缝隙连接在左旋多巴诱发异动症发生机制中的作用

发布时间：2018-03-10 10:10

本文选题：帕金森病　切入点：左旋多巴诱发异动症　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景左旋多巴诱发异动症(levodopa-induced dyskinesia,LID)是帕金森病药物治疗常见运动并发症，慢性不规则多巴胺刺激是LID形成的重要诱因，其发生与基底节神经元异常同步化和振荡活动关系密切。缝隙连接（gap junction，GJ）是由连接相邻2个细胞之间的连接通道排列而成的一种特殊膜结构，参与细胞间信息、能量和物质的交换、代谢偶联和电信号偶联。GJ是神经元同步化活动重要机制，其功能受到多巴胺能刺激的调节。这些线索提示缝隙连接功能异常可能参与左旋多巴诱发异动症发生机制。我们前期的实验通过免疫组化证实了LID组纹状体和运动皮层Cx36表达较PD组和正常对照组显著增多，支持了该设想。本实验将进一步探讨缝隙连接在左旋多巴诱发异动症发生机制中的作用。目的通过了解不同浓度的缝隙连接阻断剂甘珀酸（carbenoxolon,CBX)及奎宁（quinine,QN）对左旋多巴诱发异动症大鼠异动行为的影响，观察异动症大鼠缝隙连接蛋白Cx36在不同类型纹状体神经元的表达差异和基底节运动皮层蛋白水平的变化，探讨缝隙连接功能异常在异动症发生机制中的作用。方法6-羟多巴胺偏侧帕金森病大鼠模型重复腹腔注射左旋多巴（20mg/kg）和卞丝肼（10mg/kg）共21天，以建立左旋多巴诱发异动症模型。将大鼠分为三组：LID组、PD组、正常对照组。①通过LID大鼠腹腔注射不同剂量的CBX与侧脑室注射奎宁来观察缝隙连接阻断剂对异动症大鼠行为学的影响。②免疫双标法分析各组脑啡肽（ENK）阳性纹状体传出神经元及小清蛋白（parvalbumin）阳性中间神经元CX36表达情况。③Western-blot检测各组纹状体及运动皮层Cx36蛋白表达。结果①行为学研究显示大剂量(60mg/kg)的甘珀酸腹腔注射和奎宁侧脑室注射（2.5μmol）能减少异动症大鼠的AIM评分（P0.05）。②免疫双标显示异动症大鼠损毁侧纹状体脑啡肽阳性神经元Cx36表达(57.59%±5.36%)较正常对照组（32.67%±4.22%）和PD组(37.24%±0.86%)增强(F=78.060,P0.01)，小清蛋白阳性中间神经元Cx36表达(68.49%±11.60%)也较正常对照组（40.43%±2.30%）和PD组（31.92%±5.68%）增强（F=39.567,P0.01）。③白质印迹检测显示异动症大鼠损毁侧纹状体和运动皮质Cx36表达水平分别为（219.56%±18.12%）、（226.03%±16.33%），高于正常对照组的(104.05%±3.82%)(t=15.389,P＜0.01）、（105.27%±2.82%）(t=8.074,P＜0.01）和PD组的(119.31%±8.92%)(t=13.356,P＜0.01）、（138.20%±17.88%）(t=5.872,P＜0.01）。结论左旋多巴诱发的异动症大鼠损伤侧纹状体及运动皮层的Cx36蛋白表达水平增强，纹状体ENK阳性传出神经元Cx36表达水平上调，且Parvalbumin阳性中间神经元Cx36表达水平增高，缝隙连接阻断剂能有效减少大鼠的异动行为，提示缝隙连接异常可能参与了异动症的发生机制。
[Abstract]:Background levodopa-induced dyskinesia (LIDs) is a common motor complication in the treatment of Parkinson's disease. Chronic irregular dopamine stimulation is an important inducement for the formation of LID. Its occurrence is closely related to abnormal synchronization and oscillatory activity of basal ganglion neurons. Gap junction junction GJ is a special membrane structure arranged by connecting the connecting channels between two adjacent cells and participates in intercellular information. The exchange of energy and matter, metabolic coupling and electrical coupling. GJ are important mechanisms of neuronal synchronization. Its function is regulated by dopaminergic stimulation. These clues suggest that gap junction dysfunction may be involved in the pathogenesis of levodopa-induced dyskinesia. Our previous experiment confirmed the striatum and motility of LID group by immunohistochemistry. Compared with PD group and normal control group, the expression of Cx36 in the arteriocortex was significantly increased. This experiment will further explore the role of gap junction in the pathogenesis of levodopa-induced hyperactivity. Objective to investigate the effects of different concentrations of gap junction blockers carbenoxolonium (CBX) and quinine quinine (QN) on the abnormal behavior induced by levodopa in rats with dyskinesia. To observe the difference of gap junction protein (Cx36) expression in different types of striatum neurons and the changes of basal ganglia motor cortex protein level in rats with dyskinesia, and to explore the role of gap junction dysfunction in the pathogenesis of dyskinesia. Methods the rat model of 6-hydroxydopamine hemiparkinsonism was repeatedly injected intraperitoneally with levodopa (20 mg / kg) and Bensarazide (10 mg / kg) for 21 days in order to establish the model of levodopa induced hyperactivity disorder. The rats were divided into three groups: control group (n = 3) and PD group (n = 10). To observe the effect of gap junction blocker on behavior of rats with dyskinesia by intraperitoneal injection of different doses of CBX and intracerebroventricular injection of quinine in normal control group. 2 Immuno-double standard method for analysis of enkephalin positive striatum in each group. The expression of Cx36 protein in striatum and motor cortex was detected by Western-blot. Results 1Behavioral studies showed that intraperitoneal injection of high-dose carbenic acid (60 mg / kg) and intracerebroventricular injection of quinine (2.5 渭 mol) decreased the AIM score of rats with dyskinesia (P0.050.2-immunoreactive double labeling showed that dyskinetic rats had dyskinesia with dyskinetic rat striatum enkephalin positive neurons. The expression of Cx36 was 57.59% 卤5.36% and 32.67% 卤4.22% in control group and 37.24% 卤0.86 in PD group, respectively.) the expression of Cx36 was increased by 78.060% and 68.49% 卤11.601% respectively. The expression of Cx36 in striatum was also significantly higher than that in normal control group (40.43% 卤2.30%) and PD group (31.92% 卤5.68%). The expression level of Cx36 in the cortex was 219.56% 卤18.12 卤226.03% 卤16.33, which was higher than that in the normal control group (104.05% 卤3.82% 卤3.82) and in the PD group (105.27% 卤2.82% 卤8.074 P < 0.01) and in PD group (138.20% 卤17.888.872P < 0.01). Conclusion the expression of Cx36 protein in the injured striatum and motor cortex of rats induced by levodopa-induced dyskinesia is increased, the Cx36 expression of ENK positive efferent neurons in striatum is up-regulated, and the Cx36 expression of Parvalbumin positive intermediate neurons is increased. Gap junction blockers can effectively reduce the abnormal behavior of rats, suggesting that abnormal gap junction may be involved in the pathogenesis of hyperactivity.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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