本文選題：腦缺血 + 腦卒中　； 參考：《青島大學》2017年碩士論文

【摘要】：目的:缺血后處理(ischemic post-conditioning,IPO)能夠激發(fā)機體內源性保護作用,減輕缺血再灌注(ischemia reperfusion,I/R)后的炎癥反應,但具體作用機制目前尚不明確。本課題旨在探討IPO對局灶性腦缺血再灌注大鼠Toll樣受體4(Toll-like receptor 4,TLR4)-Toll/IL-1受體結構域接頭分子(Toll-interleukin 1 receptor domain-containing adapter-inducing interferon-b,TRIF)信號轉導途徑的影響,進一步闡述IPO的神經保護機制,為臨床應用IPO治療缺血性卒中提供理論依據。方法:研究選用130只成年健康雄性Sprague-Dawley大鼠并隨機分為假手術組(sham組)30只,模型組和干預組各50只,sham組根據手術后,模型組和干預組根據再灌注后時間點隨機分為6h、12h、24h、48h和72h 5個亞組,sham組每個亞組各6只,模型組和干預組每個亞組各10只。后兩組采用Zea-longa線栓法建立大鼠大腦中動脈閉塞(middle cerebral artery occlusion,MCAO)模型。sham組僅手術暴露頸總動脈及分叉處,不阻斷大腦中動脈。在再灌注開始時即模型建立后2h對干預組大鼠進行IPO處理(即術后2 h將栓線拔出至頭部位于頸總動脈分叉處,10s后再將栓線置入初始位置10s,如此重復6次)。對模型組大鼠僅拔出栓線,不給予IPO處理。于再灌注后6h、12h、24h、48h、72h等時間點,采用Menzies法進行神經功能缺損評分;2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色觀察腦梗死體積;原位末端標記法(terminal-deoxynucleoitidyl transferase mediated nick end labeling,TUNEL)法檢測缺血側腦組織細胞凋亡;實時定量熒光PCR(Real-time quantitative PCR,qPCR)檢測大鼠缺血側顳、頂葉皮質TLT4-TRIF信號轉導途徑的特異性結構分子及關鍵細胞因子TLR4、TRIF、TRIF相關的接頭分子(TRIF-related adaptor molecule,TRAM)、干擾素調節(jié)因子3(interferon regulatory factor 3,IRF-3)及β干擾素(interferon-β,INF-β)等的m RNA表達;免疫組織化學(immunohistochemistry)檢測大鼠缺血側腦組織中TLR4、TRIF、TRAM、IRF-3及IFN-β等結構分子及關鍵細胞因子蛋白陽性細胞的分布與表達;免疫印跡法(western blotting)定量檢測大鼠缺血側顳、頂葉皮質中TLR4、TRIF、TRAM、IRF-3及IFN-β等結構分子及關鍵細胞因子的蛋白表達。結果:模型組、干預組大鼠都出現不同程度的神經功能缺損及缺血側大腦半球梗死。相比模型組,干預組大鼠神經功能缺損評分得到顯著改善(t=2.963~5.262,P0.05),腦梗死體積明顯減少(t=3.341~3.875,P0.05),凋亡細胞計數明顯減少(t=2.332~3.643,P0.05)。模型組和干預組TLR4、TRIF、TRAM、IRF-3及IFN-βm RNA和蛋白表達于再灌注6h時已顯著升高。qPCR結果顯示干預組TLR4、TRIF、TRAM、IRF-3及IFN-βm RNA表達較模型組各對應時間點顯著降低(t=2.240~6.587,P0.05),免疫組織化學檢測結果顯示TLR4、TRIF、TRAM、IRF-3及IFN-β蛋白陽性細胞主要位于缺血側額、顳葉皮質,干預組陽性細胞計數較模型組各對應時間點顯著降低(t=2.256~8.180,P0.05)。Western blotting檢測結果顯示干預組TLR4、TRIF、TRAM、IRF-3及IFN-β蛋白表達較模型組各對應時間點顯著降低(t=2.943~8.227,P0.05)。結論:本研究證實IPO能夠通過抑制細胞凋亡、減少腦梗死體積發(fā)揮神經保護作用,改善MCAO大鼠的神經功能。IPO還可能通過抑制TLR4-TRIF信號轉導途徑減少通路下游IRF-3、IFN-β等炎癥相關因子的表達,減輕I/R后的局灶性炎癥反應,進而激發(fā)內源性神經保護作用。
[Abstract]:Objective: ischemic post-conditioning (IPO) can stimulate the endogenous protective effect of the body and reduce the inflammatory response after ischemia reperfusion (ischemia reperfusion, I/R), but the specific mechanism is not yet clear. The aim of this study is to explore Toll like receptor 4 (Toll-like receptor 4, TLR) in focal cerebral ischemia-reperfusion rats (Toll-like receptor 4, TLR). 4) the effect of -Toll/IL-1 receptor domain junction molecule (Toll-interleukin 1 receptor domain-containing adapter-inducing interferon-B, TRIF) signal transduction pathway, further expounds the neuroprotective mechanism of IPO, and provides a theoretical basis for the clinical application of IPO to treat ischemic stroke. Methods: 130 adult healthy male Sprague-D were selected. AWLEY rats were randomly divided into 30 rats in sham operation group (Group sham), model group and intervention group, and group sham were randomly divided into 5 subgroups of 6h, 12h, 24h, 48h and 72h, 6 in each group of sham group, 10 in each group of the model group and in each subgroup of intervention group after the operation. The latter two groups were treated with Zea-longa thread. The rat model of middle cerebral artery occlusion (MCAO) model was established in group.Sham only to expose the common carotid artery and the bifurcation, and did not block the middle cerebral artery. At the beginning of the reperfusion, the model was established by 2H to the rats in the intervention group (that is, 2 h after the operation was pulled out to the head of the common carotid artery, and the 10s after the operation. " Then the thrombus line was placed in the initial position 10s, so repeated 6 times). The rats in the model group were only pulled out of the thrombus line and did not give IPO treatment. After the reperfusion, 6h, 12h, 24h, 48h, 72h and other time points were measured by Menzies method, and the volume of cerebral infarction was observed by 2,3,5- chlorination of three phenyl tetrazolium (2,3,5-triphenyltetrazolium chloride, TTC). In situ terminal labeling (terminal-deoxynucleoitidyl transferase mediated nick end labeling, TUNEL) detection of apoptosis in ischemic brain tissue; real-time quantitative fluorescent PCR (Real-time quantitative PCR, qPCR) detection of rat ischemic side temporomandibular, parietal cortex, specific structural molecules and key cytokines R4, TRIF, TRIF related joint molecules (TRIF-related adaptor molecule, TRAM), interferon regulatory factor 3 (interferon regulatory factor 3, IRF-3) and interferon beta (interferon- beta, beta), etc. The distribution and expression of the positive cell and key cell factor protein positive cells; Western blotting were used to detect the protein expression of TLR4, TRIF, TRAM, IRF-3 and IFN- beta in the temporal and temporal cortex of the rat, and the protein expression of the key cytokines in the parietal cortex. Results: the model group, the rats in the intervention group had different degrees of nerve function defect and the rats in the intervention group. Compared with the model group, the neurological deficit score of the rats in the intervention group was significantly improved (t=2.963~5.262, P0.05), the volume of cerebral infarction decreased significantly (t=3.341~3.875, P0.05), the number of apoptotic cells decreased significantly (t=2.332~3.643, P0.05). The model group and the intervention group were TLR4, TRIF, TRAM, IRF-3 and IFN- beta m and protein expression and protein expression. The TLR4, TRIF, TRAM, IRF-3 and IFN- beta m RNA expression in the intervention group was significantly lower than that of the model group (t=2.240~6.587, P0.05) in the intervention group. The results of immunohistochemical detection showed that TLR4, TLR4, and beta protein positive cells were mainly located in the ischemic side, temporal lobe cortex, and the intervention group. The cell count decreased significantly at the corresponding time points of the model group (t=2.256~8.180, P0.05).Western blotting detection results showed that the expression of TLR4, TRIF, TRAM, IRF-3 and IFN- beta protein in the intervention group was significantly lower than the corresponding time points in the model group (t=2.943~8.227, P0.05). Conclusion: this study confirmed that IPO can reduce the volume of cerebral infarction by inhibiting apoptosis and reducing the volume of cerebral infarction. To improve the neuroprotective effect and improve the neural function of MCAO rats,.IPO may also reduce the expression of inflammatory related factors such as IRF-3, IFN- beta, and so on by inhibiting the TLR4-TRIF signal transduction pathway, and reduce the focal inflammatory response after I/R, and then stimulate endogenous neuroprotective effect.

【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743.3

【參考文獻】

相關期刊論文 前5條

1 Wang-sheng Duanmu;Liu Cao;Jing-yu Chen;Hong-fei Ge;Rong Hu;Hua Feng;;Ischemic postconditioning protects against ischemic brain injury by up-regulation of acid-sensing ion channel 2a[J];Neural Regeneration Research;2016年04期

2 王鵬;趙仁亮;呂敬雷;隋雪琴;高翔;;缺血后處理下調大鼠腦缺血再灌注時TLR4和NF-кB表達[J];中風與神經疾病雜志;2012年06期

3 呂敬雷;王國峰;王鵬;趙仁亮;;缺血后處理下調腦缺血再灌注大鼠白細胞介素-1β和腫瘤壞死因子-α表達[J];國際腦血管病雜志;2012年02期

4 郭朝暉;李峰;王維治;;腦缺血的損傷機制及其保護性干預(英文)[J];Neuroscience Bulletin;2009年03期

5 孫靜;董海龍;欒琪;趙，

本文編號：1891844