本文選題：缺血性腦卒中　切入點(diǎn)：大腦中動(dòng)脈栓塞模型　出處：《南京醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的研究缺血性腦卒中恢復(fù)期神經(jīng)血管單元的作用及三條細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路(絲裂原活化蛋白激酶P38-MAPK通路、細(xì)胞膜ATP敏感鉀通道、聚腺苷酸二磷酸核糖轉(zhuǎn)移酶-1 介導(dǎo)的細(xì)胞凋亡和炎癥反應(yīng))在神經(jīng)功能恢復(fù)中的價(jià)值,證明可能潛在的治療靶點(diǎn)。方法線栓法構(gòu)建大鼠大腦中動(dòng)脈栓塞(MCAO)模型,實(shí)驗(yàn)分三部分完成。實(shí)驗(yàn)一：隨機(jī)抓取分成對(duì)照組(假手術(shù)組)、MCAO組和P38拮抗劑組,各6只。假手術(shù)組只暴露相關(guān)解剖,不進(jìn)行線栓,直接全層縫合。P38拮抗劑組在造模前30min給予腹腔注射SB203580 (5mg/kg,溶于5mg/ml DMSO液)。造模成功后3d, TTC染色法計(jì)算腦梗死面積,免疫組織化學(xué)染色檢測(cè)p38α和p38β陽性率,Western Blot法檢測(cè)p38總蛋白表達(dá)水平。實(shí)驗(yàn)二：隨機(jī)抓取分成對(duì)照組(假手術(shù)組)、MCAO組、KATP阻斷劑組和KATP開放劑組,各6只。造模成功后3d,KATP阻斷劑組給予腹腔注射5-hydroxydecanoate (5-HD,40mg/Kg),開放劑組給予腹腔注射二氮嗪(40mg/Kg),假手術(shù)組和MCAO組給予等量生理鹽水注射；正常飼養(yǎng)3d后離斷取材。TTC染色法計(jì)算梗死面積,RT-PCR法檢測(cè)KATP三個(gè)亞基kir6.1、SUR1和SUR2 mRNA的表達(dá)水平。實(shí)驗(yàn)三：隨機(jī)抓取分成對(duì)照組(假手術(shù)組)、MCAO組、PARP-1抑制劑組各6只。PARP-1抑制劑組在造模前30min給予腹腔注射PARP-1抑制劑(AG14361,5mg/kg,溶于5mg/ml DMSO液)；正常飼養(yǎng)3d后離斷取材。采用平衡木實(shí)驗(yàn)評(píng)價(jià)大鼠神經(jīng)功能,TUNEL法檢測(cè)組織凋亡率,Western blot法檢測(cè)組織P53、Fas、NF-κB蛋白的表達(dá)水平。結(jié)果第一部分：拮抗劑組的梗死面積明顯小于模型組,差異有統(tǒng)計(jì)學(xué)意義[(26.5±2.4)vs(43.8±2.6)%,P0.05]。模型組p38a和p38p的陽性表達(dá)率明顯高于拮抗劑組,差異有統(tǒng)計(jì)學(xué)意義[(41.7±3.3)vs(28.3±1.6%,P0.05；(40.3 ±2.4)vs(28.2±2.6)%,P0.05].模型組p38的表達(dá)水平明顯高于拮抗劑組,差異有統(tǒng)計(jì)學(xué)意義[(0.44±0.02)曬(0.344±0.01),P0.05]。第二部分：開放劑組的梗死面積顯著少于模型組和阻斷劑組,阻斷劑組梗死面積最大,差異有統(tǒng)計(jì)學(xué)意義[開放劑組(24.3±3.1),模型組(48.8±2.0),阻斷劑組(70.7±3.5),假手術(shù)組(0.08±0.02),P0.05]。開放劑組的KATP三個(gè)亞基mRNA表達(dá)水平最高,其次為模型組和阻斷劑組,對(duì)照組最低,差異有統(tǒng)計(jì)學(xué)意義[Kir6.1：開放劑組(1.0534±0.0362),模型組(0.8524±0.0632),阻斷劑組(0.6529±0.0452),假手術(shù)組(0.3951±0.0286),P0.05;SUR1:開放劑組(1.6257±0.0965),模型組(1.2548±0.0462),阻斷劑組(0.7548±0.0629),假手術(shù)組(0.4562±0.0862),P0.05;SUR2:開放劑組(1.4528±0.0624),模型組(1.3263±0.0864),阻斷劑組(0.8021±0.0632),假手術(shù)組(0.5123±0.0623),P0.05].第三部分：抑制劑的神經(jīng)功能評(píng)分明顯高于模型組,差異有統(tǒng)計(jì)學(xué)意義義[假手術(shù)組：(5.8±0.2),抑制劑組(3.9±0.1),模型組(2.4±0.1)分,P0.05]。抑制劑的組織凋亡率明顯小于模型組,差異有統(tǒng)計(jì)學(xué)意義[(37.0±1.3)vs(60.0±2.8)%,P0.05]。抑制劑的P53、Fas、NF-κB蛋白水平明顯低于模型組,差異有統(tǒng)計(jì)學(xué)意義[(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].結(jié)論1.神經(jīng)血管單元包括神經(jīng)元、血腦屏障、小膠質(zhì)細(xì)胞以及細(xì)胞外基質(zhì)成分,各組分間存在廣泛的信號(hào)聯(lián)系,是保證神經(jīng)元功能和正常腦血流的物質(zhì)基礎(chǔ)。2.生物體內(nèi)MARK通路是介導(dǎo)體外的信號(hào)分子或遞質(zhì)轉(zhuǎn)運(yùn)至細(xì)胞核內(nèi)部的重要傳遞者,屬于絲／蘇氨酸殘基的蛋白激酶；KATP通道廣泛分布于中樞神經(jīng)系統(tǒng),屬于配體門控的電壓非依賴性內(nèi)向整流K+通道,主要功能是將細(xì)胞能量代謝和生物電活動(dòng)偶聯(lián)起來,在腦卒中的發(fā)生、缺血預(yù)適應(yīng)和再灌注損傷中均發(fā)揮重要作用；PARP-1是一種重要的轉(zhuǎn)錄調(diào)控因子,接受受損DNA的誘導(dǎo)活化,但是過量的PARP-1又反過來促使更多的NAD+無效增加、細(xì)胞內(nèi)ATP的大量消耗殆盡及凋亡誘導(dǎo)因子的核轉(zhuǎn)位等,啟動(dòng)細(xì)胞死亡和凋亡程序,在腦卒中后神經(jīng)血管單元的損傷和重塑中扮演重要角色等。3.P38-MAPK通路、KATP通道及PARP-1可能均在缺血性腦卒中恢復(fù)期神經(jīng)血管單元功能的損傷及修復(fù)中發(fā)揮重要作用,干預(yù)其過程可能成為潛在的治療靶點(diǎn)。
[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嫻嬬粍緇囧噵浜＄巼,Western blot娉曟嫻嬬粍緇嘝53,Fas,NF-魏B铔嬬櫧鐨勮〃杈炬按騫，

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