妊娠期高血压疾病尿液差异蛋白质组学研究
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摘要
妊娠高血压疾病在我国发病率为9.4%-10.4%,该疾病可导致孕妇全身各脏器损伤以及胎儿生长受限、胎死宫内等严重并发症,是引起孕产妇和围产儿死亡的主要原因之一,在发展中国家因子痫前期死亡的孕妇占20%-80%,发达国家子痫前期孕妇围产期死亡率为未患子痫前期疾病孕妇的5倍。
近百年的研究使人类对该疾病有了一定认识,但其病因和发病机制错综复杂,至今仍不明确,因此目前仍无有效的预防、早期筛查以及预测疾病严重程度的方法,寻找能早期筛查该疾病并预测其严重程度的办法一直是围产医学研究的重点和热点。对于妊娠期高血压疾病,目前公认的病因学说有：血管内皮受损学说、免疫学说、滋养细胞异常入侵学说、氧化应激学说、遗传学说以及一元化学说等。尽管目前的研究在血管内皮受损、免疫以及分子遗传方面发现了一些潜在的生物标志物,如血清血管内皮生长因子、肿瘤坏死因子α、一氧化氮、内皮素、胎盘蛋白13、血浆血管性假血友病因子、胎盘整合素等,这些物质被实验证实参与了疾病发生、发展的过程,其含量存在一定的变化,可以可用来反映子痫前期病情的严重程度,有希望成为该疾病的检测指标。但是,由于导致该疾病的多种病因并不独立而是彼此紧密联系,始终没能找到特异性的检测指标和方法作为该疾病的早期筛查和疾病严重程度预测的办法,有效的应用于临床。
传统蛋白质功能研究大多是在已有的工作基础上对某几个蛋白质进行分析,从理论上推导出对疾病发生发展可能具有重要意义的蛋白质,继而进行深入细致的功能研究。而这种“假设驱动”的研究策略往往存在片面性、主观性和低效率等缺点。随着人类基因组计划(human genomic project, HGP)的提前完成,蛋白质组学的理论系统和技术方法已经成为后基因组时代的主旋律。由于蛋白质组学研究具有大规模、高通量、高灵敏度的特点,因此可以有效地、客观地分析细胞内的蛋白质整体。差异蛋白质组学能通过比较样本间蛋白质表达水平的变化,寻找差异分子,用以解决各种各样的生物学问题,在临床上可以从整体上研究与疾病相关的差异蛋白质组,寻找疾病的标记蛋白,已广泛应用于疾病的早期筛查和诊断。目前己涌现出许多新的差异蛋白质组分析方法,其中荧光差异凝胶电泳(difference gel electrophoresis, DIGE)技术由于继承了二维凝胶电泳(two-dimensional electrophoresis,2-DE)的高分辨率,同时又具备高重现性、高灵敏度、高通量和高动态范围等优势,使得DIGE日益受到关注,成为最受欢迎的定量蛋白质组学研究手段之一。DIGE是一种在2D电泳之前进行荧光染料标记蛋白样品的方法,通过对不同的蛋白样品用不同的荧光染料进行标记,在同一块双向凝胶中可同时分离多至三种不同的蛋白样品,并且每块胶上均引入了内标,内标的利用可以进一步增加可信度,确保结果能反映出真实的生物学差异,避免了系统误差对实验结果的影响,从而能够对样品间蛋白质丰度差异进行精确分析。
联合应用DIGE分离蛋白和质谱技术鉴定蛋白为差异蛋白质组学研究提供了最为有效的技术平台,实现了快速、高效、高通量检测,目前该种技术在恶性肿瘤(鼻咽癌、乳腺癌、胃癌、前列腺癌、肝癌、膀胱癌)、自身免疫性疾病、感染性疾病等的早期诊断上显示了不可估量的价值,其诊断的敏感性和特异性均可达90%以上。近年来一些学者已将其用于产科领域研究,通过对孕妇附属物及孕妇体液,如胎盘、羊水、血液、脑脊液、尿液等的差异蛋白质谱的分析,有望阐明妊娠及其严重并发症的相关的分子生物学基础,通过比较正常妊娠妇女和患有产科合并症的孕妇的胎盘组织及体液的差异蛋白质谱,能发现一些潜在的特异性的分子生物学标志蛋白。
尿液是血液经肾小球滤过、肾小管和集合管重吸收、排泄及分泌产生的终末代谢产物,其组成、数量与性状的变化携带有泌尿系统疾病发生、发展及预后的各种信息,也可反映机体整体代谢状态,与血清等其他体液样本相比,其获取无创、收集便利,并且蛋白组成相对简单、易于分析。国内外学者目前热衷于对生理和疾病状态下的尿液进行蛋白质组学分析,并已经获得了正常尿液标准蛋白图谱,同时也着手开始研究疾病状态下尿蛋白的变化。
因此本研究拟选用正常妊娠女性和妊娠期高血压疾病孕妇的尿液作为研究对象,利用基于DIGE-基质辅助激光解吸离子化-飞行时间／飞行时间质谱(matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry, MALDI-TOF/TOF)-生物信息学(bioinformatics)的差异蛋白质组学技术平台,来寻找并分析在妊娠期高血压疾病发生发展中的尿液中的差异蛋白质,然后采用临床上经典且易操作的酶联免疫吸附测定法(enzyme-linked immonosorbent asssay, ELISA)检测妊娠期高血压疾病孕妇和正常妊娠女性尿液中特定差异蛋白质的含量进行验证实验,确定特异性的蛋白标记物,从而早期筛查妊娠期高血压疾病的高危人群,对其进行产前监控及早期预防,早期诊断该疾病同时预测其严重程度,及时阻止该病的发生、发展,减少子痫、HELLP综合征等严重并发症的发生,降低孕产妇及围产儿死亡率。
第一部分妊娠期高血压疾病尿液差异蛋白质组的鉴定与生物信息学分析
[目的]
妊娠期高血压疾病病因和发病机制不明确,各种不同病因之间相互联系和影响导致目前仍无特异性的检测指标和方法早期筛查、诊断该疾病并预测疾病严重程度。本研究利用差异蛋白质组学技术平台,寻找妊娠期高血压疾病发生发展过程中的特异性蛋白标记物。
[方法]
选择符合入选标准的5例妊娠期高血压患者、5例轻度子痫前期患者、5例重度子痫前期患者和5例正常妊娠女性,收集其尿液标本,进行双向差异凝胶电泳分析,然后利用全自动切胶仪Spot picker挖取差异蛋白胶粒,并进行样品胶内酶解,再使用MALDI-TOF/TOF质谱仪进行分析,寻找出正常妊娠女性和妊娠期高血压疾病孕妇尿液中的差异蛋白质,最后通过Mascot软件检索SwissProt数据库对蛋白进行鉴定。
[结果]
根据DIGE的差异图谱并结合Decyder差异分析软件进行差异蛋白点分析,妊娠期高血压组和正常妊娠组相比较,共找到了44个差异蛋白点,其中上调的有22个,下调的有22个；轻度子痫前期组和妊娠期高血压组相比较,共找到了50个差异蛋白点,其中上调的有15个,下调的有35个；重度子痫前期组和轻度子痫前期组相比较,共找到了45个差异蛋白点,其中上调的有9个,下调的有36个。有些差异蛋白点在几组之间均有变化,共分析出65个差异蛋白质点。经质谱分析后共鉴定出包括前列腺素-H2D-异构酶(prostaglandin-H2D-isomerase,又称为Lipocalin型前列腺素D合成酶(lipocalin-prostaglandin D synthase, L-PGDS)).基底膜特异的硫酸乙酰肝素蛋白多糖核心蛋白(basement membrane-specific heparan sulfate proteoglycan core protein,又称为Perlecan)、激肽原和妊娠特异性β-1-糖蛋白11等的30种蛋白质,蛋白质相互作用分析显示这30种蛋白质中其中19种存在直接或间接的相互作用。
[结论]
利用基于DIGE-MALDI-TOF/TOF-生物信息学的差异蛋白质组学技术平台,发现妊娠期高血压疾病和正常妊娠女性尿液中存在差异表达的蛋白质,且这些蛋白质之间存在相互作用,这些差异蛋白质可能与该疾病的发生、发展密切相关,有望成为其特异性蛋白标记物。
第二部分妊娠期高血压疾病相关差异蛋白质的初步定量研究
第一章L-PGDS在妊娠期高血压疾病孕妇尿液中的含量变化
[目的]
蛋白组学分析发现L-PGDS在妊娠期高血压疾病尿液中存在差异表达。L-PGDS可催化前列腺素(prostaglandin, PG) H2转变为PGD2,后者具有抑制血小板凝集、诱导血管舒张等功能。本研究拟通过比较正常妊娠组、妊娠期高血压组、轻度子痫前期组和重度子痫前期组尿液中L-PGDS浓度来证实其是否存在变化,可用来诊断妊娠期高血压疾病并预测该疾病的严重程度。
[方法]
确诊时收集正常妊娠女性、妊娠期高血压、轻度子痫前期和重度子痫前期患者无菌尿液10ml,每组各20例,应用ELISA方法检测尿液标本中L-PGDS的浓度,然后进行对比分析。
[结果]
经Levene检验显示四组方差齐性(P=0.385),选用One-Way ANOVA进行方差分析,结果显示四组尿液中L-PGDS浓度存在显著差异(F=27.610,P<0.001),进一步使用LSD检验方法进行两两比较发现妊娠期高血压组患者尿液中L-PGDS浓度(1.32±0.16μg/mL)和正常妊娠组(1.26±0.25μg/mL)无显著差异(P=0.399)；而轻度子痫前期组(0.94±0.23μmL)和重度子痫前期组(0.78±0.23μg/mL)患者尿液中L-PGDS浓度则显著低于正常妊娠组(P均<0.001)。相对于妊娠期高血压组,轻度子痫前期组和重度子痫前期组尿液中L-PGDS浓度均显著降低(P均<0.001)。重度子痫前期组患者尿液L-PGDS浓度显著低于轻度子痫前期组(P=0.023)。由此可见,相对于正常妊娠女性,子痫前期时患者血浆中L-PGDS经肾小球滤过减少导致尿液中含量降低,并且随着疾病的严重程度其浓度逐渐递减。而对于妊娠期高血压患者,其尿液中L-PGDS浓度没有出现下降,可能提示此时患者肾脏功能正处于代偿状态。
[结论]
尿液中L-PGDS浓度在妊娠期高血压疾病不同分类中存在差异,可以很好区别妊娠期高血压和子痫前期,并反映子痫前期的严重程度,可作为尿液中一种较好的蛋白标记物来判断该疾病的发生发展情况。
第二章Perlecan在妊娠期高血压疾病孕妇尿液中的含量变化
[目的]
蛋白组学分析发现Perlecan在妊娠期高血压疾病尿液中存在差异表达。Perlecan作为肾小球基底膜的重要组成成分,主要参与其负电荷屏障的维持。本研究拟通过比较正常妊娠组、妊娠期高血压组、轻度子痫前期组和重度子痫前期组尿液中Perlecan浓度来证实其是否存在差异,可用来诊断妊娠期高血压疾病并预测该疾病的严重程度。
[方法]
确诊时收集正常妊娠、妊娠期高血压、轻度子痫前期和重度子痫前期患者无菌尿液10ml,每组各20例,应用ELISA方法检测尿液标本中Perlecan的浓度,然后进行对比分析。
[结果]
经Levene检验显示四组方差不齐(P<0.001),选用类似方差分析Welch检验进行统计分析,结果显示四组尿液中Perlecan浓度存在显著差异(Weich F值=23.934,P<0.001),进一步使用DunnettT3检验方法进行两两比较发现妊娠期高血压组患者尿液中Perlecan浓度(12.73±3.71nmol/L)高于正常妊娠组(9.99±1.85nmol/L),存在显著差异(P=0.036)；轻度子痫前期组(8.02±1.66nmol/L)和重度子痫前期组(6.63±1.39nmol/L)患者尿液中Perlecan浓度则显著低于正常妊娠组(P=0.006;P<0.001)。相对于妊娠期高血压组,轻度子痫前期组和重度子痫前期组尿液中Perlecan浓度均显著降低(P均<0.001)。重度子痫前期组患者尿液Perlecan浓度显著低于轻度子痫前期组(P=0.039)。由此可见,相对于正常妊娠女性,子痫前期时患者肾小球基底膜上Perlecan分子结构和功能发生变化导致尿液中含量降低,并且随着疾病的严重程度其浓度逐渐递减,此时大量蛋白从肾小球漏出形成蛋白尿。而对于妊娠期高血压患者,其尿液中Perlecan浓度不下降反而上升,说明此时肾小球基底膜滤过屏障仍处于正常状态,无尿蛋白发生。
[结论]
同L-PGDS一样,尿液中Perlecan浓度在妊娠期高血压疾病不同分类中也存在差异,可以很好区别妊娠期高血压和子痫前期,并反映子痫前期的严重程度,可作为尿液中一种较好的蛋白标记物来判断该疾病的发生发展情况
In China, the morbidity of hypertensive disorders in pregnancy (HDP) is about9.4%-10.4%. It can causes organ injuries of the patient and fetal growth restriction, even fetal death, being one of the important reasons of maternal and fetal death. In developing countries,20%-80%maternal death contributes to preeclampsia and in developed, the maternal death of preeclampsia is5times of that of without preeclampsia.
Based on hundred years research on HDP, people have known more about this disease, however, the etiology and pathogenesis are still unclear. Therefore, there still be no good method to prevent HDP and make an early diagnosis. Finding a good measure to make diagnosis early and estimate its severity of HDP is an important and hot spot in Obstetrics. For HDP, there are many etiological theries, such as vascular epithial injury; immunological theory; oxidative theory and so on. Although several potential biomarkers exist in aspect of vascular epithial injury and immunology and genetics, for example, vascular epithial growth factor, tumor necrosis factor, nitrogen monoxidum, endothelin, placental protein13, von Wilebrand factor, placental integrin.These materials were identified that they participated in the generation and development of HDP. The concentrations changed in the patients of HDP when comparing to normal pregnacies, making possible promising biomakers in predicting the severity of hypertensive disorders of pregnancy. However, because these reasons causing HDP are not independent and the estimating technology are limited, now we still have not a specific biomarker to make early diagnosis of HDP and predict its severity in clinic.
Traditional study of protein function was mostly focus at a few proteins on the basis of the existing work and theoretically deduced proteins which may have important implications on the development of the disease, and then followed by in-depth and detailed functional study. But this kind of "hypothesis driven"research strategy often exist shortcomings, such as unilateralism, subjectivity and low efficiency. As the human genome project (human genomic project, HGP) completed ahead, proteomics theory and the technical method has become the theme of post genome era. As a large-scale, high throughput, high sensitivity method, proteomics can be effectively, objectively analyze the overall intracellular proteins. Through comparing the protein expression level between differences samples, differential expression proteins were identified, which can be used to solve various biological problems. At present there are many novel differential proteomics analysis methods, among which the difference gel electrophoresis (difference gel electrophoresis, DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. DIGE is a method which can labele protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the real biological differences, while avoid influence of systematic errors on experimental results.
Using DIGE to separate proteins and mass spectrometry to identify the protein together provides most effective technology platform for differential proteome, with the advantage of fast, high effective and high throughout detection. Recently, this technology has revealed important value on early diagnosis of malignant tumors, such as nasopharyngeal carcinoma, breast cancer, gastric cancer, liver cancer, autoimmune disease and infectious diseases, with the specificity and sensitivity both up to90%. In nearly these years, some authors use this technology to study the obstetrical diseases. Basing on differential proteome analysis of maternal accessory and body fluid, such as placenta, amniotic fluid, blood, cerebrospinal fluid and urine, it is possible to explain the molecular biology of pregnancy and its complications. From comparing differential proteome of normal pregnancy and obstetrical complications, we can find some potential specific molecular biomarkers.
Urine is the last metabolic product of blood after glomerular filtration and renal tubule resorption. The change of its composition, quantity and characteristics can reflect the information of urinary system diseases and even the metabolism condition of whole body. Comparing with the other body fluid, such as blood, urine is easy to get in the clinic and the protein composition are more simple. Studying and analyzing the proteome of urine is under normal and abnormal situations is a hot spot for medical experts. Now, the protein spectrum of the normal urine has obtained and people are ready to study the protein spectrum under the situation of disease. The proteome analysis using mass spectrometry of the urine were applied in screening and diagnosing many diseases.
Therefore, this study intends to find the different proteins between the urine of hypertensive disorders of pregnancy and normal pregnancy using a technology platform based on difference gel electrophoresis, matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI TOF/TOF)and bioinformatics analysis. It can provide the specific different proteins for screening the high risk group of HDP and early diagnosis and ultimately decrease the morbidity of severe complication, such as eclampsia and HELLP syndrome, and the death rate of the maternal and fetus.
Part One Urine differential proteome identification and bioinfornatics analysis of hypertensive disorders in pregnancy
[Purpose]
The etiology and pathogenesis of HDP were unclear. Different causes were not independent, but connected and influenced with each other, therefore we still have not a specific biomarker to make early diagnosis of HDP and predict its severity. This study intended to find the specific protein biomarker using the technology platform of differential proteome.
[Method]
Urine samples were collected from5gestational hypertension patients,5mild preeclampsia patients,5severe preeclampsia and5normal pregnancies. DIGE were used to separate the proteins. Protein spots of interest were excised by automatic Spot picker, and then the picked spots were carried out with in-gel digestion, followed by using ABI4800MALDI TOF/TOF mass spectrometry. Retrieving SWISSPROT database through the mascot software, then differential proteins were identified.
[Results]
According to the analysis of differential protein spots combining different maps of DIGE and Decyder software, we set these which was up-regulated or down-regulated1.5times as differential protein spots. Comparing the gestaional hypertension group with the normal pregnancy group,44differential protein spots were found, The number of up-regulated is22and on the contrary are22. Comparing the mild preeclampsia group with the gestational hypertension group,50differential protein spots were found, The number of up-regulated are15and on the contrary are30. Comparing with the severe preeclampsia group with the mild preeclampsia group,45differential protein spots were found, The number of up-regulated are9and on the contrary are36. Some differential protein spots between a few groups all change, we finally analyse65differential protein spots. After mass spectrometry analysis,28differential proteins were identified, containing Prostaglandin-H2D-isomeraseand Basement membrane-specific heparan sulfate proteoglycan core protein.
[Conclusion]
Using a technology platform based on DIGE-MALDI TOF/TOF and bioinformatics analysis, we can find the differential preoteins in urine between HDP and normal pregnancies, which are closely associated with the generation and development of HDP. These proteins have the possibility to be the specific biomaekers of this disease.
Part two Initial quantitative study of some associated differential proteins of hypertensive disorders in pregnancy
Chapter1The urine L-PGDS level in hypertensive disorders in pregnancy
[Purpose]
There is differential expression of L-PGDS in hypertensive disorders in pregnancy based on proteomic analysis. L-PGDS can change PGH2to PGD2, and the latter can inhibit platelet aggregation and have the function of vasodilatation. This study intends to copare the urine L-PGDS levels among normal pregnancies, gestational hypertension, mild preeclampsia and severe preeclampsia to diagnosis this disease.
[Method]
Collect10ml urine from every normal normal pregnancy, gestational hypertension, mild preeclampsia and severe preeclampsia case. Every group have20cases. Evaluate the L-PGDS concentrations of the urine samples using ELISA mathod and then analysis the results.
[Results]
ANOVA analysis indicted that there exited significant difference of the urine L-PGDS concentrations between the four groups(F=27.610, P<0.001). Comparing with the normal pregnancy group, we found that preoperative and postoperative urodynamics of Group RH, we found that the urine L-PGDS concentration of Group gestational hypertension was higher(1.32±0.16ug/mL), however, the L-PGDS levels of Group mild preeclampsia(0.94+0.23μg/mL) and Group severe preeclampsia (0.78±0.23Mg/mL)were significantly lower than that of normal pregnancy Group. Compared with Group gestational hypertension, both of the urine L-PGDS levels of Group mild preeclampsia and Group severe preeclampsia were lower. And the urine L-PGDS concentration of Group severe preeclampsia was significantly lower than that of Group mild preeclampsia. From these results, we know that the urine L-PGDS level decrease in preeclampsia patients because of glomerular filtration decrease. But for gestational hypertension, the increase of urine L-PGDS in contrary indict that the renal function maybe in the situation of compensation.
[Conclusion]
The concentration of urine L-PGDS exits difference in hypertensive disorders in pregnancy. It can use to distinct the gestational hypertension and preeclampsia and evaluate the severity of preeclampsia. It can be a good marker to judge the development of this disease.
Chapter2The urine Perlecan level in hypertensive disorders in pregnancy
[Purpose]
There is differential expression of Perlecan in hypertensive disorders in pregnancy based on proteomic analysis. Perlecan is the important constitution of glomerular basement membrane, having the function of meintain the electroststic barrier. This study intends to copare the urine Perlecan levels among normal pregnancies, gestational hypertension, mild preeclampsia and severe preeclampsia to diagnosis this disease.
[Method]
Collect10ml urine from every normal normal pregnancy, gestational hypertension, mild preeclampsia and severe preeclampsia case. Every group have20cases. Evaluate the Perlecan concentrations of the urine samples using ELISA mathod and then analysis the results.
[Results]
ANOVA analysis indicted that there exited significant difference of the urine Perlecan concentrations between the four groups(Welch F=23.934, P<0.001). Comparing with the normal pregnancy group, we found that preoperative and postoperative urodynamics of Group RH, we found that the urine Perlecan concentration of Group gestational hypertension was significantly higher(12.73±3.71nmol/L), however, the Perlecan levels of Group mild preeclampsia(8.02±1.66nmol/L) and Group severe preeclampsia (6.63±1.39nmol/L)were significantly lower than that of normal pregnancy Group. Compared with Group gestational hypertension, both of the urine Perlecan levels of Group mild preeclampsia and Group severe preeclampsia were lower. And the urine Perlecan concentration of Group severe preeclampsia was significantly lower than that of Group mild preeclampsia. From these results, we know that the urine Perlecan level decrease in preeclampsia patients because of its molecular constitution and function changed and then cause proteinuria. But for gestational hypertension, the increase of urine Perlecan in contrary indict that the filtration barrier of glomerular basement membrane is still normal without proteinuria.
[Conclusion]
As L-PGDS, the concentration of urine Perlecan exits difference in hypertensive disorders in pregnancy. It can use to distinct the gestational hypertension and preeclampsia and evaluate the severity of preeclampsia. It can be a good marker to judge the development of this disease.
近百年的研究使人类对该疾病有了一定认识,但其病因和发病机制错综复杂,至今仍不明确,因此目前仍无有效的预防、早期筛查以及预测疾病严重程度的方法,寻找能早期筛查该疾病并预测其严重程度的办法一直是围产医学研究的重点和热点。对于妊娠期高血压疾病,目前公认的病因学说有：血管内皮受损学说、免疫学说、滋养细胞异常入侵学说、氧化应激学说、遗传学说以及一元化学说等。尽管目前的研究在血管内皮受损、免疫以及分子遗传方面发现了一些潜在的生物标志物,如血清血管内皮生长因子、肿瘤坏死因子α、一氧化氮、内皮素、胎盘蛋白13、血浆血管性假血友病因子、胎盘整合素等,这些物质被实验证实参与了疾病发生、发展的过程,其含量存在一定的变化,可以可用来反映子痫前期病情的严重程度,有希望成为该疾病的检测指标。但是,由于导致该疾病的多种病因并不独立而是彼此紧密联系,始终没能找到特异性的检测指标和方法作为该疾病的早期筛查和疾病严重程度预测的办法,有效的应用于临床。
传统蛋白质功能研究大多是在已有的工作基础上对某几个蛋白质进行分析,从理论上推导出对疾病发生发展可能具有重要意义的蛋白质,继而进行深入细致的功能研究。而这种“假设驱动”的研究策略往往存在片面性、主观性和低效率等缺点。随着人类基因组计划(human genomic project, HGP)的提前完成,蛋白质组学的理论系统和技术方法已经成为后基因组时代的主旋律。由于蛋白质组学研究具有大规模、高通量、高灵敏度的特点,因此可以有效地、客观地分析细胞内的蛋白质整体。差异蛋白质组学能通过比较样本间蛋白质表达水平的变化,寻找差异分子,用以解决各种各样的生物学问题,在临床上可以从整体上研究与疾病相关的差异蛋白质组,寻找疾病的标记蛋白,已广泛应用于疾病的早期筛查和诊断。目前己涌现出许多新的差异蛋白质组分析方法,其中荧光差异凝胶电泳(difference gel electrophoresis, DIGE)技术由于继承了二维凝胶电泳(two-dimensional electrophoresis,2-DE)的高分辨率,同时又具备高重现性、高灵敏度、高通量和高动态范围等优势,使得DIGE日益受到关注,成为最受欢迎的定量蛋白质组学研究手段之一。DIGE是一种在2D电泳之前进行荧光染料标记蛋白样品的方法,通过对不同的蛋白样品用不同的荧光染料进行标记,在同一块双向凝胶中可同时分离多至三种不同的蛋白样品,并且每块胶上均引入了内标,内标的利用可以进一步增加可信度,确保结果能反映出真实的生物学差异,避免了系统误差对实验结果的影响,从而能够对样品间蛋白质丰度差异进行精确分析。
联合应用DIGE分离蛋白和质谱技术鉴定蛋白为差异蛋白质组学研究提供了最为有效的技术平台,实现了快速、高效、高通量检测,目前该种技术在恶性肿瘤(鼻咽癌、乳腺癌、胃癌、前列腺癌、肝癌、膀胱癌)、自身免疫性疾病、感染性疾病等的早期诊断上显示了不可估量的价值,其诊断的敏感性和特异性均可达90%以上。近年来一些学者已将其用于产科领域研究,通过对孕妇附属物及孕妇体液,如胎盘、羊水、血液、脑脊液、尿液等的差异蛋白质谱的分析,有望阐明妊娠及其严重并发症的相关的分子生物学基础,通过比较正常妊娠妇女和患有产科合并症的孕妇的胎盘组织及体液的差异蛋白质谱,能发现一些潜在的特异性的分子生物学标志蛋白。
尿液是血液经肾小球滤过、肾小管和集合管重吸收、排泄及分泌产生的终末代谢产物,其组成、数量与性状的变化携带有泌尿系统疾病发生、发展及预后的各种信息,也可反映机体整体代谢状态,与血清等其他体液样本相比,其获取无创、收集便利,并且蛋白组成相对简单、易于分析。国内外学者目前热衷于对生理和疾病状态下的尿液进行蛋白质组学分析,并已经获得了正常尿液标准蛋白图谱,同时也着手开始研究疾病状态下尿蛋白的变化。
因此本研究拟选用正常妊娠女性和妊娠期高血压疾病孕妇的尿液作为研究对象,利用基于DIGE-基质辅助激光解吸离子化-飞行时间／飞行时间质谱(matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry, MALDI-TOF/TOF)-生物信息学(bioinformatics)的差异蛋白质组学技术平台,来寻找并分析在妊娠期高血压疾病发生发展中的尿液中的差异蛋白质,然后采用临床上经典且易操作的酶联免疫吸附测定法(enzyme-linked immonosorbent asssay, ELISA)检测妊娠期高血压疾病孕妇和正常妊娠女性尿液中特定差异蛋白质的含量进行验证实验,确定特异性的蛋白标记物,从而早期筛查妊娠期高血压疾病的高危人群,对其进行产前监控及早期预防,早期诊断该疾病同时预测其严重程度,及时阻止该病的发生、发展,减少子痫、HELLP综合征等严重并发症的发生,降低孕产妇及围产儿死亡率。
第一部分妊娠期高血压疾病尿液差异蛋白质组的鉴定与生物信息学分析
[目的]
妊娠期高血压疾病病因和发病机制不明确,各种不同病因之间相互联系和影响导致目前仍无特异性的检测指标和方法早期筛查、诊断该疾病并预测疾病严重程度。本研究利用差异蛋白质组学技术平台,寻找妊娠期高血压疾病发生发展过程中的特异性蛋白标记物。
[方法]
选择符合入选标准的5例妊娠期高血压患者、5例轻度子痫前期患者、5例重度子痫前期患者和5例正常妊娠女性,收集其尿液标本,进行双向差异凝胶电泳分析,然后利用全自动切胶仪Spot picker挖取差异蛋白胶粒,并进行样品胶内酶解,再使用MALDI-TOF/TOF质谱仪进行分析,寻找出正常妊娠女性和妊娠期高血压疾病孕妇尿液中的差异蛋白质,最后通过Mascot软件检索SwissProt数据库对蛋白进行鉴定。
[结果]
根据DIGE的差异图谱并结合Decyder差异分析软件进行差异蛋白点分析,妊娠期高血压组和正常妊娠组相比较,共找到了44个差异蛋白点,其中上调的有22个,下调的有22个；轻度子痫前期组和妊娠期高血压组相比较,共找到了50个差异蛋白点,其中上调的有15个,下调的有35个；重度子痫前期组和轻度子痫前期组相比较,共找到了45个差异蛋白点,其中上调的有9个,下调的有36个。有些差异蛋白点在几组之间均有变化,共分析出65个差异蛋白质点。经质谱分析后共鉴定出包括前列腺素-H2D-异构酶(prostaglandin-H2D-isomerase,又称为Lipocalin型前列腺素D合成酶(lipocalin-prostaglandin D synthase, L-PGDS)).基底膜特异的硫酸乙酰肝素蛋白多糖核心蛋白(basement membrane-specific heparan sulfate proteoglycan core protein,又称为Perlecan)、激肽原和妊娠特异性β-1-糖蛋白11等的30种蛋白质,蛋白质相互作用分析显示这30种蛋白质中其中19种存在直接或间接的相互作用。
[结论]
利用基于DIGE-MALDI-TOF/TOF-生物信息学的差异蛋白质组学技术平台,发现妊娠期高血压疾病和正常妊娠女性尿液中存在差异表达的蛋白质,且这些蛋白质之间存在相互作用,这些差异蛋白质可能与该疾病的发生、发展密切相关,有望成为其特异性蛋白标记物。
第二部分妊娠期高血压疾病相关差异蛋白质的初步定量研究
第一章L-PGDS在妊娠期高血压疾病孕妇尿液中的含量变化
[目的]
蛋白组学分析发现L-PGDS在妊娠期高血压疾病尿液中存在差异表达。L-PGDS可催化前列腺素(prostaglandin, PG) H2转变为PGD2,后者具有抑制血小板凝集、诱导血管舒张等功能。本研究拟通过比较正常妊娠组、妊娠期高血压组、轻度子痫前期组和重度子痫前期组尿液中L-PGDS浓度来证实其是否存在变化,可用来诊断妊娠期高血压疾病并预测该疾病的严重程度。
[方法]
确诊时收集正常妊娠女性、妊娠期高血压、轻度子痫前期和重度子痫前期患者无菌尿液10ml,每组各20例,应用ELISA方法检测尿液标本中L-PGDS的浓度,然后进行对比分析。
[结果]
经Levene检验显示四组方差齐性(P=0.385),选用One-Way ANOVA进行方差分析,结果显示四组尿液中L-PGDS浓度存在显著差异(F=27.610,P<0.001),进一步使用LSD检验方法进行两两比较发现妊娠期高血压组患者尿液中L-PGDS浓度(1.32±0.16μg/mL)和正常妊娠组(1.26±0.25μg/mL)无显著差异(P=0.399)；而轻度子痫前期组(0.94±0.23μmL)和重度子痫前期组(0.78±0.23μg/mL)患者尿液中L-PGDS浓度则显著低于正常妊娠组(P均<0.001)。相对于妊娠期高血压组,轻度子痫前期组和重度子痫前期组尿液中L-PGDS浓度均显著降低(P均<0.001)。重度子痫前期组患者尿液L-PGDS浓度显著低于轻度子痫前期组(P=0.023)。由此可见,相对于正常妊娠女性,子痫前期时患者血浆中L-PGDS经肾小球滤过减少导致尿液中含量降低,并且随着疾病的严重程度其浓度逐渐递减。而对于妊娠期高血压患者,其尿液中L-PGDS浓度没有出现下降,可能提示此时患者肾脏功能正处于代偿状态。
[结论]
尿液中L-PGDS浓度在妊娠期高血压疾病不同分类中存在差异,可以很好区别妊娠期高血压和子痫前期,并反映子痫前期的严重程度,可作为尿液中一种较好的蛋白标记物来判断该疾病的发生发展情况。
第二章Perlecan在妊娠期高血压疾病孕妇尿液中的含量变化
[目的]
蛋白组学分析发现Perlecan在妊娠期高血压疾病尿液中存在差异表达。Perlecan作为肾小球基底膜的重要组成成分,主要参与其负电荷屏障的维持。本研究拟通过比较正常妊娠组、妊娠期高血压组、轻度子痫前期组和重度子痫前期组尿液中Perlecan浓度来证实其是否存在差异,可用来诊断妊娠期高血压疾病并预测该疾病的严重程度。
[方法]
确诊时收集正常妊娠、妊娠期高血压、轻度子痫前期和重度子痫前期患者无菌尿液10ml,每组各20例,应用ELISA方法检测尿液标本中Perlecan的浓度,然后进行对比分析。
[结果]
经Levene检验显示四组方差不齐(P<0.001),选用类似方差分析Welch检验进行统计分析,结果显示四组尿液中Perlecan浓度存在显著差异(Weich F值=23.934,P<0.001),进一步使用DunnettT3检验方法进行两两比较发现妊娠期高血压组患者尿液中Perlecan浓度(12.73±3.71nmol/L)高于正常妊娠组(9.99±1.85nmol/L),存在显著差异(P=0.036)；轻度子痫前期组(8.02±1.66nmol/L)和重度子痫前期组(6.63±1.39nmol/L)患者尿液中Perlecan浓度则显著低于正常妊娠组(P=0.006;P<0.001)。相对于妊娠期高血压组,轻度子痫前期组和重度子痫前期组尿液中Perlecan浓度均显著降低(P均<0.001)。重度子痫前期组患者尿液Perlecan浓度显著低于轻度子痫前期组(P=0.039)。由此可见,相对于正常妊娠女性,子痫前期时患者肾小球基底膜上Perlecan分子结构和功能发生变化导致尿液中含量降低,并且随着疾病的严重程度其浓度逐渐递减,此时大量蛋白从肾小球漏出形成蛋白尿。而对于妊娠期高血压患者,其尿液中Perlecan浓度不下降反而上升,说明此时肾小球基底膜滤过屏障仍处于正常状态,无尿蛋白发生。
[结论]
同L-PGDS一样,尿液中Perlecan浓度在妊娠期高血压疾病不同分类中也存在差异,可以很好区别妊娠期高血压和子痫前期,并反映子痫前期的严重程度,可作为尿液中一种较好的蛋白标记物来判断该疾病的发生发展情况
In China, the morbidity of hypertensive disorders in pregnancy (HDP) is about9.4%-10.4%. It can causes organ injuries of the patient and fetal growth restriction, even fetal death, being one of the important reasons of maternal and fetal death. In developing countries,20%-80%maternal death contributes to preeclampsia and in developed, the maternal death of preeclampsia is5times of that of without preeclampsia.
Based on hundred years research on HDP, people have known more about this disease, however, the etiology and pathogenesis are still unclear. Therefore, there still be no good method to prevent HDP and make an early diagnosis. Finding a good measure to make diagnosis early and estimate its severity of HDP is an important and hot spot in Obstetrics. For HDP, there are many etiological theries, such as vascular epithial injury; immunological theory; oxidative theory and so on. Although several potential biomarkers exist in aspect of vascular epithial injury and immunology and genetics, for example, vascular epithial growth factor, tumor necrosis factor, nitrogen monoxidum, endothelin, placental protein13, von Wilebrand factor, placental integrin.These materials were identified that they participated in the generation and development of HDP. The concentrations changed in the patients of HDP when comparing to normal pregnacies, making possible promising biomakers in predicting the severity of hypertensive disorders of pregnancy. However, because these reasons causing HDP are not independent and the estimating technology are limited, now we still have not a specific biomarker to make early diagnosis of HDP and predict its severity in clinic.
Traditional study of protein function was mostly focus at a few proteins on the basis of the existing work and theoretically deduced proteins which may have important implications on the development of the disease, and then followed by in-depth and detailed functional study. But this kind of "hypothesis driven"research strategy often exist shortcomings, such as unilateralism, subjectivity and low efficiency. As the human genome project (human genomic project, HGP) completed ahead, proteomics theory and the technical method has become the theme of post genome era. As a large-scale, high throughput, high sensitivity method, proteomics can be effectively, objectively analyze the overall intracellular proteins. Through comparing the protein expression level between differences samples, differential expression proteins were identified, which can be used to solve various biological problems. At present there are many novel differential proteomics analysis methods, among which the difference gel electrophoresis (difference gel electrophoresis, DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. DIGE is a method which can labele protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the real biological differences, while avoid influence of systematic errors on experimental results.
Using DIGE to separate proteins and mass spectrometry to identify the protein together provides most effective technology platform for differential proteome, with the advantage of fast, high effective and high throughout detection. Recently, this technology has revealed important value on early diagnosis of malignant tumors, such as nasopharyngeal carcinoma, breast cancer, gastric cancer, liver cancer, autoimmune disease and infectious diseases, with the specificity and sensitivity both up to90%. In nearly these years, some authors use this technology to study the obstetrical diseases. Basing on differential proteome analysis of maternal accessory and body fluid, such as placenta, amniotic fluid, blood, cerebrospinal fluid and urine, it is possible to explain the molecular biology of pregnancy and its complications. From comparing differential proteome of normal pregnancy and obstetrical complications, we can find some potential specific molecular biomarkers.
Urine is the last metabolic product of blood after glomerular filtration and renal tubule resorption. The change of its composition, quantity and characteristics can reflect the information of urinary system diseases and even the metabolism condition of whole body. Comparing with the other body fluid, such as blood, urine is easy to get in the clinic and the protein composition are more simple. Studying and analyzing the proteome of urine is under normal and abnormal situations is a hot spot for medical experts. Now, the protein spectrum of the normal urine has obtained and people are ready to study the protein spectrum under the situation of disease. The proteome analysis using mass spectrometry of the urine were applied in screening and diagnosing many diseases.
Therefore, this study intends to find the different proteins between the urine of hypertensive disorders of pregnancy and normal pregnancy using a technology platform based on difference gel electrophoresis, matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI TOF/TOF)and bioinformatics analysis. It can provide the specific different proteins for screening the high risk group of HDP and early diagnosis and ultimately decrease the morbidity of severe complication, such as eclampsia and HELLP syndrome, and the death rate of the maternal and fetus.
Part One Urine differential proteome identification and bioinfornatics analysis of hypertensive disorders in pregnancy
[Purpose]
The etiology and pathogenesis of HDP were unclear. Different causes were not independent, but connected and influenced with each other, therefore we still have not a specific biomarker to make early diagnosis of HDP and predict its severity. This study intended to find the specific protein biomarker using the technology platform of differential proteome.
[Method]
Urine samples were collected from5gestational hypertension patients,5mild preeclampsia patients,5severe preeclampsia and5normal pregnancies. DIGE were used to separate the proteins. Protein spots of interest were excised by automatic Spot picker, and then the picked spots were carried out with in-gel digestion, followed by using ABI4800MALDI TOF/TOF mass spectrometry. Retrieving SWISSPROT database through the mascot software, then differential proteins were identified.
[Results]
According to the analysis of differential protein spots combining different maps of DIGE and Decyder software, we set these which was up-regulated or down-regulated1.5times as differential protein spots. Comparing the gestaional hypertension group with the normal pregnancy group,44differential protein spots were found, The number of up-regulated is22and on the contrary are22. Comparing the mild preeclampsia group with the gestational hypertension group,50differential protein spots were found, The number of up-regulated are15and on the contrary are30. Comparing with the severe preeclampsia group with the mild preeclampsia group,45differential protein spots were found, The number of up-regulated are9and on the contrary are36. Some differential protein spots between a few groups all change, we finally analyse65differential protein spots. After mass spectrometry analysis,28differential proteins were identified, containing Prostaglandin-H2D-isomeraseand Basement membrane-specific heparan sulfate proteoglycan core protein.
[Conclusion]
Using a technology platform based on DIGE-MALDI TOF/TOF and bioinformatics analysis, we can find the differential preoteins in urine between HDP and normal pregnancies, which are closely associated with the generation and development of HDP. These proteins have the possibility to be the specific biomaekers of this disease.
Part two Initial quantitative study of some associated differential proteins of hypertensive disorders in pregnancy
Chapter1The urine L-PGDS level in hypertensive disorders in pregnancy
[Purpose]
There is differential expression of L-PGDS in hypertensive disorders in pregnancy based on proteomic analysis. L-PGDS can change PGH2to PGD2, and the latter can inhibit platelet aggregation and have the function of vasodilatation. This study intends to copare the urine L-PGDS levels among normal pregnancies, gestational hypertension, mild preeclampsia and severe preeclampsia to diagnosis this disease.
[Method]
Collect10ml urine from every normal normal pregnancy, gestational hypertension, mild preeclampsia and severe preeclampsia case. Every group have20cases. Evaluate the L-PGDS concentrations of the urine samples using ELISA mathod and then analysis the results.
[Results]
ANOVA analysis indicted that there exited significant difference of the urine L-PGDS concentrations between the four groups(F=27.610, P<0.001). Comparing with the normal pregnancy group, we found that preoperative and postoperative urodynamics of Group RH, we found that the urine L-PGDS concentration of Group gestational hypertension was higher(1.32±0.16ug/mL), however, the L-PGDS levels of Group mild preeclampsia(0.94+0.23μg/mL) and Group severe preeclampsia (0.78±0.23Mg/mL)were significantly lower than that of normal pregnancy Group. Compared with Group gestational hypertension, both of the urine L-PGDS levels of Group mild preeclampsia and Group severe preeclampsia were lower. And the urine L-PGDS concentration of Group severe preeclampsia was significantly lower than that of Group mild preeclampsia. From these results, we know that the urine L-PGDS level decrease in preeclampsia patients because of glomerular filtration decrease. But for gestational hypertension, the increase of urine L-PGDS in contrary indict that the renal function maybe in the situation of compensation.
[Conclusion]
The concentration of urine L-PGDS exits difference in hypertensive disorders in pregnancy. It can use to distinct the gestational hypertension and preeclampsia and evaluate the severity of preeclampsia. It can be a good marker to judge the development of this disease.
Chapter2The urine Perlecan level in hypertensive disorders in pregnancy
[Purpose]
There is differential expression of Perlecan in hypertensive disorders in pregnancy based on proteomic analysis. Perlecan is the important constitution of glomerular basement membrane, having the function of meintain the electroststic barrier. This study intends to copare the urine Perlecan levels among normal pregnancies, gestational hypertension, mild preeclampsia and severe preeclampsia to diagnosis this disease.
[Method]
Collect10ml urine from every normal normal pregnancy, gestational hypertension, mild preeclampsia and severe preeclampsia case. Every group have20cases. Evaluate the Perlecan concentrations of the urine samples using ELISA mathod and then analysis the results.
[Results]
ANOVA analysis indicted that there exited significant difference of the urine Perlecan concentrations between the four groups(Welch F=23.934, P<0.001). Comparing with the normal pregnancy group, we found that preoperative and postoperative urodynamics of Group RH, we found that the urine Perlecan concentration of Group gestational hypertension was significantly higher(12.73±3.71nmol/L), however, the Perlecan levels of Group mild preeclampsia(8.02±1.66nmol/L) and Group severe preeclampsia (6.63±1.39nmol/L)were significantly lower than that of normal pregnancy Group. Compared with Group gestational hypertension, both of the urine Perlecan levels of Group mild preeclampsia and Group severe preeclampsia were lower. And the urine Perlecan concentration of Group severe preeclampsia was significantly lower than that of Group mild preeclampsia. From these results, we know that the urine Perlecan level decrease in preeclampsia patients because of its molecular constitution and function changed and then cause proteinuria. But for gestational hypertension, the increase of urine Perlecan in contrary indict that the filtration barrier of glomerular basement membrane is still normal without proteinuria.
[Conclusion]
As L-PGDS, the concentration of urine Perlecan exits difference in hypertensive disorders in pregnancy. It can use to distinct the gestational hypertension and preeclampsia and evaluate the severity of preeclampsia. It can be a good marker to judge the development of this disease.
引文
1. Baha S, Gus D, Michael K. Pre-eclamPsia. Lancet,2005,365(9461):785-799.
2. Endresen JR, Jonathan M, Ariadnec N, et al. Serum from preeclamptic woman induces vascular cell adhesion molecule-1 expression on human endothelial cells invitro:A possible role of increased circulating levels of free fatty acids[J]. AmJ Obstet Gynecol,1998,179:665-669.
3. Deng L, Bremme K, Hansson LO, et al. Plasma levels of vonwillebrand factor and fibronectin as markers of persisting endothelial damage in preeclampsia. Obstet Gynecol,1994,84:941-945.
4. Yallampalli CDVM, Garfield R. Inhibitionof nitricoxide synthesis in rats during pregancy producs signs similiar to those of preeclampsia[J].AmJ Obstet Gynecol, 1996,169:1316-1321.
5. Silver RK, Kupfermine MJ, Russell TLBA, et al. Evaluationof nitricoxideas amediator of severepreeclampsia[J]. AmJ Obstet Gynecol,1996,175:1013-1017.
6. Livengston JC, Chin R, Haddad B et all Eeductons of vascular endothelial growth factor and placental growth factor concentration in severe preeclampsia.AmJ Obster Gyneco,2000,183 (6):1554-1557.
7.乔福元,吴媛媛 妊娠期高血压疾病病因的研究现状及进展[J]华中医学杂志,2005,29(6)：339-400.
8. Goldman WDS,Ariel I,Greenfeild C,et al.Lack of human Leukocyte antigen -G expression in extravillous trophoblasts is associated with preeclampsia.Mol Hum Reprod,2000,6(1):88-95.
9.吴媛媛,乔福元 妊娠期高血压疾病的分子遗传与病因研究最新进展[J]中国优生与遗传杂志,2005,13(11)：8-9.
10.王晨虹,胡冬煦,张铨富,等.子痫前期患者胎盘整合素α1与β1及肿瘤坏死因子α的表达及其临床意义[J].中国妇幼保健,2007,2：4164-4166.
11. Ishihara N, Matsuo H, Murakoshi H et all Increased apoptosis in the syncytiotrohoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. Am J Obstet Gynecol,2002,186 (1):158-166.
12. Reister F, Frank HG, Kingdom J C et allMacrophage-induced apoptosis limits dovascular trophoblast invasion in the uterine wall of preeclamptic women. Lab Invest,2001,81 (8):1143-1152.
13. Wei Y S, Zheng Y H, Liang W B, et al. Identification of serum biomarkers for nasopharyngeal carcinoma by proteomic analysis [J]. Cancer,2008,112(3): 544-551.
14. Suzuki M, Ross G F, Wiers K, et al. Identification of a urinary proteomic signature for lupus nephritis in children[J]. Pediatr Nephrol,2007,22(12): 2047-2057.
1. Baha S, Gus D, Michael K. Pre-eclamPsia. Lancet,2005,365(9461):785-799.
2. Alban A, David SO, Bjorkesten L, et al. A novel experimental design for comparative two-dimensional gel analysis:two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 2003; 3(1): 36-44.
3. Amersham Biosciences Ettan DIGE User manual, http://www. amershambiosciences.com.
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5. Suzuki M, Ross G F, Wiers K, et al. Identification of a urinary proteomic signature for lupus nephritis in children[J]. Pediatr Nephrol,2007,22(12): 2047-2057.
6. Buhimschi IA, Christner R, Buhimschi CS. Proteomic biomarker analysis of amniotic fluid for identification of intra-amniotic inflammation. BJOG. 2005;l 12:173-181.
7. Buhimschi CS, Bhandari V, Hamar BD, Bahtiyar MO, Zhao GM, Sfakianaki AK, et al. Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis. PLoS Med.2007 January; 4(1):e18.
8. Vuadens F, Benay C, Crettaz D, et al. Identification of biologic markers of the premature rupture of fetal membranes[J]. Proteomic approach Proteomics,2003, 3,1521-1525.
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11. Vaseotto C, Salzano AM, D'Ambrosio C, Fruscalzo A, Marchesoni D, di Loreto C, et al. Oxidized transthyretin in amniotic fluid as an early marker of Preeclampsia.J Proteome Res.2007,6(1):160-70.
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14.林晶,何晓宇,曾芳.SELDI-TOF-MS技术检测妊娠期高血压疾病血清差异蛋白的表达[J].实用医学杂志,2008,24(14)：2413-2415.
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1. Knox S M, Whitelock J M. Perlecan:how does one molecu le do so many things? Cell Mol Life Sci,2006,63:2435-2445
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2. Endresen JR, Jonathan M, Ariadnec N, et al. Serum from preeclamptic woman induces vascular cell adhesion molecule-1 expression on human endothelial cells invitro:A possible role of increased circulating levels of free fatty acids[J]. AmJ Obstet Gynecol,1998,179:665-669.
3. Deng L, Bremme K, Hansson LO, et al. Plasma levels of vonwillebrand factor and fibronectin as markers of persisting endothelial damage in preeclampsia. Obstet Gynecol,1994,84:941-945.
4. Yallampalli CDVM, Garfield R. Inhibitionof nitricoxide synthesis in rats during pregancy producs signs similiar to those of preeclampsia[J].AmJ Obstet Gynecol, 1996,169:1316-1321.
5. Silver RK, Kupfermine MJ, Russell TLBA, et al. Evaluationof nitricoxideas amediator of severepreeclampsia[J]. AmJ Obstet Gynecol,1996,175:1013-1017.
6. Livengston JC, Chin R, Haddad B et all Eeductons of vascular endothelial growth factor and placental growth factor concentration in severe preeclampsia.AmJ Obster Gyneco,2000,183 (6):1554-1557.
7.乔福元,吴媛媛 妊娠期高血压疾病病因的研究现状及进展[J]华中医学杂志,2005,29(6)：339-400.
8. Goldman WDS,Ariel I,Greenfeild C,et al.Lack of human Leukocyte antigen -G expression in extravillous trophoblasts is associated with preeclampsia.Mol Hum Reprod,2000,6(1):88-95.
9.吴媛媛,乔福元 妊娠期高血压疾病的分子遗传与病因研究最新进展[J]中国优生与遗传杂志,2005,13(11)：8-9.
10.王晨虹,胡冬煦,张铨富,等.子痫前期患者胎盘整合素α1与β1及肿瘤坏死因子α的表达及其临床意义[J].中国妇幼保健,2007,2：4164-4166.
11. Ishihara N, Matsuo H, Murakoshi H et all Increased apoptosis in the syncytiotrohoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. Am J Obstet Gynecol,2002,186 (1):158-166.
12. Reister F, Frank HG, Kingdom J C et allMacrophage-induced apoptosis limits dovascular trophoblast invasion in the uterine wall of preeclamptic women. Lab Invest,2001,81 (8):1143-1152.
13. Wei Y S, Zheng Y H, Liang W B, et al. Identification of serum biomarkers for nasopharyngeal carcinoma by proteomic analysis [J]. Cancer,2008,112(3): 544-551.
14. Suzuki M, Ross G F, Wiers K, et al. Identification of a urinary proteomic signature for lupus nephritis in children[J]. Pediatr Nephrol,2007,22(12): 2047-2057.
1. Baha S, Gus D, Michael K. Pre-eclamPsia. Lancet,2005,365(9461):785-799.
2. Alban A, David SO, Bjorkesten L, et al. A novel experimental design for comparative two-dimensional gel analysis:two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 2003; 3(1): 36-44.
3. Amersham Biosciences Ettan DIGE User manual, http://www. amershambiosciences.com.
4. Wei Y S, Zheng Y H, Liang W B, et al. Identification of serum biomarkers for nasopharyngeal carcinoma by proteomic analysis [J]. Cancer,2008,112(3): 544-551.
5. Suzuki M, Ross G F, Wiers K, et al. Identification of a urinary proteomic signature for lupus nephritis in children[J]. Pediatr Nephrol,2007,22(12): 2047-2057.
6. Buhimschi IA, Christner R, Buhimschi CS. Proteomic biomarker analysis of amniotic fluid for identification of intra-amniotic inflammation. BJOG. 2005;l 12:173-181.
7. Buhimschi CS, Bhandari V, Hamar BD, Bahtiyar MO, Zhao GM, Sfakianaki AK, et al. Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis. PLoS Med.2007 January; 4(1):e18.
8. Vuadens F, Benay C, Crettaz D, et al. Identification of biologic markers of the premature rupture of fetal membranes[J]. Proteomic approach Proteomics,2003, 3,1521-1525.
9. Irina B, Lawrence T, Joong SP, et al. Proteomic profiling of cerebrospinal fluid (CSF) in patients with severe preeclampsia (sPE) suggests subclinical microhemorrhage with functional consequence [J]. American Journal of Obsterics and Gynecology,2004,191:S32.
10. Park JS, Oh KJ, Norwitz ER, Han JS, Choi HJ, Seong HS, et al. Identification of Proteomic biomarkers of Preeelampsia in amniotic fluid using SELDI-TOF mass Spectrometry. Reprod Sci.2008,15(5):457-68.
11. Vaseotto C, Salzano AM, D'Ambrosio C, Fruscalzo A, Marchesoni D, di Loreto C, et al. Oxidized transthyretin in amniotic fluid as an early marker of Preeclampsia.J Proteome Res.2007,6(1):160-70.
12.吕雅洁,李力,俞丽丽,许洋.妊娠期高血压疾病患者特异性血清标志物筛查的初步探讨[J].中华妇产科杂志,2005,40(5)：306-308.
13.俞丽丽,李力,吕雅洁,王云,许洋.应用血清蛋白质谱图筛查妊娠期高血压疾病血清学指标[J].中国实用妇科与产科杂志,2006,22(11)：855-856.
14.林晶,何晓宇,曾芳.SELDI-TOF-MS技术检测妊娠期高血压疾病血清差异蛋白的表达[J].实用医学杂志,2008,24(14)：2413-2415.
15. Buhimschi I A, Guomao Z,Funai, et al. Proteomics analysis of urine in preeclampsia:A novel diagnosis for an old disease[J].Am J Obstet Gynecol,2006,193(6):S 15.
16. Stemmler MP. Cadherins in development and cancer [J]. Mol Bio-syst,2008,4(8): 835-850.
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