护方法，但其确切机制未明。文献表明：Notch信号通路在脑I/R后被激活，诱导神经干细胞增殖，抑制其凋亡，促进脑缺血后神经再生、修复。我们以前的研究发现：CIP能促进Notch活化分子NICD的增高。我们推测：Notch信号激活可能是CIP的重要机制之一，CIP可能通过Notch信号促进神经干细胞增殖分化，减轻神经细胞凋亡产生脑保护作用。.课题组利用RT-PCR、Western-blot技术检测了CIP后Notch信号通路关键蛋白Notch1、Jagged1、NICD和Hes1的表达，用Brdu/NSE双标抗体染色法检测海马区神经干细胞增殖，Tunel法检测缺血区神经细胞凋亡；并通过原代培养神经干细胞和神经元细胞，利用过表达和siRNA技术观察Notch信号对神经干细胞增殖和神经元细胞凋亡的影响，最后通过γ分泌酶阻断剂组阻断大鼠Notch信号通路，验证I/R大鼠在脑缺血预处理后神经干细胞增殖和神经细胞凋亡的情况。.结果表明：CIP后，Notch信号关键蛋白较I/R组增高，CIP+I/R后2h较单纯I/R组Notch信号明显增加，而24h后即有所下降，而I/R组在2-24hNotch都处于增加状态，但均低于CIP+I/R后2h，在I/R7天后，两组均降低，表明Notch信号在CIP+I/R后较I/R高峰前移。体内、外实验表明：随着Notch信号的增加，神经干细胞增殖增加，神经细胞凋亡减少，而阻断Notch信号后神经干细胞增殖减少，神经细胞凋亡增加。.由此，我们得出结论：Notch信号是CIP对MACO脑保护作用的重要保护机制；CIP后可以较早启动Notch信号参与神经再生修复，减少神经损伤；CIP通过Notch信号抑制了缺血区神经细胞凋亡，促进了神经干细胞的增殖和分化，从而产生脑保护作用。