【作者】 谢宗义；

【导师】 程远；

【作者基本信息】 重庆医科大学 ， 外科学， 2008， 博士

【摘要】 目的建立激光捕获显微切割(laser capture microdissection, LCM)技术分离脑微血管和微血管蛋白质组双向凝胶电泳(two-dimensional gelelectrophoresis,2-DE)的技术平台，运用比较蛋白质组学分析鉴定蛛网膜下腔出血(subarachnoid hemorrhage, SAH)后不同时间点脑微血管的差异表达蛋白质，为在蛋白质水平进一步阐明SAH后微血管损伤的分子机制奠定基础。方法1建立SAH模型：采用视交叉前池注血法建立大鼠SAH模型。2分析SAH后脑微血管的动态改变：应用荧光分光光度计和荧光显微镜方法分别检测SAH后不同时间点血脑屏障(blood–brain barrier,BBB)通透性的动态改变，同时利用透射电镜观察其超微结构变化，并确定微血管改变的时相。3LCM分离SAH后皮质微血管：采用免疫组织化学染色(CD31单克隆抗体)标记微血管内皮细胞，使用LCM分离皮质微血管。4运用比较蛋白质组学分析SAH后不同时间点脑微血管的差异表达蛋白谱：通过优化2-DE的各种条件和参数，确定最佳的样品上样量、等电聚焦(isoclectfic focusing, IEF)、染色方法等。在此基础上，对SAH后不同时间点脑微血管蛋白质组进行2-DE分离。使用PDQuest软件对2-DE图像进行分析，筛选出不同时间点脑微血管的差异表达蛋白点。候选差异表达蛋白斑点经胶内酶解、基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析，得到肽指纹图谱(peptide mass fingerprint,PMF)，使用Mascot软件在Swiss-Prot或NCBInr数据库中检索鉴定差异表达蛋白质，并对差异蛋白质进行生物信息学分析。5验证候选差异表达蛋白：使用Western blotting验证感兴趣的蛋白质。结果1．SAH后6h时，可见血液广泛分布在前颅窝底、Willis环、基底池周围以及大脑半球和小脑。2．SAH引起大脑皮质BBB通透性增加。BBB通透性开始增加出现在SAH后24h，于36h达最高峰，然后在48h开始逐渐减少，至72h恢复正常。透射电镜观察，在SAH后36h，毛细血管周围水肿明显，管腔受压；星形胶质细胞足突肿胀，线粒体轻度肿胀，但其细胞膜尚保存；内皮细胞管腔面不光滑，表面微绒毛增多，但紧密连接未见开放；基膜连续且完整。3．优化PEN切片的固定、染色、脱水等制备方法和LCM切割参数。应用LCM技术分离SAH6h组、SAH36h组皮质微血管，分别得到32000、32500条微血管。4．通过优化2-DE条件，最终确定合适的样品上样量为120μg、非线性pH3-l0IPG胶条、最佳染色方法为银染。通过对SAH后6h、36h脑微血管蛋白质组的2-DE图像进行对比分析，发现有81个蛋白质斑点表达有差异，其中有48个蛋白点表达明显上调，有33个蛋白点表达明显下调。对其中30个差异蛋白点进行MALDI-TOF-MS和生物信息学分析后，鉴定出了24种可能的差异表达蛋白。5．Western blotting法对VDAC1、MAPK10两个感兴趣的蛋白质进行了鉴定，结果与2-DE分析的结果一致：与SAH6h组相比，SAH36h组脑微血管中VDAC1、MAPK10蛋白含量均明显增加。结论1视交叉前池注血模型更好地模拟临床SAH，重复性好，适合于前循环动脉瘤性SAH后病理生理学机制的研究。2SAH引起BBB结构和功能最显著的变化出现在36h，而且，这些病理改变对SAH后继发性脑损伤和不良预后有着重要的影响。3LCM技术是一种强有力的分离方法，能从脑组织中分离出纯度较高的微血管，可用于基因组学和蛋白质组学研究。4鉴定出的24种差异表达蛋白可能在SAH后脑微血管损伤过程中起重要作用。更多还原

【Abstract】 ObjectiveTo establish a technical platform of cerebral microvessels isolated bylaser capture microdissection (LCM) and their proteome analyzed bytwo-dimensional gel electrophoresis (2-DE). Further, to investigatedifferentially expressed proteins in cortical microvessels followingsubarachnoid hemorrage in rats by comparative proteomics in order toelucidate the molecular mechanism underlying the microvessel injuryfollowing subarachnoid hemorrage at the protein level.Methods1Induction of SAH model in rat: SAH models were induced by aprechiasmatic blood injection technique.2Analysis of dynamic changes in cortical microvessels after SAH:Blood–brain barrier (BBB) permeability was determined at different timepoints by fluorescence spectrophotometer and fluorescence microscopy,respectively. The ultrastructural changes in BBB were observed withtransmission electron microscope.3Isolation of cortical microvessels by LCM after SAH: Immunohistochemical staining (antibody to CD31) was employed to labelendothelial cells throughout the cerebral microvascular tree. Selectivemicrodissection of labeled cortical microvessels in frozen tissue sections byLCM.4Analysis of differentially expressed proteins in cortical microvesselsat different time points following subarachnoid hemorrage in rats bycomparative proteomics: Firstly, optimal parameters and conditions of2-DE were ascertained, including suitable loading quantity of sample, IEF,staining method etc. Secondly, microvessels proteome at various timepoints post-SAH were separated by2-DE. Next, PDQuest software (7.4)was applied to analyze2-DE images to screen differentially expressedprotein spots. Differentially expressed protein spots were subjected todigest with trypsin, and then identified by matrix assisted laserdesorption/isonization time of flying mass spectrometry (MALDI-TOF-MS)to obtain peptide mass fingerprint (PMF). PMF was used to search theSwiss-Prot or NCBInr database with MASCOT search engine.5Validation of candidate differential proteins: Interesting proteinswere selected and subjected to Western blotting analysis.Results1The subarachnoid blood was widely distributed throughout theanterior cranial fossa base, Willis circle, basal cisternal system and over thehemispheres and cerebellum at6h after SAH induction. 2SAH brought about a significant increase in cortical BBBpermeability. BBB permeability began to increase at24hours after SAH,peaked at36hours, and significantly declined at later observations period,normalized at72hours after SAH. Electron micrograph demonstrated at36hours post-SAH, a notable perivascular edema combined with a collapse ofthe capillary. Astrocytic end feet and mitochondria were swollen. Thelumina of the endothelial cells appeared unsmooth. The tight junctions andthe basal laminas were identified normal.3Preparation methods of PEN tissue sections (including cutting,fixation, immunostaining and dehydration) and microdissection parameterswere optimized before isolation of microvessels by LCM. Approximately32000cortical microvessels from6h group and32500cortical microvesselsfrom36h group were successfully isolated by immuno-LCM.4Based on optimal conditions and parameters of2-DE, the loadingquantity of sample, category of IPG strips and staining methods weredetermined as120μg, nonlinear IPG strip (pH3-10，17cm) and silverstaining, respectively. Compared with SAH6h group, eighty-onedifferentially expressed protein spots were showed in SAH36h group.Among them, forty-eight protein spots were up-regulated markedly, whilethirty-three protein spots were down-regulated significantly. Thirtydifferential proteins were randomly selected and subjected to identify byMALDI-TOF-MS and bioinformatics analysis. Twenty-four differential proteins were successfully identified.5To validate the2-DE results, two interesting proteins, VDAC1andMAPK10, were selected and subjected to Western blotting analysis. Theresults showed that both VDAC1and MAPK10had an increasedabundance in SAH36h group as compared with SAH6h group.Conclusions1The prechiasmatic blood injection model resembles clinical SAH. Itis very reproducible and appropriate for the study on thepathophysiological mechanisms of aneurysmal SAH in the anteriorcirculation.2SAH could induce rapid changes in microvascular function andstructure at36hours. Moreover, microvascular dysfunction may play acrucial role in the development of secondary brain injury and unfavorableoutcome.3LCM technique is a powerful method which could isolate purermicrovessels from complicated brain tissue than other methods, and couldbe applied in the fields of genomics and proteomics.4Twenty-four differentially expressed proteins identified in this studymight be strongly associated with the development of cerebral microvesselinjury following SAH.更多还原