【摘要】：在許多國家,卒中已成為了主要的致死原因。在中國縣鄉(xiāng),腦血管病為第二位致死原因;在中國的大城市,腦血管病是第三位致死原因。腦卒中后的殘疾及功能障礙給患者與家庭帶來了極大的負擔。卒中患者中大部分為缺血性卒中,少部分為出血性卒中。分布于免疫細胞、血管內(nèi)皮細胞以及腦組織固有細胞等表面的跨膜TLRs可能介導腦缺血后的炎癥反應。研究顯示,TLR2在腦缺血后數(shù)小時即上調,可能參與了腦缺血后神經(jīng)元損傷。Ziegler等研究表明,MCAO后注射抗TLR2封閉性單克隆抗體T2.5,免疫組化發(fā)現(xiàn)CD1 1b陽性細胞數(shù)量下降,還能抑制白細胞聚集以及小膠質細胞遷移;同時導致腦缺血后NeuN陽性細胞數(shù)量增加,提示抑制TLR2具有神經(jīng)保護作用。髓樣分化因子(MyD88)是TLR信號通路中的重要轉導蛋白,其依賴的信號通路以及調控的基因產(chǎn)物在固有免疫和適應性免疫中發(fā)揮著關鍵作用�；|金屬蛋白酶9(MMP-9)可降解腦血管周圍基膜的成分從而導致BBB通透性增加,白細胞侵潤、腦水腫及出血轉化。目的:探討大鼠腦缺血再灌注后,Toll樣受體2(Toll-like receptor 2,TLR2)、髓樣分化因子88(Myeloid differentiation factor 88,MyD88)與基質金屬蛋白酶9(matrix metalloproteinase-9,MMP-9)三者之間的變化關系,以及三者在腦缺血再灌注后腦損傷形成過程中的可能作用機制。方法:制作Sprague-Dawley大鼠(250-280g)右側大腦中動脈栓塞(middle cerebral artery occlusion,MCAO)2小時再灌注模型。實驗共分為三組:缺血組、sham組(對照組)、T2.5處理組(大鼠MCAO 2h再灌注開始時,經(jīng)頸靜脈注射T2.5,劑量為0.1212μg/g)。在腦缺血再灌注后不同時間點,選取缺血組、sham組和T2.5處理組缺血側腦組織來進行以下實驗:1、MCAO 再灌注 1h、2h、3h、6h、12h、24h 六個時間點,利用 western blot來測定sham組和缺血組缺血側大腦皮層中TLR2、MyD88及MMP-9的蛋白表達水平變化情況(每個時間點五只老鼠,n=5)。2、MCAO再灌注開始時,給予TLR2拮抗劑T2.5處置。缺血再灌注24h時,利用western blot測定缺血組和T2.5處理組缺血側大腦皮層中TLR2、MyD88及MMP-9的蛋白表達水平變化情況(該時間點五只老鼠,n=5)。3、MCAO再灌注24h后,測定三組中的腦梗死體積(TTC染色法)、腦水腫(干濕重法)、血腦屏障(blood-brain barrier,BBB)通透性(Evan's藍法)以及神經(jīng)功能缺損評分(每種測量五只老鼠,n=5)。結果:1、Western blot結果顯示,在大鼠MCAO再灌注6h,缺血組缺血側皮層中TLR2較sham組即開始升高,且有顯著性差異(p0.05),持續(xù)至24h(p0.05)。2、Western blot結果顯示,在大鼠MCAO再灌注6h,缺血組缺血側皮層中MyD88較sham組即開始升高,且有顯著性差異(p0.01),持續(xù)至24h(p0.05)。3、Western blot結果顯示,在大鼠MCAO再灌注24h,缺血組缺血側皮層中MMP-9較sham組升高,具有顯著性差異(p0.05)。4、大鼠MCAO再灌注24h,缺血組缺血側腦組織BBB通透性,腦水腫程度,腦梗死體積及神經(jīng)功能缺損評分均較sham組升高,且有顯著性差異(p0.01、p0.01、p0.001、p0.001)。5、Western blot結果顯示,大鼠MCAO再灌注24h,T2.5處理組與缺血組比較,TLR2、MyD88及MMP-9表達水平均降低(p0.05)。大鼠MCAO再灌注24h,T2.5處理組BBB通透性、腦水腫程度、腦梗死體積、神經(jīng)功能缺損評分均較缺血組下降,且均有顯著性差異(p0.01、p0.05、p0.01、p0.01)。結論:1、腦缺血再灌注后6h,TLR2及MyD88即開始上升;腦缺血再灌注后24h,MMP-9才開始上升,以上因素可能通過增加BBB通透性,參與了腦水腫、腦梗死及神經(jīng)功能損傷。2、TLR2拮抗劑T2.5可能通過拮抗TLR2-MyD88信號通路,減少MMP-9的表達,從而減輕BBB通透性,緩解腦水腫、減少腦梗死及修復神經(jīng)功能,最終改善缺血再灌注腦損傷。
[Abstract]:Stroke has become a major cause of death in many countries. In China, cerebrovascular disease is the second cause of death; in big cities in China, cerebrovascular disease is the third cause of death. Disability and dysfunction after stroke have a great burden on the patient and the family. Most of stroke patients are ischemic stroke and a small part is hemorrhagic stroke. Transmembrane TLRs distributed on the surface of immune cells, vascular endothelial cells and native cells of brain tissue may mediate inflammatory responses following cerebral ischemia. The study showed that TLR2 was upregulated after focal cerebral ischemia and may be involved in neuronal damage after cerebral ischemia. Ziegler et al. showed that the number of CD1b positive cells decreased and the number of CD1b positive cells decreased, and the number of N positive cells increased after cerebral ischemia. It is suggested that inhibition of TLR2 has neuroprotective effect. The myeloid differentiation factor (MyD88) is an important transduction protein in TLR signaling pathway, and its dependent signaling pathway and regulated gene products play a key role in innate immunity and adaptive immunity. Matrix metalloproteinase-9 (MMP-9) can degrade the components of basement membrane around cerebral vessels, which leads to increased permeability of the brain, invasion of leucocytes, cerebral edema and hemorrhagic transformation. Objective: To investigate the changes of Toll-like receptor 2 (TLR2), myeloid differentiation factor (88, MyD88) and matrix metalloproteinase-9 (MMP-9) after cerebral ischemia-reperfusion in rats. Methods: A 2-hour reperfusion model was established in the right cerebral artery of Sprague-Dawley rats (250-280g). The experiment was divided into three groups: ischemic group, sham group (control group), and T2. 5 treatment group (at the beginning of reperfusion in the rat heart O 2h, the dose was 0. 1212 ug/ g). At different time points after cerebral ischemia-reperfusion, ischemia group, sham group and T2. 5 treatment group were selected for the following experiments: 1, 1h, 2h, 3h, 6h, 12h and 24h were reperfusion 1h, 2h, 3h, 6h, 12h and 24h, and the TLR2 in cerebral cortex of ischemic side was determined by western blot. The levels of protein expression in MyD88 and MMP-9 (five rats per time point, n = 5). 2. At the start of reperfusion, TLR2 antagonist T2. 5 was administered. The levels of TLR2, MyD88 and MMP-9 in cerebral cortex of ischemic-side cerebral cortex were determined by western blot. The levels of TLR2, MyD88 and MMP-9 in ischemic-side cerebral cortex were determined by western blot. Brain edema (dry-wet weight method), blood-brain barrier (BBB) permeability (Evan's blue method) and neurological deficit score (five mice each, n = 5). Results: 1. Western blot analysis showed that there was a significant difference (P0.05) in the ischemia-side cortex of rats after reperfusion 6h, and there was significant difference (P0.05). There was a significant difference in the expression of MMP-9 in the ischemic side cortex of the ischemic group (P0.05). The results of Western blot showed that the MMP-9 was higher in the ischemic side cortex of the ischemia group than in the sham group (P0.05). The permeability of brain tissue, the degree of cerebral edema, the volume of cerebral infarction and neurological deficit were higher in ischemic group than sham group, and there was a significant difference (P. 01, No. 01, No. 001, V1.001). The results of Western blot showed that the reperfusion 24h, T2. 5 treatment group in rats were compared with ischemia group and TLR2. The expression levels of MyD88 and MMP-9 decreased (P0.05). The permeability, degree of brain edema, volume of cerebral infarction and neurological deficit were all lower than that in ischemia group. Conclusion: 1. After cerebral ischemia and reperfusion, 6h, TLR2 and MyD88 started to rise; after cerebral ischemia and reperfusion 24h, MMP-9 began to rise. The above factors may be involved in cerebral edema, cerebral infarction and nerve function injury by increasing permeability of TLR2. The TLR2 antagonist, T2. 5, may antagonize the signal pathway of TLR2-MyD88. the expression of MMP-9 is reduced, so that the permeability of the brain is reduced, the cerebral edema is relieved, the cerebral infarction is reduced, the nerve function is restored, and the brain injury of the ischemia reperfusion is finally improved.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743

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