2008 年中华医学会全国麻醉学术年会

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    ·优秀论文·

    Akt 信号通路与二氮嗪预处理抗缺氧复氧后大鼠海马神经元

    凋亡作用关系的研究

    福建省立医院麻醉科(福建福州，350001)

    李捷萌 陈彦青 刘荣国 吴晓丹

    目的： 探讨 Akt 信号通路与二氮嗪预处理抗缺氧复氧后大鼠海马神经元凋亡作用的关系。 方法：

    取离体培养的大鼠海马神经元，随机分为 6 组：对照组(A组) 、缺氧组(B 组) 、缺氧+二氮嗪 100

    μmolL 预处理组(C 组) 、缺氧+二氮嗪 100 μmolL+5-羟癸酸 100 μmolL 预处理组(D组) 、缺

    氧+5-羟癸酸 100 μmolL 预处理组(E 组) ，缺氧+二氮嗪 100 μmolL+LY294002 50μmolL 预处理

    组(F组) ，各组神经元每天给予相应药物预处理 1 h，连续3 d，继而缺氧 4 h 复氧24 h，观察神经

    元的活力、凋亡率、Akt、Bcl-2、Bax 蛋白的表达水平。结果：C 组与 B、D、E 3 组缺氧组比较，海马神经元的活力增强，凋亡率降低，Akt、Bcl-2 蛋白表达增强，Bax 蛋白表达减弱(P<0.01) ；F

    组与 C 组比较，海马神经元的活力降低，凋亡率增高，Akt、Bcl-2 蛋白表达降低，Bax 蛋白表达增

    强(P<0.01) 。结论：Akt信号通路参与或部分参与了二氮嗪预处理抗缺氧复氧后大鼠海马神经元凋

    亡的作用。

    关键词：药物预处理；缺氧耐受性；Akt；Bcl-2；Bax；LY294002

    Relation of Akt signal transduction passageway and the mechanism of diazoxide preconditioning for

    anti-apoptosis in cultured hippocampal neurons by anoxia- reoxygenation in rats

    Li Jiemeng, Chen Yanqing, Liu Rongguo, Wu Xiaodan. Department of Anaesthesiology, Fujian Provincial

    Hospital, Fuzhou 350001 CHINA

    Abstract Objective To investigate relation of Akt signal transduction passageway and the mechanism

    of diazoxide preconditioning for anti-apoptosis in cultured hippocampal neurons by anoxia-reoxygenation

    in rats. Methods Isolated cultured hippocampal neurons of rat were assigned into the following six groups

    randomly: control group (Group A), diazoxide 0μmolL group (Group B), diazoxide 100 μmolL group

    (Group C), diazoxide 100 μmolL and 5-hydroxydecanoate 100 μmolL group (Group D),5-hydroxydecanoate 100 μmolL group (Group E), diazoxide 100 μmolL and LY294002 50 μmolL

    group (Group F). The hippocampal neurons were treated with diazoxide 1 h, per day for 3 days before

    being subjected to 4 h oxygen deprivation followed by reoxygenation. The neuronal vitality was assayed

    and apoptosis rate determined after 24 h reoxygenation. The expression of Akt, Bcl-2 and Bax protein was

    determined by western blotting. Results The vitality of hippocampal neurons in Group C is significant

    higher than that in Group B, D and E, whereas the apoptotic rate was opposite (P<0.01); Akt, Bcl-2

    protein in Group C expressed more intensively than that in Group B, D and E, while Bax protein was

    weaken (P<0.01). The vitality of hippocampal neurons in Group F is significant lower than that in Group

    C, whereas the apoptotic rate was opposite (P<0.01); Akt, Bcl-2 protein in Group F expressed significant

    lower than that in Group C, while Bax protein was strengthen (P<0.01). Conclusions Akt signal

    transduction passageway completely or partly mediated the process of diazoxide preconditioning for 2008 年中华医学会全国麻醉学术年会

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    anti-apoptosis in cultured hippocampal neurons by anoxia-reoxygenation in rats.

    Key words Pharmacological preconditioning Hypoxia-tolerance Akt Bcl-2 Bax LY294002

    研究发现，开放线粒体内膜 ATP敏感钾通道(Mitochondrial ATP-sensitive Potassiun Channel，mitoKATP)能模拟出脑的缺血预处理效应[1,2]

    ，这为临床治疗缺血缺氧性脑损伤提供了理想的治疗靶

    点[3]。但其机制目前并未完全阐明。本实验采用 SD 大鼠海马神经元离体培养模型，应用四唑蓝比

    色法、流式细胞术、Western blot 诸方法，拟观察二氮嗪预处理对缺氧复氧后大鼠海马神经元凋亡

    相关蛋白的影响，探讨其脑保护的可能机制。

    材料与方法

    原代 SD大鼠海马神经元的培养 出生<24 h 新生SD大鼠，体重 5~6 g，由北京大学实验动物

    中心提供。75%乙醇消毒，在无菌条件下断头，去除头部皮肤等外层组织，将全脑取出，在放大

    15 倍的解剖显微镜下取出海马组织，剪碎(约 1×1×l mm组织块) ，加入含 0.125%胰蛋白酶(Gibeo

    公司，美国)的解剖液，在 37℃下，于 5%CO2 培养箱中消化 30 min，以 0.5×106

    个ml 的神经元

    密度接种在 35 mm无菌培养皿或 96 孔无菌培养板(Coster公司，美国) ，2 ml皿或 100 μl孔 ......