【作者】 张星；

【导师】 史继新；

【作者基本信息】 南京大学 ， 外科学， 2011， 博士

【摘要】 本课题通过建立新西兰大白兔蛛网膜下腔二次注血模型,主要研究以下内容：(1)SAH后p38MAPK在血管壁组织中的表达及其时相；(2)给予p38MAPK特异性抑制剂SB203580对SAH后血管壁炎症反应的抑制作用；(3)给予p38MAPK特异性抑制剂SB203580对SAH后血管内皮细胞凋亡的抑制作用。第一部分p38MAPK信号转导通路在蛛网膜下腔出血后脑血管痉挛中的作用目的：细胞内信号转导通路是近年蛛网膜下腔出血后脑血管痉挛发病机制中的研究热点。p38MAPK信号通路广泛参与组织细胞的生长、存活、分化、凋亡及炎症反应等病理生理过程。本实验旨在研究实验性SAH后p38MAPK在血管壁组织中的表达及其时相,以及应用p38MAPK抑制剂SB203580后脑血管痉挛是否得到改善。方法：第一组实验中,48例成年新西兰兔,随机分为4组：对照组(n=6),SAH3天组(n=6),SAH5天组(n=6),SAH7天组(n=6)。采用SAH模型为枕大池二次注血模型。分别于第一次注血后第3、5、7天,处死SAH组动物,取基底动脉标本。第二组实验中,24例成年新西兰兔,随机分为4组：对照组(n=6),SAH组(n=6),SAH+DMSO组(n=6),SAH+SB203580组(n=6)。动物模型同第一组实验,于第7天处死所有动物,获得基底动脉标本。Western Blot法测定血管壁组织中总p38MAPK、磷酸化p38MAPK (p-p38MAPK)、总ATF2及磷酸化ATF2的水平。活体灌注后HE染色测量基底动脉管腔面积。结果：(1)SAH后兔基底动脉p-p38MAPK与P-ATF2的水平在SAH后第3天即有明显升高,第5天达到高峰,第7天略有回落；(2) SB203580可明显降低SAH后p38MAPK、ATF2磷酸化水平,抑制了p38MAPK通路的活性；(3)SB203580明显缓解了脑血管痉挛。结论：p38MAPK的活性在SAH后痉挛血管中明显上调,其介导的信号通路在SAH后脑血管痉挛的病理过程中起重要作用。第二部分p38MAPK信号转导通路对蛛网膜下腔出血后脑血管壁炎症反应的作用目的：血管壁的炎症反应是导致蛛网膜下腔出血后脑血管痉挛的发生以及维持的重要因素。p38MAPK信号通路是参与调控组织细胞炎症反应的重要通路。本实验旨在研究应用p38MAPK特异性抑制齐(?)SB203580对实验性SAH后基底动脉血管壁组织炎症反应的抑制作用。方法：48例成年新西兰兔,随机分为4组：对照组(n=6),SAH组(n=6),SAH+DMSO组(n=6),SAH+SB203580组(n=6)。动物模型同第一组实验,于第一次注血后第7天,处死所有动物,获得基底动脉标本。RT-PCR法测定血管壁组织中炎性细胞因子IL-1β、TNF-α、ICAM-1、VCAM-1的转录活性,免疫组化法观察血管壁组织中炎症细胞相关抗原CD4、CD68、MPO的表达。结果：SAH后第7天兔基底动脉组织中炎性细胞因子IL-1β、TNF-α、ICAM-1、 VCAM-1的转录活性均明显增高,炎症细胞相关抗原CD4、CD68、MPO的表达增多。SB203580可明显降低SAH后血管壁组织炎性细胞因子的转录活性和炎症细胞相关抗原的表达。结论：SAH后血管壁组织存在明显的炎症反应,可能与p38MAPK信号通路的活化有关,应用p38MAPK特异性抑制剂SB203580可明显抑制血管壁炎症反应水平。第三部分p38MAPK信号转导通路与蛛网膜下腔出血后的脑血管内皮细胞凋亡目的：血管内皮细胞凋亡是蛛网膜下腔出血后脑血管痉挛发病机制中的重要环节。p38MAPK信号通路是调控组织细胞凋亡的重要通路。本实验旨在研究应用p38MAPK特异性抑制剂SB203580对实验性SAH后基底动脉血管内皮细胞凋亡的抑制作用。方法：48只成年新西兰兔,随机分为4组：对照组(n=6),SAH组(n=6),SAH+DMSO组(n=6),SAH+SB203580组(n=6)。动物模型同第一组实验,于第一次注血后第7天,处死所有动物,获得基底动脉标本。Western-blot法测定血管壁组织中半胱氨酸蛋白酶-3(caspase-3)的表达,TUNEL法观察血管内皮细胞凋亡情况。结果：SAH后第7天兔基底动脉组织中;aspase-3的表达明显增高,TUNEL法染色血管内皮细胞阳性染色明显增多。SB203580可明显降低SAH后血管壁组织中caspase-3的活性并抑制血管内皮细胞的凋亡。结论：SAH后血管内皮细胞存在明显的凋亡,可能与p38MAPK信号通路的活化有关,应用p38MAPK特异性抑制剂SB203580可明显抑制SAH后血管内皮细胞的凋亡。更多还原

【Abstract】 PART Ⅰ:Potential role of p38MAPK pathway in cerebral vasospasm after experimental subarachnoid hemorrhage in rabbitsObjective:Previous studies have demonstrated signal transduction pathways play a critical role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage and p38MAPK pathway has been shown to be involved in numerous physiological processes, such as cell proliferation,cell survival, apoptosis, inflammation, and embryonic development. This work was conducted to investigate the role of p38MAPK on cerebral vasospasm in a rabbit model of SAH.Methods:In experiment1,24rabbits were assigned randomly to four groups:control, SAH day3, SAH day5, and SAH day7groups,and were killed on days3,5, and7. The time course of the total p38MAPK,p-p38MAPK,total ATF2and p-ATF2activation in the basilar artery after SAH was analyzed by Wester blot.In experiment2,24rabbits were assigned randomly to four groups:control, SAH, SAH+DMSO,SAH+SB203580,and were killed on day7. The blood vessel cross-sectional area was measured by hematoxylin-eosin staining.Results:As a result, the elevated expression of activated p-p38MAPK and p-ATF2was detected in the basilar artery after SAH from day3,peaked on day5. After SB203580intracisternal administration, the level of p-p38MAPK and p-ATF2were decreased and the vasospasm was markedly attenuated in the basilar arteries.Conclusion:p38MAPK pathway was activated in the arterial wall after SAH and contribute to vasospasm development. Administration of p38MAPK inhibitor may attenuate cerebral vasospasm in a rabbit model of SAH.PART Ⅱ:Potential role of p38MAPK pathway in the inflammatory reaction after experimental subarachnoid hemorrhageObjective:Inflammatory recaction is involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was conducted to examine whether SB203580,a p38MAPK inhibitor,would suppress inflammatory recaction after experimental SAH.Methods:48rabbits were assigned randomly to four groups:control, SAH, SAH+DMSO,SAH+SB203580,and were killed on day7. The gene expression levels of cytokines and adhesion molecules in the basilar artery were measured by RT-PCR. Immunohistochemical study was performed to assess the expression and localization of CD4、CD68and myeloperoxidase (MPO).Results:SAH could induce increases of the gene expression levels of IL-1β、TNF-α、ICAM-1and VCAM-1, which were reduced in the SAH+SB203580 group.Immunohistochemical study demonstrated that the expression levels of CD4、 CD68and and MPO were all increased in the SAH group, but these increases were attenuated in the SAH+SB203580group.Conclusion:Inflammatory recaction was evoked by SAH,maybe due to the activation of p38MAPK pathway,and could be suppressed by p38MAPK inhibitor SB203580.PART HI:Potential role of p38MAPK pathway in the endothelial apoptosis after experimental subarachnoid hemorrhageObjective:Previous study have demonstrated that p38mitogen-activated protein kinase(MAPK) plays an important role in apoptosis, which is involved in the development of cerebral vasospasm after SAH. This study was conducted to examine whether SB203580,a selective p38MAPK inhibitor could reduce cerebral vasospasm through the suppression of apoptosis.Methods:48rabbits were assigned randomly to four groups:control, SAH, SAH+DMSO,SAH+SB203580,and were killed on day7. The the endothelial apoptosis was examined by was examined by Western blot analysis of caspase-3activity and TUNEL staining.Results:Elevated expression of cleaved caspase-3was detected in the basilar artery after SAH and suppresed after SB203580administration. Apoptosis was not detected in the control group. Strong positive cells were visualized in the SAH and SAH+DMSO groups. Weak positive cells were observed in the SAH+SB203580group. Conclusion:Endothelial apoptosis was evoked by SAH,maybe due to the activation of p38MAPK pathway,and could be suppressed by p38MAPK inhibitor SB203580.更多还原