缺血性腦損傷中TLR2介導(dǎo)的MyD88信號(hào)調(diào)控機(jī)制的研究
發(fā)布時(shí)間：2018-10-29 09:15
【摘要】：在許多國(guó)家,卒中已成為了主要的致死原因。在中國(guó)縣鄉(xiāng),腦血管病為第二位致死原因;在中國(guó)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(lái)了極大的負(fù)擔(dān)。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(xì)胞、血管內(nèi)皮細(xì)胞以及腦組織固有細(xì)胞等表面的跨膜TLRs可能介導(dǎo)腦缺血后的炎癥反應(yīng)。研究顯示,TLR2在腦缺血后數(shù)小時(shí)即上調(diào),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(yáng)性細(xì)胞數(shù)量下降,還能抑制白細(xì)胞聚集以及小膠質(zhì)細(xì)胞遷移;同時(shí)導(dǎo)致腦缺血后NeuN陽(yáng)性細(xì)胞數(shù)量增加,提示抑制TLR2具有神經(jīng)保護(hù)作用。髓樣分化因子(MyD88)是TLR信號(hào)通路中的重要轉(zhuǎn)導(dǎo)蛋白,其依賴(lài)的信號(hào)通路以及調(diào)控的基因產(chǎn)物在固有免疫和適應(yīng)性免疫中發(fā)揮著關(guān)鍵作用�；|(zhì)金屬蛋白酶9(MMP-9)可降解腦血管周?chē)さ某煞謴亩鴮?dǎo)致BBB通透性增加,白細(xì)胞侵潤(rùn)、腦水腫及出血轉(zhuǎn)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(zhì)金屬蛋白酶9(matrix metalloproteinase-9,MMP-9)三者之間的變化關(guān)系,以及三者在腦缺血再灌注后腦損傷形成過(guò)程中的可能作用機(jī)制。方法:制作Sprague-Dawley大鼠(250-280g)右側(cè)大腦中動(dòng)脈栓塞(middle cerebral artery occlusion,MCAO)2小時(shí)再灌注模型。實(shí)驗(yàn)共分為三組:缺血組、sham組(對(duì)照組)、T2.5處理組(大鼠MCAO 2h再灌注開(kāi)始時(shí),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(shí)間點(diǎn),選取缺血組、sham組和T2.5處理組缺血側(cè)腦組織來(lái)進(jìn)行以下實(shí)驗(yàn):1、MCAO 再灌注 1h、2h、3h、6h、12h、24h 六個(gè)時(shí)間點(diǎn),利用 western blot來(lái)測(cè)定sham組和缺血組缺血側(cè)大腦皮層中TLR2、MyD88及MMP-9的蛋白表達(dá)水平變化情況(每個(gè)時(shí)間點(diǎn)五只老鼠,n=5)。2、MCAO再灌注開(kāi)始時(shí),給予TLR2拮抗劑T2.5處置。缺血再灌注24h時(shí),利用western blot測(cè)定缺血組和T2.5處理組缺血側(cè)大腦皮層中TLR2、MyD88及MMP-9的蛋白表達(dá)水平變化情況(該時(shí)間點(diǎn)五只老鼠,n=5)。3、MCAO再灌注24h后,測(cè)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(lán)法)以及神經(jīng)功能缺損評(píng)分(每種測(cè)量五只老鼠,n=5)。結(jié)果:1、Western blot結(jié)果顯示,在大鼠MCAO再灌注6h,缺血組缺血側(cè)皮層中TLR2較sham組即開(kāi)始升高,且有顯著性差異(p0.05),持續(xù)至24h(p0.05)。2、Western blot結(jié)果顯示,在大鼠MCAO再灌注6h,缺血組缺血側(cè)皮層中MyD88較sham組即開(kāi)始升高,且有顯著性差異(p0.01),持續(xù)至24h(p0.05)。3、Western blot結(jié)果顯示,在大鼠MCAO再灌注24h,缺血組缺血側(cè)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(cè)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(píng)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(jié)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(dá)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(píng)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(jié)論:1、腦缺血再灌注后6h,TLR2及MyD88即開(kāi)始上升;腦缺血再灌注后24h,MMP-9才開(kāi)始上升,以上因素可能通過(guò)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(guò)拮抗TLR2-MyD88信號(hào)通路,減少M(fèi)MP-9的表達(dá),從而減輕BBB通透性,緩解腦水腫、減少腦梗死及修復(fù)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(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R743
本文編號(hào)：2297272
[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(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R743
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 章烈;李園;周總光;;髓樣分化因子88的研究進(jìn)展:把握TLR信號(hào)通路中的瓶頸[J];中國(guó)普外基礎(chǔ)與臨床雜志;2013年06期
2 方麗娟;孟民杰;;髓樣分化因子88研究進(jìn)展[J];廣東藥學(xué)院學(xué)報(bào);2011年02期
3 陳興洲,陸兵勛;基質(zhì)金屬蛋白酶與腦血管病[J];國(guó)外醫(yī)學(xué)(腦血管疾病分冊(cè));1999年04期
相關(guān)碩士學(xué)位論文 前2條
1 丁曉蔚;腦缺血再灌注后TLR-2與MMP-2表達(dá)的變化關(guān)系及其在缺血性腦損傷中的作用機(jī)制[D];昆明理工大學(xué);2016年
2 王帥;TLR2介導(dǎo)的MyD88信號(hào)通路在HO-1保護(hù)肝臟IRI中的作用及機(jī)制[D];昆明醫(yī)科大學(xué);2013年
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