缺血性脑卒中恢复期神经血管单元的治疗靶点研究
发布时间：2018-04-01 00:17
本文选题：缺血性脑卒中 切入点：大脑中动脉栓塞模型 出处：《南京医科大学》2016年博士论文
【摘要】：目的研究缺血性脑卒中恢复期神经血管单元的作用及三条细胞信号转导通路(丝裂原活化蛋白激酶P38-MAPK通路、细胞膜ATP敏感钾通道、聚腺苷酸二磷酸核糖转移酶-1 介导的细胞凋亡和炎症反应)在神经功能恢复中的价值,证明可能潜在的治疗靶点。方法线栓法构建大鼠大脑中动脉栓塞(MCAO)模型,实验分三部分完成。实验一：随机抓取分成对照组(假手术组)、MCAO组和P38拮抗剂组,各6只。假手术组只暴露相关解剖,不进行线栓,直接全层缝合。P38拮抗剂组在造模前30min给予腹腔注射SB203580 (5mg/kg,溶于5mg/ml DMSO液)。造模成功后3d, TTC染色法计算脑梗死面积,免疫组织化学染色检测p38α和p38β阳性率,Western Blot法检测p38总蛋白表达水平。实验二：随机抓取分成对照组(假手术组)、MCAO组、KATP阻断剂组和KATP开放剂组,各6只。造模成功后3d,KATP阻断剂组给予腹腔注射5-hydroxydecanoate (5-HD,40mg/Kg),开放剂组给予腹腔注射二氮嗪(40mg/Kg),假手术组和MCAO组给予等量生理盐水注射；正常饲养3d后离断取材。TTC染色法计算梗死面积,RT-PCR法检测KATP三个亚基kir6.1、SUR1和SUR2 mRNA的表达水平。实验三：随机抓取分成对照组(假手术组)、MCAO组、PARP-1抑制剂组各6只。PARP-1抑制剂组在造模前30min给予腹腔注射PARP-1抑制剂(AG14361,5mg/kg,溶于5mg/ml DMSO液)；正常饲养3d后离断取材。采用平衡木实验评价大鼠神经功能,TUNEL法检测组织凋亡率,Western blot法检测组织P53、Fas、NF-κB蛋白的表达水平。结果第一部分：拮抗剂组的梗死面积明显小于模型组,差异有统计学意义[(26.5±2.4)vs(43.8±2.6)%,P0.05]。模型组p38a和p38p的阳性表达率明显高于拮抗剂组,差异有统计学意义[(41.7±3.3)vs(28.3±1.6%,P0.05；(40.3 ±2.4)vs(28.2±2.6)%,P0.05].模型组p38的表达水平明显高于拮抗剂组,差异有统计学意义[(0.44±0.02)晒(0.344±0.01),P0.05]。第二部分：开放剂组的梗死面积显著少于模型组和阻断剂组,阻断剂组梗死面积最大,差异有统计学意义[开放剂组(24.3±3.1),模型组(48.8±2.0),阻断剂组(70.7±3.5),假手术组(0.08±0.02),P0.05]。开放剂组的KATP三个亚基mRNA表达水平最高,其次为模型组和阻断剂组,对照组最低,差异有统计学意义[Kir6.1：开放剂组(1.0534±0.0362),模型组(0.8524±0.0632),阻断剂组(0.6529±0.0452),假手术组(0.3951±0.0286),P0.05;SUR1:开放剂组(1.6257±0.0965),模型组(1.2548±0.0462),阻断剂组(0.7548±0.0629),假手术组(0.4562±0.0862),P0.05;SUR2:开放剂组(1.4528±0.0624),模型组(1.3263±0.0864),阻断剂组(0.8021±0.0632),假手术组(0.5123±0.0623),P0.05].第三部分：抑制剂的神经功能评分明显高于模型组,差异有统计学意义义[假手术组：(5.8±0.2),抑制剂组(3.9±0.1),模型组(2.4±0.1)分,P0.05]。抑制剂的组织凋亡率明显小于模型组,差异有统计学意义[(37.0±1.3)vs(60.0±2.8)%,P0.05]。抑制剂的P53、Fas、NF-κB蛋白水平明显低于模型组,差异有统计学意义[(0.32±0.01)vs(0.53±0.02),P0.05；(0.28±0.02)vs(0.49 ±0.02),P0.05;(0.23±0.02)vs(0.45±0.02),P0.05].结论1.神经血管单元包括神经元、血脑屏障、小胶质细胞以及细胞外基质成分,各组分间存在广泛的信号联系,是保证神经元功能和正常脑血流的物质基础。2.生物体内MARK通路是介导体外的信号分子或递质转运至细胞核内部的重要传递者,属于丝／苏氨酸残基的蛋白激酶；KATP通道广泛分布于中枢神经系统,属于配体门控的电压非依赖性内向整流K+通道,主要功能是将细胞能量代谢和生物电活动偶联起来,在脑卒中的发生、缺血预适应和再灌注损伤中均发挥重要作用；PARP-1是一种重要的转录调控因子,接受受损DNA的诱导活化,但是过量的PARP-1又反过来促使更多的NAD+无效增加、细胞内ATP的大量消耗殆尽及凋亡诱导因子的核转位等,启动细胞死亡和凋亡程序,在脑卒中后神经血管单元的损伤和重塑中扮演重要角色等。3.P38-MAPK通路、KATP通道及PARP-1可能均在缺血性脑卒中恢复期神经血管单元功能的损伤及修复中发挥重要作用,干预其过程可能成为潜在的治疗靶点。
[Abstract]:During the period of neurovascular unit and three cell signal transduction pathway to study the recovery of ischemic stroke (apoptosis and inflammatory reaction of P38-MAPK mitogen activated protein kinase pathway, the cell membrane of ATP sensitive potassium channel, poly two phosphoribosyl transferase mediated -1) on neural function recovery in the value of therapeutic targets may prove potential. Construction of the middle cerebral artery occlusion (MCAO) method of suture model experiment is divided into three parts. Experiment one: random samples were divided into control group (sham operation group), MCAO group and P38 antagonist group, 6 rats each. The sham operation group only exposed related anatomy, without suture. Direct suture.P38 antagonist group rats in 30min received intraperitoneal injection of SB203580 (5mg/kg, dissolved in 5mg/ml DMSO solution). After the success of the model 3D, calculate the cerebral infarction area by TTC staining, immunohistochemical staining of p38 alpha and p38 beta. The rate of Western, Blot detected the expression of p38 protein level. Experiment two: grab randomly divided into control group (sham operation group), MCAO group, KATP group and KATP blocker open agent group, 6 rats in each. After the success of the model 3D, KATP blocker group were given intraperitoneal injection of 5-hydroxydecanoate (5-HD, 40mg, /Kg) opener diazoxide group were given intraperitoneal injection of two (40mg/Kg), sham operation group and MCAO group were given normal saline injection; normal feeding after 3D transection were calculated.TTC infarction area staining and detection of KATP RT-PCR three subunit Kir6.1 expression levels of SUR1 and SUR2 mRNA. Experiment three: random crawl into control group (sham operation group), MCAO group, PARP-1 inhibitor group with 6 rats in each group were given intraperitoneal injection of.PARP-1 inhibitor, PARP-1 inhibitor before modeling 30min (AG14361,5mg/kg, dissolved in 5mg/ml DMSO solution); normal feeding after 3D transection. The material balance beam experimental evaluation of neurological function in rats, TUNEL娉曟娴嬬粍缁囧噵浜＄巼,Western blot娉曟娴嬬粍缁嘝53,Fas,NF-魏B铔嬬櫧鐨勮〃杈炬按骞，
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