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胶质细胞谷氨酸转运体--1反义寡核苷酸对脑缺血预处理神经保护效应的抑制作用

耿进霞, 蔡劲松, 张敏, 李淑琴, 孙晓彩, 羡晓辉, 胡玉燕, 李文斌, 李清君

河北医科大学病理生理学教研室.河北,石家庄 050017

摘要

该研究应用胶质细胞谷氨酸转运体--1(glial glutamate transporter--1,GLT--1)的反义寡核苷酸(antisense oligo--deoxynucleotides,AS--ODNs)抑制Wistar大鼠GLT--1蛋白的表达,观察其对脑缺血预处理(cerebral ischemic preconditioning,CIP)增强脑缺血耐受作用的影响,探讨GLT--1在CIP诱导的脑缺血耐受中的作用。将凝闭双侧椎动脉的Wistar大鼠随机分为7组:(1)Sham组:只暴露双侧颈总动脉,不阻断血流;(2)CIP组:夹闭双侧颈总动脉3min;(3)脑缺血打击组:夹闭双侧颈总动脉8min;(4)CIP+脑缺血打击组:夹闭双侧颈总动脉3min作为CIP,再灌注2d后,夹闭双侧颈总动脉8min;(5)双蒸水组:于分离暴露双侧颈总动脉(但不夹闭)前12h、后12h及后36h右侧脑室注射双蒸水,每次5#mu#L,其它同sham组;(6)AS--ODNs组:于分离暴露双侧颈总动脉(但不夹闭)前12h、后12h及后36h右侧脑室注射GLT--1 AS--ODNs溶液,每次5#mu#L,其它同sham组,再根据AS--ODNs的剂量进一步分为9nmol和18nmol2个亚组;(7)AS-ODNs+CIP+脑缺血打击组:于CIP前12h、后12h及后36h右侧脑室注射GLT--1 AS--ODNs溶液,每次5#mu#L,其它同CIP+脑缺血打击组,根据AS--ODNs的剂量进一步分为9nmol和18nmol2个亚组。Western blot分析法观察GLT--1蛋白的表达,硫堇染色观察海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)情况。Western blot分析显示,侧脑室注射GLT--1 AS--ODNs可剂量依赖性地抑制大鼠海马CA1区GLT--1蛋白表达。硫堇染色显示,sham组和CIP组海马CA1区未见明显的DND;脑缺血打击组海马CA1区有明显的DND;预先给予CIP可显著对抗脑缺血打击引起的DND,表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗脑缺血打击引起的DND;而在GLT--1AS--ODNs+CIP+脑缺血打击组,侧脑室注射GLT--1 AS--ODNs后,大鼠海马CA1区出现了明显的DND,表明GLT--1AS--ODNs通过抑制大鼠GLT--1蛋白表达从而减弱CIP对抗脑缺血打击的神经保护作用。以上结果进一步证实了GLT--1参与CIP诱导的脑缺血耐受。

关键词： 脑缺血预处理; 脑缺血耐受; 海马; 胶质细胞谷氨酸转运体-1; 反义寡核苷酸; 大鼠

Antisense oligodeoxynucleotides of glial glutamate transporter--1 inhibits the neuro--protection of cerebral ischemic preconditioning in rats

Geng Jinxia, Cai Jingsong, Zhang Min, Li Shuqin, Sun Xiaocai, Xian Xiaohui, Hu Yuyan, Li Wenbin, Li Qingjun

Department of Pathophysiology,Hebei Medical University.Shijiazhuang 050017,Hebei

Abstract

The present study was undertaken to investigate the role of glial glutamate transporter-1 (GLT-1) in the brain ischemic tolerance induced by cerebral ischemic preconditioning (CIP) by observing the effect of GLT-1 antisense oligodeoxynucleotides (AS-ODNs) on the neuro-protection of CIP against brain ischemic insult in rats. Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 7 groups: (1) Sham group: the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow; (2) CIP group: the BCCA were clamped for 3 min; (3) Brain ischemic insult group: the BCCA were clamped for 8 min;(4) CIP + brain ischemic insult group: 3 min CIP was preformed 2 d prior to 8 min ischemic insult; (5) Double distilled water group: 5 #mu#L double distilled water was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after the BCCA was separated (but without occluding the blood flow), respectively; (6) AS-ODNs group: 5 #mu#L AS-ODNs solution was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after the BCCA was separated (but without occluding the blood flow), respectively. This group was further divided into 9 nmol and 18 nmol subgroups according to the doses of AS-ODNs; (7) AS-ODNs + CIP + brain ischemic insult group: 5 #mu#L AS-ODNs solution was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after CIP, respectively. This group was also further divided into 9 nmol and 18 nmol subgroups according to the doses of AS-ODNs. The other treatments were the same as those in CIP + brain ischemic insult group. The effect of the AS-ODNs on the expression of GLT-1 was assayed by using Western blot analysis. The profile of delayed neuronal death (DND) of pyramidal neurons in the CA1 hippocampus was evaluated by using thionin staining under light microscope by determining the neuronal density (ND) and histological grade (HG). Western blot analysis showed that AS-ODNs injected into the lateral cerebroventricle inhibited the expression of GLT-1 in the CA1 hippocampus in a dose-dependent manner. Neuropathological evaluation showed that there was no apparent DND in sham and CIP groups. Obvious DND of pyramidal neurons was found in brain ischemic insult group, which was represented by an increase in HG and a decrease in ND. CIP effectively protected the pyramidal neurons in the CA1 hippocampus against DND normally induced by ischemic insult, which indicating that CIP induced ischemic tolerance on the pyramidal neurons in the CA1 hippocampus. However, the injection of AS-ODNs into the lateral cerebroventricle blocked the neuro-protection of CIP against DND induced by brain ischemic insult. These results further proved the role of GLT-1 in the brain ischemic tolerance induced by CIP in rats.

Key words: cerebral ischemic preconditioning;brain ischemic tolerance;Hippocampus;glial glutamate transporter-1;antisense oligodeoxynucleotides;Rats

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引用本文：

耿进霞, 蔡劲松, 张敏, 李淑琴, 孙晓彩, 羡晓辉, 胡玉燕, 李文斌, 李清君. 胶质细胞谷氨酸转运体--1反义寡核苷酸对脑缺血预处理神经保护效应的抑制作用[J]. 生理学报 2008; 60 (4): 497-503.

Geng Jinxia, Cai Jingsong, Zhang Min, Li Shuqin, Sun Xiaocai, Xian Xiaohui, Hu Yuyan, Li Wenbin, Li Qingjun. Antisense oligodeoxynucleotides of glial glutamate transporter--1 inhibits the neuro--protection of cerebral ischemic preconditioning in rats. Acta Physiol Sin 2008; 60 (4): 497-503 (in Chinese with English abstract).