小鼠脑梗死后脑组织Pannexin1与HDAC9的表达改变及其意义
发布时间：2018-08-17 12:52
【摘要】：研究背景与目的根据全球流行病学的调查,脑卒中(Cerebral Stroke)是导致人类死亡的第二大病因,其中缺血性脑卒中是脑血管病中最常见的一种类型,由于其高致残率与高死亡率等原因,脑卒中仍然对人类生命健康构成重大的威胁。现今缺血性脑卒中的治疗手段十分有限,至今仍缺乏有效的临床治疗药物。深入研究缺血性脑卒中的发病机制以及寻找其潜在的治疗靶点是现在研究的热点。Pannexin1广泛分布于哺乳动物的中枢神经系统中,主要位于细胞膜上,形成膜通道结构。已有研究发现基于Pannexin1的脑梗死治疗策略在部分动物实验中得到验证,但对于Pannexin1蛋白在脑梗死中的表达情况尚未有较深入研究。因此本课题通过小鼠脑缺血模型探讨Pannexin1表达的改变,希望为后续Pannexin1在脑梗死的作用与机制的研究提供新的理论依据。最近研究发现,表观遗传学机制组蛋白去乙酰化等在缺血性脑梗死的病理机制中也起到重要的作用。在动物脑梗死模型上多种组蛋白去乙酰化酶抑制剂(HDACs Inhibitors)也被证实具有神经保护作用,HDACs家族具有多个亚型,部分亚型的作用与机制已被探讨,其中组蛋白去乙酰化酶9(HDAC9)在脑梗死中的作用尚未深入研究。因此,本课题通过观察HDAC9在脑梗死后表达的情况,探索其与脑梗死发生的关系,从而进一步了解HDAC9在缺血性脑卒中的相关机制。第一部分小鼠脑梗死后脑组织Pannexin1的表达改变及其意义目的:检测线栓法大脑中动脉阻塞(MCAO)后小鼠不同部位脑组织Pannexin1在卒中后不同时间点的表达情况,探讨Pannexin1与缺血性脑卒中的关系。方法:健康雄性C57BL/6小鼠随机分为两组:假手术组与模型组,模型组按照Koizumi' s线栓法进行小鼠脑梗死模型的构建,假手术组只进行颈部正中纵行剖开后缝合。模型组根据术后不同时间(4h,8h,12h,1d,3d,7d and 14d)提取健侧与患侧脑组织。采用免疫荧光染色观察Pannexin1脑组织内分布位置,以及脑梗死后Pannexin1分别与MAP2和GFAP的关系,采用Western免疫印迹法与实时荧光定量PCR法检测各组不同部位(梗死侧/对侧皮层//假手术组皮层)Pannexin1的表达变化。结果:1.免疫荧光染色:梗死灶周围脑组织Pannexin1的荧光强度较其他部位明显增强。2.免疫荧光双标染色:Pannexin1与MAP2存在共定位,与GFAP不存在共定位表达。3.蛋白与mRNA表达检测:(1)小鼠梗死侧脑组织Pannexin1的表达比健侧大脑及对照组高,并具有统计学意义(P0.05),mRNA与蛋白表达的情况相似。(2)脑梗死后4小时,Pannexin1的表达已出现上调,到梗死后3天达到高峰,而后逐步下降,到14天Pannexin1的表达仍高于对照组。(3)进一步检测Pannexin蛋白家族另一亚型Pannexin2表达情况:脑梗死后Pannexin2蛋白并没有发生上调或下调。结论:1.脑梗死后Pannexin1表达的上调及随时间表达改变的情况,进一步说明Pannexin1与缺血性卒中密切相关。2.小鼠脑梗死后Pannexin1主要在神经元内表达,而非星形胶质细胞上。3.Pannexin2蛋白可能不与Pannexin1协同参与脑梗死的病理过程。第二部分小鼠脑梗死后脑组织HDAC9的表达改变及其意义目的:检测线栓法大脑中动脉阻塞(MCAO)后小鼠不同部位脑组织HDCA9的表达及胞内分布变化,探讨HDAC9与缺血性脑卒中的关系。方法:健康雄性C57BL/6小鼠随机分为两组:假手术组与模型组,模型组按照Koizumi' s线栓法进行小鼠脑梗死模型的构建,假手术组只进行颈部正中纵行剖开后缝合。采用免疫荧光染色观察HDAC9脑组织内分布位置与胞内分布变化,采用Western免疫印迹法与实时荧光定量PCR法检测各组不同部位(梗死侧/对侧皮层/MCAO组小脑/假手术组皮层/假手术组小脑)HDAC9的表达变化。结果:1.免疫荧光染色:(1)脑梗死后,梗死灶周围脑组织HDAC9的荧光强度较其他部位明显增强。(2)高倍镜下与假手术组对比,梗死灶周围HDAC9胞浆内表达增多,核内表达减少。2.蛋白与mRNA表达检测:与各组对比,梗死侧脑组织HDAC9表达显著升高,具有统计学差异(均P0.05),蛋白与mRNA表达情况相一致。结论:HDAC9表达的上调以及细胞内表达的改变与缺血性卒中密切相关,可能参与其病理生理过程。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
本文编号：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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
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