【作者】 张旋；

【导师】 周政；

【作者基本信息】 第三军医大学 ， 外科学， 2011， 硕士

【摘要】 研究背景和目的:迟发性脑血管痉挛(DCVS)作为蛛网膜下腔出血(SAH)后的严重并发症在临床中发生率较高,是导致患者致死及致残的最主要原因之一,不但影响患者预后的生活质量,同时给社会和家庭带来沉重的经济负担,因此,研究如何预防及逆转脑血管痉挛具有重要意义[1, 2]。关于SAH后脑血管痉挛发生、发展机制的研究延续数十年,但对其本质尚存在着诸多疑问和争论。回顾近年来的基础研究表明:血管内皮功能障碍、血管平滑肌收缩及增殖、免疫炎性反应、基因调控等多因素环节都参与其中。虽然CVS发生是多因素多环节共同作用的结果的观点已被多数研究者所接受,但关于触发、维持痉挛的关键环节尚未有统一的定论。许多研究发现:受累动脉痉挛的持续存在不但与平滑肌的持续收缩有关,血管壁结构的改变包括内皮细胞的凋亡、血管平滑肌细胞(VSMCs)的增殖也参与其中。尤其是在迟发性脑血管痉挛(DCVS)发生过程中,平滑肌细胞的增殖可能起着关键性作用。Caveolae是细胞膜上特定微区域,由caveolin蛋白、胆固醇、鞘磷脂及糖基鞘磷脂构成,在介导细胞信号转导、胞吞胞饮、膜磷脂代谢等生理过程中发挥重要作用。Caveolin是caveolae的表面标记蛋白,富集于该区域,包括caveolin-1、caveolin-2、caveolin-3三种。Caveolin-1在平滑肌细胞上有着丰富的表达,与诸如Src家族酪氨酸激酶、生长因子受体、内皮源性一氧化氮合酶(eNOS)、G蛋白及G蛋白偶联受体(GPCRs)等众多分子结合,且被认为在细胞信号转导过程中起负性调节作用。在研究其它血管性疾病例如动脉粥样硬化、高血压时发现caveolin-1与血管平滑肌的增殖、表型改变关系密切。作为VSMCs信号转导的“总阀门”,caveolin-1在DCVS发生、发展过程中所发挥的重要作用是可以合理预期,但观察DCVS病理改变过程中caveolin-1的表达变化迄今鲜有报道。本研究旨在观察caveolin-1在SAH后受累动脉组织中的表达规律以及与PDGF表达水平、基底动脉中膜厚度的动态关系,探讨其在脑血管痉挛中的可能作用及机制。方法:1、采用穿刺兔枕大池首次枕大池注入自体动脉血,48h二次注血的方法,建立兔SAH后DCVS模型;2、将实验动物随机分为正常对照组、假手术组和SAH组;3、观察模型动物SAH后行为学变化;4、按照设定时间点于术后1d、3d、5d、7d、14d随机抽取实验动物行心脏灌注,行基底动脉普通HE染色,明确SAH后基底动脉血管病理学变化;5、取各时间点组织切片,应用测量软件测量血管管径变化及中膜厚度改变;6、采集各时间点动物基底动脉组织标本,western blot法检测caveolin-1、PDGF蛋白变化趋势;7、采集各时间点实验动物基底动脉组织标本,PCR法检测caveolin-1、PDGF mRNA变化趋势;结果:1、经皮枕大池穿刺二次注血后可成功建立兔脑血管痉挛动物模型;2、SAH组模型动物行为学表现不同于正常对照组与假手术组,出现毛发蓬松及自洁性差,摄食减少,呈头低位蜷缩状,活动明显减少;3、HE染色显示SAH后基底动脉血管壁增厚,管腔狭窄,内皮细胞不同程度的变性、肿胀;内弹性膜迂曲皱褶或断裂,厚薄不均;中膜增厚,VSMCs排列紊乱、增殖明显;4、对照组和假手术组SAH组各时间点动脉中膜厚度无明显统计学差异(P>0.05);SAH组中膜厚度随时间逐渐增厚,于二次注血后7d较对照组增厚最为明显(P<0.01);5、SAH组caveolin-1蛋白表达逐渐减低,于二次注血后7d降至最低(P<0.01);PDGF蛋白表达则逐渐增加,于二次注血后7d达峰(P<0.01);对照组和假手术组caveolin-1、PDGF蛋白表达水平无统计学差异(P>0.05);6、SAH组caveolin-1 mRNA转录水平随时间推移逐渐上调,于二次注血后5d达峰(P<0.01);PDGF mRNA亦逐渐上调,于二次注血后3d达峰(P<0.01);对照组和假手术组caveolin-1、PDGF mRNA转录水平无明显改变(P>0.05);7、SAH组caveolin-1蛋白表达水平与血管痉挛程度呈显著负相关(r=-0.89,P<0.01)。结论:1、枕大池二次注血法成功建立兔DCVS动物模型,其动物行为学较正常对照组有特征性表现;2、SAH模型动物的基底动脉形态学发生显著改变:血管壁增厚、管腔狭窄,伴有内皮细胞变性及平滑肌细胞增殖等病理学表现;3、SAH后动脉组织caveolin-1蛋白表达逐步下降,7d最为显著,但mRNA转录水平则逐步上调,5d达峰;4、SAH后动脉组织PDGF蛋白表达逐渐增加,7d达峰,mRNA转录水平亦上调明显,3d达峰;5、SAH后动脉组织中caveolin-1蛋白表达水平呈下降趋势,与DCVS程度呈显著负相关;而PDGF蛋白表达水平与血管中膜厚度同步增加,呈显著正相关;提示Caveolin-1可能在DCVS病理演变过程中扮演着负性调节因子的角色。更多还原

【Abstract】 Backgroud and Purposes:As a seriuouly complication of subarachnoid hemorrhage (SAH), delayed cerebral vasospasm (DCVS) has a high incidence and is the main occasion leading a poor mobility and mortality of the SAH patients. The prevention and reversion of CVS has become a tough problem in front of our neurosurgeon and neurobiologist.The mechanism research of CVS after SAH has already been in progress for decades, but the origin is still a mystery. The past researches showed that the functional disorder of vascular endothelial cells, proliferation of vascular smooth muscle cells(VSMCs), inflammatory reaction, gene regulation and so on all take part in the mechanism. Although the conception that CVS is reduced by multifactors and multi-links has been widely accepted, the key point has not been fully recognized.Many experiments had indicated that the continuous vasospasm was related with the apoptosis of endothelial cells, reconstruction of vessel wall, constriction and proliferation of VSMCs. Especially, VSMCs may play an important role in the DCVS.Caveolae is a special micro-domain on the cell membrane, consist of caveolin, cholesterol, sphingomyelin and glycosyl sphingomyelin. Caveolae participate in the physiological processes of celluar signal transduction, endocytosis and pinocytosis, metabolism of membrane phospholipid. Caveolin-1 is rich in the membrane of the VSMCs, it combines with many adaptor molecule such as tyrosine kinase, growth factor receptors, endothelial nitric oxide synthase, G protein and G protein-coupled receptors as a negative regulator.Caveolin-1 has been found a close relationship with the phenotypic alternation of VSMCs in the research field of cardiovascular diseases. As the“main valve”of signal transduction of VSMCs, we can predict logically the significance of caveolin-1 in the mechanisms of CVS. So, this study is to determine the effects and mechanisms of Caveolin-1 on the proliferation of VSMCs of basilar artery in SAH animal model.Methods:1. The method of twice fresh autologous arterial blood injection into the cisterna magna of rabbit has been taken to set up the experimental cerebral vasospasm animal model.2. All the intervention animals were divided into SAH group, sham group and control group randomly.3. The observation of behavioral changes of the SAH animal.4. The animals were executed to harvest the basilar artery from each group an the 1st, 3rd, 5th, 7th, 14th after the second injection. HE staining and microscopic observation had been taken to evaluate the animal model.5. Measurement of the thickness of arterial tunica media.6. The protein expression of caveolin-1 and PDGF were detected by western blot. 7. The mRNA transcription of caveolin-1 and PDGF were detected by PCR.Results:1. The experimental SAH model has been established successfully by the twice fresh autologous arterial blood injection into the cisterna magna of rabbit.2. The animals in SAH group have some ethological change such as huddling, anorexia and decrease of activity compared with sham group and control group.3. The HE staining slices showed vessel stenosis, vessel wall incrassation, swelling and denaturation of endothelial cells, crease and fracture of internal elastic membrane, structural derangement and proliferation of VSMCs.4. The thickness of the arterial tunica media was thickening gradually and peak at the 7th (P<0.01) compared with other groups.5. Protein expression of caveolin-1 had undergone down-regulated after SAH and reach the valley bottom on the 7th day compared with sham group and control group (P< 0.01); PDGF increased and peaked on the 7th day (P<0.01).6. Transcription of Caveolin-1 mRNA peaked on 5th day compared with other groups (P<0.01); the PDGF mRNA peaked on 3rd day compared with other groups (P<0.01).7. Caveolin-1 protein is significantly negative correlate with the thickness of basilar artery (r =-0.89, P<0.01).Conclusions:1. The experimental SAH animal model leads a notable behavioral changes compare with control group and sham group.2. The basilar artery from the SAH group shows a marked morphological changes similar to the clinical specimens from SAH patients.3. The protein of caveolin-1 is down-regulated after SAH, but the mRNA shows a tendency in contrast.4. The protein and mRNA of PDGF show a similar trend and be up-regulated after SAH.5. The expression of Caveolin-1 is down-regulated continuously during the period of VMSCs proliferation, and, Caveolin-1 may be a potential inhibitor for cerebral vasospasm.更多还原