【作者】 王玉萍；

【导师】 牛秀敏；

【作者基本信息】 天津医科大学 ， 妇产科学， 2009， 博士

【摘要】 子痫前期(pre-eclampsia)是妊娠期特有的疾病，为妊娠期高血压疾病的最常见类型，也是导致孕产妇死亡和婴儿病率及死亡率的重要原因之一。目前仍然没有理想的标志物早期诊断子痫前期，且具体的发病机制仍未阐明。蛋白质组学是近年来发展的新技术，主要包含蛋白质分离、蛋白质鉴定和生物信息学三大部分，具有高通量和高灵敏度等优点。胎盘是子痫前期发生的关键环节，直接与子宫蜕膜接触，参与母胎免疫耐受形成及维持妊娠。为进一步阐明子痫前期的发病机制和寻找该病的蛋白标志物，本实验利用蛋白质组学技术，通过比较重度子痫前期患者和正常妊娠胎盘组织的蛋白表达差异，探讨引起子痫前期发生的可能因素，同时寻找子痫前期血清中的标志蛋白，为实现子痫前期的早期诊断提供依据。目的：探讨重度子痫前期和正常妊娠的胎盘组织差异蛋白，筛选出子痫前期胎盘相关蛋白。此外，在血清中寻找与子痫前期胎盘抗原相关的抗体蛋白，进一步检测子痫前期母体外周血中抗体蛋白浓度的变化，为发现特异性标记物提供线索。方法：1收集子痫前期患者、正常孕妇胎盘组织标本，提取胎盘总蛋白，双向凝胶电泳的第一向电泳采用固相pH梯度电泳，第二向采用SDS-聚丙烯酰胺凝胶电泳分离胎盘组织总蛋白质成份，然后进行硝酸银和考马斯亮蓝染色。通过PDQuest7．4双向电泳图像分析软件对2-DE图谱进行数字化分析匹配与对比，并通过统计学分析方法评价双向凝胶电泳结果。2通过比较子痫前期患者、正常孕妇胎盘组织的双向电泳图谱，筛选出有差异的蛋白质点进行胶内胰蛋白酶解，经过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析肽质量指纹图谱，数据库搜索鉴定蛋白质。应用生物信息学技术对差异蛋白进行结构和生物学功能分析和预测。3将双向电泳提取的子痫前期胎盘组织总蛋白转移至PVDF膜上，分别以患者的血清和正常孕妇的血清进行免疫印迹杂交，找出差异反应蛋白质点，筛选出可能引起子痫前期孕妇机体抗原抗体反应的胎盘抗原，经过质谱鉴定出蛋白名称，在大样本的子痫前期患者和正常孕妇的血清标本中应用ELISA方法进行抗体蛋白表达水平的检测。结果：1获得了图像清晰、重复性好的双向电泳图凝胶。2与正常妊娠相比，子痫前期患者胎盘组织丰度差异表达相差2倍以上并经Student’s t-test检验差异有统计学意义的蛋白质点有18个(P＜0．05)，其中12个点在子痫前期组表达上调，另6个点表达下调。3质谱鉴定出14个差异蛋白质点，在子痫前期胎盘中上调的蛋白包括纤维蛋白原β链、维生素D结合蛋白、肾小管间质性肾炎抗原样前体蛋白、纤维蛋白原γ链前体、α-1抗胰蛋白酶前体、膜联蛋白Al、α-烯醇酶、Ⅰ型细胞骨架蛋白9、Ⅱ型细胞骨架蛋白1。下调的蛋白包括真核翻译起始因子5A-1、铁蛋白轻链、突变子-2、ATP合酶β亚单位、雌二醇17β脱氢酶1。4免疫印迹技术证实子痫前期患者体内存在抗原抗体反应，对应双向电泳凝胶图找到了胎盘相关抗原位置。5通过胶内酶解消化，质谱分析获得了各个蛋白点的肽谱图和肽段氨基酸序列，经过数据库检索分析后，鉴定出3个胎盘抗原蛋白。子痫前期患者血清中存在纤维蛋白原蛋白β链抗体、热休克蛋白70抗体、线粒体核糖体蛋白的可溶性结构蛋白或抗角蛋白9抗体。6子痫前期患者和正常孕妇的血清中纤维蛋白原蛋白β链抗体、热休克蛋白70抗体蛋白表达水平增加，与蛋白质组学结果基本一致。结论：1子痫前期胎盘组织与正常妊娠胎盘组织存在蛋白表达差异，差异蛋白分别涉及炎症、血栓形成、参与糖酵解的酶类、分子伴侣、信号转导蛋白、细胞骨架蛋白、细胞存活和增殖、凋亡调控蛋白，以及参与细胞内物质转运和能量代谢的酶等。2子痫前期发生发展过程中，血清中存在纤维蛋白原蛋白β链抗体、热休克蛋白70抗体、线粒体核糖体蛋白的可溶性结构蛋白或抗角蛋白9的抗体。3血清中纤维蛋白原β链抗体、热休克蛋白70抗体可能在子痫前期病理过程中起重要作用，有望成为子痫前期标记物候选指标。更多还原

【Abstract】 Preeclampsia,a common type of hypertensive disorder complicating pregnancyand a kind of idiosyncratic disease in pregnancy,has been considered a majorcontributor to the maternal and neonatal mortality and morbidity.The preciseetiopathogenesis of pre-eclampsia remains to be a subject of extensive research and ithas no ideal markers.Proteomics is a new technology,which includes proteinseparation,identification and bioinformatics.It has the virtue of high output and highsensitivity.Placenta plays a crucial role in the development of pre-eclampsia,whichcontact directly with basal deciduas and participate in immune tolerance andmaintaining pregnancy.We studied total proteins of placenta by using proteomicstechnology in this study,by comparing the differences of protein expression inplacenta of pre-eclampsia and normal pregnant women in hopes of finding associatedprotein of pre-eclampsia,so as to elucidate the pathogenic mechanism ofpreeclampsia and find out specific biomarkers.Objective:To investigate comprehensively differential proteins expressiveprofiles in placenta tissues of severely pre-eclampsia patients and normal pregnancywoman in order to scalp out pre-eclampsia-associationed proteins.In addition,find outsome antibodies to placenta antigen proteins in serum of pre-eclampsia .At last ,studythe differential expression of the antibodies in maternal peripheral blood ofpre-eclampsia patients to provide some clues for the pathomechanism and find outspecific biomarkers.Methods:1.The total proteins extracted from placenta tissues of preeclampsiapatients and normal controls were separated by means of two-dimensional gelelectrophoresis (2-DE);The first dimension was carried by IPG pH gradientisoelectric focusing system,the second dimension was separated by verticalSDS-PAGE,the late step was stained by silver or coomas brilliant blue forvisualization.Acquired gel images were analized and the spots were matched andcompared by PDQuest7.4 software (Bio-Rad).The statistical method was applized toevaluate the 2-DE results.2.Differentially expressed proteins spots in 2-DE maps were chosed and incised after digested in-gel by trypsin,and the candidate proteins were identified usingmatrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) .The peptide mass spectrometry maps and amino acid sequenceswere searched from SEQUEST databases.The functions and construction of theidentified proteins were searched by bioinformatics.3.Proteins from placenta tissues of preeclampsia patients were separated bytwo-dimensional PAGE.Then proteins were transferred to PVDF membranes.Serafrom the same patients with preeclapsia and normal controls were screened for IgGautoantibodies against the proteins by Western blot analysis.Finally massspectrometric analysis system was used to identify the constituents of the positiveproteins.Some antibodies to antigen proteins were further validated by ELISA amongserum.Results:1 The good reproducible results were acquired using the optimized2-DE technique.2 Compared with the normal placenta,18 protein spots with 2 fold of differencestatistically in PE placenta were found including 12 up-regulated and 6down-regulated protein spots.3 Fourteen proteins were identified by MALDI-TOF-MS,.Whereas levels ofFibrinogen beta chain precursor ,Vitamin D-binding protein,Tubulointerstitialnephritis antigen-like precursor,Fibrinogen gamma chain precursor ,Alpha-1-antitrypsin precursor ,Annexin A1,Alpha-enolase ,Keratin,typeⅠcytoskeletal 9,Keratin,typeⅡcytoskeletal 1,were upregulated in preeclapsia groupcompared with control group,we found a significant down regulation of Eukaryotictranslation initiation factor 5A-l,Ferritin light chain ,Transgelin-2,ATP synthasesubunit beta,Estradiol 17-beta-dehydrogenase 1 in preeclampsia group.4 Western blotting validated abnomal immunity in preeclampsia group.Acording 2-DE image find the position of placenta antigen.5 The peptide mass spectrometry maps and amino acid sequences were obtainedsuccessfully by digesting in-gel by trypsin and mass spectrometric analysissystem.Three placenta antigen were identified.There are antibody proteins againstFibrinogen beta chain,Heat shock 70kDa protein and Solution Structure of the Mitochondrial Ribosomal Protein or Keratin 9.6 The concentration of antibody proteins against fibrinogen beta chain and heatshock 70kDa protein in the preeclampsia maternal peripheral blood was higher thannormal pregnancy,which were paralleled with the results ofproteomic.Conclusion:1.2-DE pattern of placenta tissue from preeclampsia is differentfrom that of normal control group.The different proteins are invoulving in theimflammation,thrombosis,enzymes of glycolysis,physiological process of signaltransduction,cytoskeleton,cell survival and proliferation,cell apoptosis regulation,enzymes of energy metabolism and substances transport.2 Western blotting validated that there are abnomal immunity response againstplacenta proteins in preeclampsia group during the development of preeclampsia.Antibody proteins against Fibrinogen beta chain,Heat shock 70kDa protein andsolution Structure of the Mitochondrial Ribosomal Protein or Keratin 9 developeduring preeclampsia3 Antibody proteins against Fibrinogen beta chain,Heat shock 70kDa proteinmight play a crucial role for the pathogenesis.Perhaps they would be a candicatedbiomark of preelampsia.更多还原