头部浅低温对大鼠脑缺血再灌注海马CA1区GLT-1表达及细胞外谷氨酸浓度的影响

发布时间：2018-01-08 04:03

本文关键词：头部浅低温对大鼠脑缺血再灌注海马CA1区GLT-1表达及细胞外谷氨酸浓度的影响　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本实验采用改良的Pulsinelli四血管阻断(4-Vessel occlusion,4VO)法制备大鼠全脑缺血再灌注模型,鼻咽腔降温法维持头部浅低温,微透析技术收集大鼠海马CA1区微透析液并测定其中谷氨酸(Glutamic acid,Glu)浓度([Glu]e),比较在头部浅低温条件下应用谷氨酸转运体(Glutamate transporter 1,GLT-1)特异性抑制剂二氢卡因酸盐(Dihydrokainate,DHK)或应用PI3K/Akt通路特异性抑制剂LY294002后,对海马CA1区GLT-1表达及细胞外谷氨酸浓度影响,评价磷脂酰肌醇-3激酶/蛋白激酶B(Phosphoinositide 3-kinase/protein kinase B,PI3K/Akt)信号通路调控GLT-1表达在脑缺血再灌注损伤中的影响,为治疗性低温在临床应用提供参考。方法:1实验动物分组健康雄性清洁级Wistar大鼠30只(由河北医科大学实验动物中心提供)。采用随机数字表法将大鼠随机分配为5组(n=6):假手术组(Sham组/S组)、常温缺血再灌注组(IN组)、低温缺血再灌注组(IH组)、GLT-1特异性抑制剂组(DHK组)、PI3K/Akt信号通路特异性抑制剂LY294002组(LY组)。2动物模型制备采用改良的Pulsinelli四血管阻断法(4-Vessel occlusion,4VO)制备全脑缺血再灌注模型,鼻咽腔降温法制备头部浅低温模型。(1)假手术组(Sham组/S组)(n=6):仅暴露双侧椎动脉但不烧灼,仅分离双侧颈总动脉但不夹闭。(2)常温缺血/再灌注组(IN组)(n=6):永久热凝闭双侧椎动脉,48小时后暴露双侧颈总动脉穿线并夹闭双侧8 min恢复灌注。(3)低温缺血再灌注组(IH组)(n=6):永久热凝闭大鼠双侧椎动脉,48小时后分离双侧颈总动脉穿线备用,鼻咽腔降温法持续低温,维持海马温度(33.0±0.5)℃,夹闭双侧颈总动脉8 min后复灌。(4)GLT-1特异性抑制剂组(DHK组)(n=6):永久热凝闭双侧椎动脉,48小时后暴露双侧颈总动脉,鼻咽腔降温至目标温度持续收集细胞外液,夹闭双侧颈总动脉前30 min,右侧脑室缓慢注射DHK溶液20μl(200nmol),夹闭双侧颈总动脉8 min复灌。(5)LY294002组(LY组)(n=6):永久热凝闭双侧椎动脉,48小时后暴露双侧颈总动脉,夹闭双侧颈总动脉前20 min,右侧脑室缓慢注射5μl LY294002(LY294002溶于DMSO中,浓度为25mmol/L),夹闭双侧颈总动脉8 min复灌。以上各组均须同时监测直肠温度,并维持其在(37±0.5)℃。3标本采集及检测方法3.1微透析液的收集时间及测定大鼠全脑缺血前20min~10min,记为t0;全脑缺血前10min~缺血前,记为t1;缺血即刻~10min,记为t2;缺血后10min~20min,记为t3;缺血后20min~30min,记为t4;缺血后30min~40min,记为t5。收集各组大鼠微透析液并密封保存于-80℃冰箱,应用高效液相色谱(HPLC)法测定谷氨酸标准品在不同浓度的峰面积,绘制标准曲线并计算标准曲线回归方程,从而得出微透析液中谷氨酸浓度([Glu]e)变化。3.2免疫组化测定海马CA1区Bax、Bcl-2、p Akt和GLT-1蛋白表达各组大鼠微透析液收集完成后,继续维持头部浅低温(33±0.5)℃,直肠温度维持在(37±0.5)℃,待2小时后自然复温。各组大鼠于缺血再灌注8 h断头取脑,使用4%多聚甲醛固定脑组织,应用免疫组化技术观察Bax、Bcl-2、GLT-1和p Akt蛋白的表达情况。结果:1大鼠海马CA1区[Glu]e比较与t0比较,各组t1、t5[Glu]e均无明显改变(P0.05),t2、t3、t4[Glu]e差异有统计学意义(P0.05)。t2时,IN组、DHK组、LY组[Glu]e明显高于S组与IH组,差异有统计学意义(P0.05);t3时,DHK组与LY组之间差异无统计学意义(P0.05),S组、IH组、DHK组、LY组[Glu]e均低于IN组,差异有统计学意义(P0.05);t4时,DHK、LY组高于Sham组[Glu]e,IH组[Glu]e低于LY组,差异有统计学意义(P0.05)。2各组大鼠海马CA1区Bax、Bcl-2表达的免疫组化比较与S组比较,IN组、DHK组、LY组Bax表上达调,Bcl-2表达下调,IH组Bcl-2表达上调,差异有统计学意义(P0.05);与IN组相比,IH组、DHK组、LY组Bax表达下调,Bcl-2表达上调,差异有统计学意义(P0.05);与IH组相比,DHK组、LY组Bax表达上调,Bcl-2表达下调,差异有统计学意义(P0.05);DHK组、LY组两组相比差异无统计学意义(P0.05)。3各组大鼠海马CA1区p Akt蛋白表达的免疫组化比较与S组比较,IN组、LY组p Akt表达下调,IH组、DHK组p Akt表达上调(P0.05);与IN组相比,IH组、DHK组p Akt蛋白表达上调,LY组p Akt表达下调(P0.05);与IH组相比,DHK组、LY组p Akt表达下调(P0.05);与DHK组相比,LY组p Akt表达下调(P0.05)。4各组大鼠海马CA1区GLT-1蛋白表达的免疫组化比较海马CA1区星形胶质细胞中GLT-1蛋白均有表达。S组有少量棕黄色GLT-1蛋白阳性免疫颗粒。与S组比较,IN组GLT-1表达下调,IH组、DHK组GLT-1表达上调(P0.05);与IN组相比,IH组、DHK组、LY组GLT-1表达上调(P0.05);与IH组相比,DHK组、LY组GLT-1表达下调(P0.05);与DHK组相比,LY组GLT-1表达下调(P0.05)。结论:1头部浅低温可有效减轻大鼠全脑缺血再灌注损伤。2头部浅低温通过上调海马CA1区GLT-1表达,降低细胞外谷氨酸浓度,减轻其兴奋性毒性作用,从而发挥脑保护作用。
[Abstract]:Objective : To evaluate the effect of modified Pulsinelli 4 - vessel occlusion ( 4VO ) method on the expression of GLT - 1 and Glu in the CA1 region of rats . ( 4 ) GLT - 1 specific inhibitor group ( DHK group ) ( n = 6 ) : After 48 hours , bilateral common carotid artery was isolated . ( 5 ) The expression of Bax , Bcl - 2 , GLT - 1 and GLT - 1 protein in hippocampus CA1 region was measured by high performance liquid chromatography ( HPLC ) . The expression of Bax and Bcl - 2 in the hippocampal CA1 region of the rats was lower than that in the sham group ( P0.05 ) . Compared with the group in the group , the expression of Bax and Bcl - 2 in the CA1 region of the group was significantly higher than that in the sham group ( P0.05 ) . The expression of GLT - 1 in the CA1 region of hippocampus was downregulated ( P0.05 ) . Compared with the group , the expression of GLT - 1 in the CA1 region of the hippocampus was down - regulated ( P0.05 ) . Compared with the group of the group , the expression of GLT - 1 in the CA1 region of the hippocampus was down - regulated ( P0.05 ) .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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