小鼠腦梗死后腦組織Pannexin1與HDAC9的表達(dá)改變及其意義
發(fā)布時(shí)間：2018-08-17 12:52
【摘要】：研究背景與目的根據(jù)全球流行病學(xué)的調(diào)查,腦卒中(Cerebral Stroke)是導(dǎo)致人類死亡的第二大病因,其中缺血性腦卒中是腦血管病中最常見的一種類型,由于其高致殘率與高死亡率等原因,腦卒中仍然對人類生命健康構(gòu)成重大的威脅�，F(xiàn)今缺血性腦卒中的治療手段十分有限,至今仍缺乏有效的臨床治療藥物。深入研究缺血性腦卒中的發(fā)病機(jī)制以及尋找其潛在的治療靶點(diǎn)是現(xiàn)在研究的熱點(diǎn)。Pannexin1廣泛分布于哺乳動物的中樞神經(jīng)系統(tǒng)中,主要位于細(xì)胞膜上,形成膜通道結(jié)構(gòu)。已有研究發(fā)現(xiàn)基于Pannexin1的腦梗死治療策略在部分動物實(shí)驗(yàn)中得到驗(yàn)證,但對于Pannexin1蛋白在腦梗死中的表達(dá)情況尚未有較深入研究。因此本課題通過小鼠腦缺血模型探討Pannexin1表達(dá)的改變,希望為后續(xù)Pannexin1在腦梗死的作用與機(jī)制的研究提供新的理論依據(jù)。最近研究發(fā)現(xiàn),表觀遺傳學(xué)機(jī)制組蛋白去乙酰化等在缺血性腦梗死的病理機(jī)制中也起到重要的作用。在動物腦梗死模型上多種組蛋白去乙酰化酶抑制劑(HDACs Inhibitors)也被證實(shí)具有神經(jīng)保護(hù)作用,HDACs家族具有多個(gè)亞型,部分亞型的作用與機(jī)制已被探討,其中組蛋白去乙�；�9(HDAC9)在腦梗死中的作用尚未深入研究。因此,本課題通過觀察HDAC9在腦梗死后表達(dá)的情況,探索其與腦梗死發(fā)生的關(guān)系,從而進(jìn)一步了解HDAC9在缺血性腦卒中的相關(guān)機(jī)制。第一部分小鼠腦梗死后腦組織Pannexin1的表達(dá)改變及其意義目的:檢測線栓法大腦中動脈阻塞(MCAO)后小鼠不同部位腦組織Pannexin1在卒中后不同時(shí)間點(diǎn)的表達(dá)情況,探討Pannexin1與缺血性腦卒中的關(guān)系。方法:健康雄性C57BL/6小鼠隨機(jī)分為兩組:假手術(shù)組與模型組,模型組按照Koizumi' s線栓法進(jìn)行小鼠腦梗死模型的構(gòu)建,假手術(shù)組只進(jìn)行頸部正中縱行剖開后縫合。模型組根據(jù)術(shù)后不同時(shí)間(4h,8h,12h,1d,3d,7d and 14d)提取健側(cè)與患側(cè)腦組織。采用免疫熒光染色觀察Pannexin1腦組織內(nèi)分布位置,以及腦梗死后Pannexin1分別與MAP2和GFAP的關(guān)系,采用Western免疫印跡法與實(shí)時(shí)熒光定量PCR法檢測各組不同部位(梗死側(cè)/對側(cè)皮層//假手術(shù)組皮層)Pannexin1的表達(dá)變化。結(jié)果:1.免疫熒光染色:梗死灶周圍腦組織Pannexin1的熒光強(qiáng)度較其他部位明顯增強(qiáng)。2.免疫熒光雙標(biāo)染色:Pannexin1與MAP2存在共定位,與GFAP不存在共定位表達(dá)。3.蛋白與mRNA表達(dá)檢測:(1)小鼠梗死側(cè)腦組織Pannexin1的表達(dá)比健側(cè)大腦及對照組高,并具有統(tǒng)計(jì)學(xué)意義(P0.05),mRNA與蛋白表達(dá)的情況相似。(2)腦梗死后4小時(shí),Pannexin1的表達(dá)已出現(xiàn)上調(diào),到梗死后3天達(dá)到高峰,而后逐步下降,到14天Pannexin1的表達(dá)仍高于對照組。(3)進(jìn)一步檢測Pannexin蛋白家族另一亞型Pannexin2表達(dá)情況:腦梗死后Pannexin2蛋白并沒有發(fā)生上調(diào)或下調(diào)。結(jié)論:1.腦梗死后Pannexin1表達(dá)的上調(diào)及隨時(shí)間表達(dá)改變的情況,進(jìn)一步說明Pannexin1與缺血性卒中密切相關(guān)。2.小鼠腦梗死后Pannexin1主要在神經(jīng)元內(nèi)表達(dá),而非星形膠質(zhì)細(xì)胞上。3.Pannexin2蛋白可能不與Pannexin1協(xié)同參與腦梗死的病理過程。第二部分小鼠腦梗死后腦組織HDAC9的表達(dá)改變及其意義目的:檢測線栓法大腦中動脈阻塞(MCAO)后小鼠不同部位腦組織HDCA9的表達(dá)及胞內(nèi)分布變化,探討HDAC9與缺血性腦卒中的關(guān)系。方法:健康雄性C57BL/6小鼠隨機(jī)分為兩組:假手術(shù)組與模型組,模型組按照Koizumi' s線栓法進(jìn)行小鼠腦梗死模型的構(gòu)建,假手術(shù)組只進(jìn)行頸部正中縱行剖開后縫合。采用免疫熒光染色觀察HDAC9腦組織內(nèi)分布位置與胞內(nèi)分布變化,采用Western免疫印跡法與實(shí)時(shí)熒光定量PCR法檢測各組不同部位(梗死側(cè)/對側(cè)皮層/MCAO組小腦/假手術(shù)組皮層/假手術(shù)組小腦)HDAC9的表達(dá)變化。結(jié)果:1.免疫熒光染色:(1)腦梗死后,梗死灶周圍腦組織HDAC9的熒光強(qiáng)度較其他部位明顯增強(qiáng)。(2)高倍鏡下與假手術(shù)組對比,梗死灶周圍HDAC9胞漿內(nèi)表達(dá)增多,核內(nèi)表達(dá)減少。2.蛋白與mRNA表達(dá)檢測:與各組對比,梗死側(cè)腦組織HDAC9表達(dá)顯著升高,具有統(tǒng)計(jì)學(xué)差異(均P0.05),蛋白與mRNA表達(dá)情況相一致。結(jié)論:HDAC9表達(dá)的上調(diào)以及細(xì)胞內(nèi)表達(dá)的改變與缺血性卒中密切相關(guān),可能參與其病理生理過程。
[Abstract]:BACKGROUND AND OBJECTIVE According to the global epidemiological survey, cerebral apoplexy (Cerebral Stroke) is the second leading cause of human death. Ischemic stroke is the most common type of cerebrovascular disease. Because of its high disability rate and high mortality, stroke still poses a major threat to human life and health. Therapeutic methods for ischemic stroke are very limited, and there is still no effective clinical drug. It is a hotspot to study the pathogenesis and potential therapeutic targets of ischemic stroke. It has been found that Pannexin-1-based cerebral infarction therapy strategy has been verified in some animal experiments, but the expression of Pannexin-1 protein in cerebral infarction has not been studied in depth. Therefore, this study explored the changes of Pannexin-1 expression in cerebral infarction model of mice, hoping to follow up the role of Pannexin-1 in cerebral infarction and Recent studies have shown that epigenetic histone deacetylation plays an important role in the pathogenesis of ischemic cerebral infarction. Many histone deacetylase inhibitors (HDACs Inhibitors) have been shown to have neuroprotective effects in animal models of cerebral infarction, HDACs expert There are many subtypes of HDAC9 family, and some subtypes of HDAC9 have been studied. The role of histone deacetylase 9 (HDAC9) in cerebral infarction has not been thoroughly studied. Part I. Expression of Pannexin 1 in brain tissue of mice after cerebral infarction and its significance Objective: To detect the expression of Pannexin 1 in different parts of brain tissue of mice after middle cerebral artery occlusion (MCAO) by thread embolization at different time points after stroke, and to explore the relationship between Pannexin 1 and ischemic stroke. The model group was divided into two groups randomly: sham operation group and model group. The model group was constructed by Koizumi's thread embolization method. The sham operation group was only performed median longitudinal neck dissection and suture. The distribution of Pannexin 1 in brain tissue and the relationship between Pannexin 1 and MAP2 and GFAP after cerebral infarction were detected by Western blot and real-time fluorescence quantitative PCR. Results: 1. Immunofluorescence staining: Pannexin 1 fluorescence in brain tissue around infarction. Immunofluorescence double labeling staining: Pannexin 1 and MAP2 co-localized, and GFAP did not co-localized expression. 3. Protein and mRNA expression detection: (1) The expression of Pannexin 1 in the infarcted brain tissue of mice was higher than that in the healthy brain and control group, and the expression of mRNA and protein was similar (P 0.05). (2) Four hours after cerebral infarction, the expression of Pannexin 1 was up-regulated, reached its peak at 3 days after infarction, then gradually decreased, and was still higher than that of the control group at 14 days. (3) Further detection of Pannexin 2 protein expression in another Pannexin protein family subtype: Pannexin 2 protein was not up-regulated or down-regulated after cerebral infarction. The up-regulation of Pannexin-1 expression after cerebral infarction and the change of Pannexin-1 expression with time further indicate that Pannexin-1 is closely related to ischemic stroke. 2. Pannexin-1 is mainly expressed in neurons after cerebral infarction in mice, but not in astrocytes. 3. Pannexin-2 protein may not participate in the pathological process of cerebral infarction with Pannexin-1. Objective: To detect the expression and intracellular distribution of HDAC9 in different brain tissues of mice after middle cerebral artery occlusion (MCAO) by thread embolization, and to explore the relationship between HDAC9 and ischemic stroke. The model of cerebral infarction in mice was established by oizumi's thread embolization, and the sham-operated group was sutured only after median and longitudinal neck dissection. Results: 1. Immunofluorescence staining showed that: (1) After cerebral infarction, the fluorescence intensity of HDAC9 in the brain tissue around the infarct was significantly higher than that in other parts. (2) Compared with the sham operation group, the expression of HDAC9 increased in the cytoplasm and decreased in the nucleus. The expression of HDAC9 in infarcted brain tissue was significantly higher than that in other groups (all P 0.05). The expression of HDAC9 protein was consistent with that of mRNA. Conclusion: The up-regulation of HDAC9 expression and the change of intracellular expression were closely related to ischemic stroke, which may be involved in its pathophysiological process.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743.3
本文編號：2187690
[Abstract]:BACKGROUND AND OBJECTIVE According to the global epidemiological survey, cerebral apoplexy (Cerebral Stroke) is the second leading cause of human death. Ischemic stroke is the most common type of cerebrovascular disease. Because of its high disability rate and high mortality, stroke still poses a major threat to human life and health. Therapeutic methods for ischemic stroke are very limited, and there is still no effective clinical drug. It is a hotspot to study the pathogenesis and potential therapeutic targets of ischemic stroke. It has been found that Pannexin-1-based cerebral infarction therapy strategy has been verified in some animal experiments, but the expression of Pannexin-1 protein in cerebral infarction has not been studied in depth. Therefore, this study explored the changes of Pannexin-1 expression in cerebral infarction model of mice, hoping to follow up the role of Pannexin-1 in cerebral infarction and Recent studies have shown that epigenetic histone deacetylation plays an important role in the pathogenesis of ischemic cerebral infarction. Many histone deacetylase inhibitors (HDACs Inhibitors) have been shown to have neuroprotective effects in animal models of cerebral infarction, HDACs expert There are many subtypes of HDAC9 family, and some subtypes of HDAC9 have been studied. The role of histone deacetylase 9 (HDAC9) in cerebral infarction has not been thoroughly studied. Part I. Expression of Pannexin 1 in brain tissue of mice after cerebral infarction and its significance Objective: To detect the expression of Pannexin 1 in different parts of brain tissue of mice after middle cerebral artery occlusion (MCAO) by thread embolization at different time points after stroke, and to explore the relationship between Pannexin 1 and ischemic stroke. The model group was divided into two groups randomly: sham operation group and model group. The model group was constructed by Koizumi's thread embolization method. The sham operation group was only performed median longitudinal neck dissection and suture. The distribution of Pannexin 1 in brain tissue and the relationship between Pannexin 1 and MAP2 and GFAP after cerebral infarction were detected by Western blot and real-time fluorescence quantitative PCR. Results: 1. Immunofluorescence staining: Pannexin 1 fluorescence in brain tissue around infarction. Immunofluorescence double labeling staining: Pannexin 1 and MAP2 co-localized, and GFAP did not co-localized expression. 3. Protein and mRNA expression detection: (1) The expression of Pannexin 1 in the infarcted brain tissue of mice was higher than that in the healthy brain and control group, and the expression of mRNA and protein was similar (P 0.05). (2) Four hours after cerebral infarction, the expression of Pannexin 1 was up-regulated, reached its peak at 3 days after infarction, then gradually decreased, and was still higher than that of the control group at 14 days. (3) Further detection of Pannexin 2 protein expression in another Pannexin protein family subtype: Pannexin 2 protein was not up-regulated or down-regulated after cerebral infarction. The up-regulation of Pannexin-1 expression after cerebral infarction and the change of Pannexin-1 expression with time further indicate that Pannexin-1 is closely related to ischemic stroke. 2. Pannexin-1 is mainly expressed in neurons after cerebral infarction in mice, but not in astrocytes. 3. Pannexin-2 protein may not participate in the pathological process of cerebral infarction with Pannexin-1. Objective: To detect the expression and intracellular distribution of HDAC9 in different brain tissues of mice after middle cerebral artery occlusion (MCAO) by thread embolization, and to explore the relationship between HDAC9 and ischemic stroke. The model of cerebral infarction in mice was established by oizumi's thread embolization, and the sham-operated group was sutured only after median and longitudinal neck dissection. Results: 1. Immunofluorescence staining showed that: (1) After cerebral infarction, the fluorescence intensity of HDAC9 in the brain tissue around the infarct was significantly higher than that in other parts. (2) Compared with the sham operation group, the expression of HDAC9 increased in the cytoplasm and decreased in the nucleus. The expression of HDAC9 in infarcted brain tissue was significantly higher than that in other groups (all P 0.05). The expression of HDAC9 protein was consistent with that of mRNA. Conclusion: The up-regulation of HDAC9 expression and the change of intracellular expression were closely related to ischemic stroke, which may be involved in its pathophysiological process.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743.3
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