【作者】 代吉文；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 目的缺血性卒中以其高发病率、高致残率和高死亡率严重危害着人类健康。缺血性脑损伤的病理生理机制极为复杂,其涉及的许多相关基因和蛋白的改变及其复杂的联系尚未研究清楚。脑缺血预处理(BIP)是指对大脑预先进行一次或多次短暂的非致死性缺血刺激后,机体获得对致死性脑缺血的耐受性。BIP的本质是通过激发内源性神经保护机制,促进新的蛋白质合成,从而阻止或减弱脑缺血引起的缺血性级联反应。HSP22是一个哺乳动物小热休克蛋白(sHSPs)家族新成员,在所有肌肉相关组织和脑组织表达,它具有分子伴侣样活性、调节细胞凋亡等作用。本研究采用沙土鼠前脑缺血再灌注模型以及应用BIP进行干预。一方面观察BIP对缺血脑组织超微结构、神经细胞凋亡、HSP22mRNA及蛋白表达以及凋亡相关因子Bcl-2、BaxmRNA及蛋白表达和活化caspases-3蛋白表达变化的影响;另一方面观察BIP对缺血脑组织MAP2、GFAPmRNA及蛋白表达变化的影响,以及通过免疫荧光共聚焦显微镜和免疫金双重标记电镜观察HSP22与MAP2是否在神经元共定位和HSP22与GFAP是否在星形胶质细胞共定位。进一步探讨BIP对沙土鼠脑缺血再灌注后HSP22表达的影响及其神经保护作用。方法1、实验动物模型的制备及分组取健康雄性沙土鼠120只,随机分成正常对照组、假手术组、缺血再灌注(I/R)组和预缺血(IPC)组,I/R组和IPC组又随机分为1d、3d和7d亚组。对照组和假手术组各18只;I/R组和IPC组1d、7d亚组各12只,3d亚组各18只。通过夹闭双侧颈总动脉10分钟的方法建立沙土鼠前脑缺血再灌注模型。IPC组沙土鼠在夹闭双侧颈总动脉10分钟前2天给予间断一天的2次2分钟缺血。2、透射电镜观察BIP对神经组织超微结构的改变。3、TUNEL染色方法检测BIP对脑组织神经细胞凋亡的影响。4、免疫荧光共聚焦方法观察BIP后HSP22分别与神经元细胞骨架蛋白MAP2和星形胶质细胞骨架蛋白GFAP共定位的情况。5、电镜免疫金双重标记方法观察BIP后HSP22分别与神经元细胞骨架蛋白MAP2和星形胶质细胞骨架蛋白GFAP共定位的情况。6、RT-PCR方法检测BIP对脑组织中HSP22、Bcl-2、Bax、MAP2、GFAP基因的mRNA表达的影响。7、Western blot方法检测BIP对脑组织中HSP22、Bcl-2、Bax、caspase-3、MAP2、GFAP蛋白表达的影响。结果1、沙土鼠前脑皮层神经细胞超微结构的变化正常对照组和假手术组前脑皮层神经细胞超微结构正常。I/R组3d亚组可观察到细胞核膜模糊,胞质内线粒体多有嵴减少,外膜局部破坏,粗面内质网轻度扩张。IPC组3d亚组也可观察到上述损伤的神经细胞,但超微结构的损伤明显减轻。2、沙土鼠前脑皮层TUNEL染色结果正常对照组和假手术组沙土鼠前脑皮层几乎未见TUNEL染色阳性细胞。I/R组1d亚组凋亡细胞数较少,3d、7d亚组凋亡细胞数明显增多,且随时间延长,凋亡细胞数逐渐增多。IPC组在1d、3d、7d亚组凋亡细胞数分别较脑缺血再灌注组明显减少(P＜0.05)。3、沙土鼠前脑皮层免疫荧光共聚焦结果HSP22的红色免疫荧光及MAP2的绿色免疫荧光在神经元细胞浆和树突内发生了明显的重叠。HSP22的红色免疫荧光及GFAP的绿色免疫荧光在星形胶质细胞的胞体和突起发生了明显的重叠。4、沙土鼠前脑皮层电镜免疫金双重标记观察结果HSP22免疫金颗粒(10nm)与MAP2免疫金颗粒(15nm)在神经元细胞浆和树突内发生了明显的重叠。HSP22免疫金颗粒(10nm)与GFAP免疫金颗粒(15nm)在星形胶质细胞的细胞浆和突起内发生了明显的重叠。5、HSP22基因的mRNA和蛋白表达结果预缺血组第3d脑组织HSP22mRNA表达与缺血再灌注组比较显著增加(p＜0.05)。预缺血组第1d、3d、7d脑组织HSP22蛋白表达与缺血再灌注组比较显著增加(p＜0.05)。6、Bcl-2、Bax基因的mRNA和蛋白表达结果预缺血组第3d脑组织Bcl-2mRNA表达与缺血再灌注组比较显著增加(p＜0.05),预缺血组第1d、3d、7d脑组织Bcl-2蛋白表达与缺血再灌注组比较显著增加(p＜0.05);缺血再灌注组第3d脑组织BaxmRNA表达与预缺血组比较显著增加(p＜0.05),缺血再灌注组第1d、3d、7d脑组织Bax蛋白表达与预缺血组比较显著增加(p＜0.05);预缺血组第3d脑组织BaxmRNA表达与正常对照组比较差异无显著性(p＞0.05),预缺血组第1d、3d、7d脑组织Bax蛋白表达与正常对照组比较差异无显著性(p＞0.05)。7、活化caspase-3蛋白表达结果预缺血组第1d、3d、7d脑组织活化caspase-3蛋白表达与缺血再灌注组比较显著降低(p＜0.05)。8、MAP2基因的mRNA和蛋白表达结果预缺血组第3d脑组织MAP2mRNA表达与缺血再灌注组比较显著增加(p＜0.05),预缺血组第1d、3d、7d脑组织MAP2蛋白表达与缺血再灌注组比较显著增加(p＜0.05)。9、GFAP基因的mRNA和蛋白表达结果预缺血组第3d脑组织GFAPmRNA表达与缺血再灌注组比较显著降低(p＜0.05);预缺血组第1d、3d、7d脑组织GFAP蛋白表达与缺血再灌注组比较显著降低(p＜0.05)。结论1、BIP能够明显地减轻沙土鼠缺血再灌注后前脑皮层神经细胞超微结构的损伤,减少缺血再灌注后前脑皮层神经细胞凋亡,增加脑缺血再灌注后前脑组织抗凋亡因子Bcl-2mRNA和蛋白的表达,降低活化caspase-3蛋白的表达,而促凋亡因子BaxmRNA和蛋白的表达则没有显著变化。2、BIP能够明显地增加沙土鼠脑缺血再灌注后前脑组织HSP22mRNA和蛋白的表达。3、BIP能够明显地增加沙土鼠脑缺血再灌注后前脑组织神经元骨架蛋白MAP2mRNA和蛋白的表达;HSP22和MAP2共定位于沙土鼠前脑皮层神经元细胞浆和树突。4、BIP能够明显地降低沙土鼠脑缺血再灌注后前脑组织星形胶质细胞骨架蛋白GFAPmRNA和蛋白的表达;HSP22和GFAP共定位于沙土鼠前脑皮层星形胶质细胞的细胞浆和突起。更多还原

【Abstract】 ObjectiveIschemic stroke severely harm the health of human being because of its higher morbidity,crippling rate,and mortality.The pathophysiologic mechanisms of cerebral ischemia damage are very complicated.It is still unclear that the mechanisms involve changes of many interrelated genes and proteins and their complicated association. Brain ischemic preconditioning(BIP) refers to that the brain is produced marked tolerance to lately lethal ischemia after one or multiple times sublethal ischemia.The essence of BIP is to promote synthesis of new proteins by arousing the mechanism of endogenous neuroprotection and prevent or attenuate ischemic cascade response which is produced by cerebral ischemia.HSP22 is a new member of mammal small heat shock proteins(sHSPs) family.HSP22 is expressed predominatly in muscle and brain tissues.It possesses molecular chaperone activity,modulators of apoptosis,and so on.In our experiments,we set up a gerbil model of bilateral carotid artery occlusion(BCAO) and intervene it by BIP in two days before BCAO.In one hand,we observe the nerve tissue ultrastructural change,the change of brain tissue nerve cell apoptosis,and the change of HSP22,Bcl-2,Bax gene mRNA and protein expression and active caspases-3 protein expression in gerbil model of bilateral carotid artery occlusion after BIR In the other hand,we observe the change of MAP2,GFAP gene mRNA and protein expression,the co-localization between HSP22 and MAP2 of cellular skeleton in neurons,and the co-localization between HSP22 and GFAP of cellular skeleton in astrocytes in gerbil model of bilateral carotid artery occlusion after BIP by confocal Immunofluorographs and immunogold double-labeling of electron microscopy.We further research the effect of expression of HSP22 and neuroprotection of brain ischemic preconditioning after cerebral ischemia/reperfusion in gerbilsMethods1.Experimental animal model preparation and grouping.120 healthy male gerbils were divided into control group,sham operation group,ischemia/reperfution (I/R) group,and ischemic preconditioning(IPC) group randomly.In I/R group and IPC group there were three subgroup-1d subgroup,3d subgroup and 7d subgroup-according to the time after I/R.In control group and sham operation group there were 18 gerbils;In I/R group and IPC group there were 12 gerbils in 1d subgroup and 7d subgroup,and 18 gerbils in 3d subgroup.Gerbil models of bilateral carotid artery occlusion(BCAO) were established by occluding both carotid arteries for 10 min.Double 2-minute periods of preconditioning ischemia,which were induced with a 1-day interval,were induced in two days before gerbils models of BCAO in IPC group.2.Observe the effect of BIP on the nerve tissue ultrastructural change by transmission electron microscopy.3.Detect the effect of BIP on the brain tissue nerve cellular apoptosis by TUNEL staining.4.Observe the co-localization between HSP22 and MAP2 of cytoskeleton in neurons,and the co-localization between HSP22 and GFAP of cytoskeleton in astrocytes after BIP by confocal Immunofluorographs.5.Observe the co-localization between HSP22 and MAP2 of cytoskeleton in neurons,and the co-localization between HSP22 and GFAP of cytoskeleton in astrocytes after BIP by immunogold double-labeling of electron microscopy.6.Detect the effect of BIP on the brain tissue HSP22,Bcl-2,Bax,MAP2,GFAP gene mRNA expression by RT-PCR method.7.Detect the effect of BIP on the brain tissue HSP22,Bcl-2,Bax,caspase-3, MAP2,GFAP protein expression by Western blot method. Results1.The nerve tissue ultrastructural change of gerbil forebrain cortexIn control group and sham operation group,the nerve cell was complete in gerbil forebrain cortex.In 3d subgroup of I/R group,cell nucleus vague of membrane,fewer crest and external membrane rupture of mitochondria and mild extension of rough endoplasmic reticulum were observed under transmission electron microscopy.In ischemic preconditioning group,the above-mentioned ultrastructural impairment of nerve cell was much lower than that of I/R group.2.TUNEL stain of gerbil forebrain cortexIn control group and sham operation group,TUNEL-positive cells almost were not seen in gerbil forebrain cortex.In 1d subgroup of I/R group,there was several TUNEL-positive cells.In 3d and 7d subgroup of I/R group,there were many apoptotic cells.With the prolong of time,the number of apoptotic cells was increased. In 1d,3d and 7d subgroup of IPC group,TUNEL-positive cells were much less than that of I/R group in gerbil forebrain cortex(p＜0.05).3.Confocal ImmunofluorographsIn perikaryon and dendrite of all cortical neurons,an extensive overlap of HSP22 immunoreactivity(red) and MAP2 immunoreactivity(green) were were observed under confocal Immunofluorographs microscopy.In all cortical astrocytes,an extensive overlap of HSP22 immunoreactivity(red) and GFAP immunoreactivity (green) were observed under confocal immunofluorographs microscopy.4.Immunogold labelingIn perikaryon and dendrite of all cortical neurons,an extensive overlap of the immunogold granules of HSP22(10nm) and the immunogold granules of MAP2(15nm) were observed under electron microscopy.In cytoplasm and prominency of all cortical astrocytes,an extensive overlap of the immunogold granules of HSP22(10nm) and the immunogold granules of GFAP(15nm) were observed under electron microscopy.5.The expression of HSP22 gene mRNA and protein In 3d subgroup of IPC group,HSP22mRNA expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group,HSP22 protein expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).6.The expression of Bcl-2、Bax gene mRNA and proteinIn 3d subgroup of IPC group,Bcl-2mRNA expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group, the expression of Bcl-2 protein was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 3d subgroup of I/R group,Bax mRNA expression was much higher than that of IPC group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of I/R group,the expression of Bax protein was much higher than that of IPC group in gerbil forebrain cortex(p＜0.05).There was no distinct difference in gerbil forebrain cortex Bax mRNA expression between IPC group and contral group in 3d subgroup(p＞0.05).There was no distinct difference in gerbil forebrain cortex Bax protein expression between IPC group and contral group in 1d,3d,7d subgroup (p＜0.05).7.The expression of active caspase-3 proteinIn 1d,3d,7d subgroup of IPC group,the expression of active caspase-3 protein was much lower than that of I/R group in gerbil forebrain cortex(p＜0.05).8.The expression of MAP2 gene mRNA and proteinIn 3d subgroup of IPC group,MAP2mRNA expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group,MAP2 protein expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).9.The expression of GFAP gene mRNA and proteinIn 3d subgroup of IPC group,GFAPmRNA expression was much lower than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group, GFAP protein expression was much lower than that of I/R group in gerbil forebrain cortex(p＜0.05). Conclusions1.BIP can significantly ease the damage of nerve cells ultrastructural organization and decrease the nerve cell apoptosis of gerbil forebrain cortex after I/R. it can obviously increase the mRNA of anti-apoptosis factor Bcl-2 and protein expression and decrease active caspase-3 protein expression.However,there was no distinct difference in gerbil forebrain cortex the mRNA of pro-apoptosis factor Bax and protein expression.2.BIP can significantly increase the mRNA of HSP22 and protein expression of gerbil forebrain cortex after I/R.3.BIP can significantly increase the mRNA of nerve cytoskeleton protein MAP2 and protein expression of gerbil forebrain cortex after I/R;HSP22 and MAP2 are co-located in perikaryon and dendrite of gerbil forebrain cortical neurons4.BIP can significantly decrease the mRNA of astrocyte cytoskeleton protein GFAP and protein expression;HSP22 and GFAP are co-located in cytoplasm and prominency of gerbil forebrain cortical astrocytes.更多还原