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本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5μL,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5μL,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5μL,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。
In this study, antisense oligo-deoxynucleotides (AS-ODNs) of glial glutamate transporter-1 (GLT-1) were used to inhibit Wistar rats GLT-1 protein To investigate the effect of cerebral ischemic preconditioning (CIP) on cerebral ischemic tolerance and to investigate the role of GLT-1 in CIP-induced cerebral ischemic tolerance. Wistar rats with bilateral vertebral artery occlusion were randomly divided into 7 groups: (1) Sham group: bilateral common carotid arteries were exposed without blocking blood flow; (2) CIP group: bilateral common carotid artery 3 min; (3) Cerebral ischemic stroke group: bilateral common carotid artery was occluded for 8 min; (4) CIP + ischemic stroke group: bilateral common carotid artery was occluded 3 min for CIP; Common carotid artery 8min; (5) double-distilled water group: bilateral carotid artery (but not clipped) were exposed 12h before, 12h after and 36h after injection into the right ventricle double distilled water, each 5μL, the other with the sham Group; (6) AS-ODNs group: GLT-1 AS-ODNs solution was injected into right ventricle at 12h, 12h and 36h after exposure to bilateral common carotid arteries (but not occluded) sham group, then further divided into 2 subgroups of 9nmol and 18nmol according to the dose of AS-ODNs; (7) AS-ODNs + CIP + ischemic stroke group: at the 12h, 1 AS-ODNs solution, each 5 μL, the other with the CIP + cerebral ischemic attack group, according to the dose of AS-ODNs is further divided into 2 sub-groups of 9nmol and 18nmol. Western blot analysis was used to observe the expression of GLT-1 protein. Thionine staining was used to observe the delayed neuronal death (DND) in hippocampal CA1 region. Western blot analysis showed that intracerebroventricular injection of GLT-1 AS-ODNs could dose-dependently inhibit GLT-1 protein expression in hippocampal CA1 region. Thionine staining showed that there was no obvious DND in hippocampal CA1 region in sham group and CIP group; obvious DND in hippocampal CA1 region of cerebral ischemic attack group; pre-administration of CIP could significantly antagonize the DND caused by cerebral ischemic attack, indicating that CIP can be induced Hippocampal CA1 region neurons produce ischemic tolerance, against the DND caused by cerebral ischemic attack; while in the GLT-1AS-ODNs + CIP + cerebral ischemic stroke group, after intracerebroventricular injection of GLT-1 AS-ODNs, the hippocampus The obvious DND appeared in the CA1 region, indicating that GLT-1AS-ODNs attenuated the neuroprotection of CIP against cerebral ischemic stroke by inhibiting the expression of GLT-1 protein in rats. The above results further confirmed that GLT-1 is involved in CIP-induced cerebral ischemic tolerance.