Proteomic Analysis Of Heparin-related Proteins From Human Serum: A Step Towards Identification Of Molecular Makers Of Preeclampsia

BackgroundHypertensive disorders in pregnancy is a special kind of disease which happens in gestation. The mobidity in our country is 9.4%～10.4% and 7%～12% in other countries. This disease emphasize the relationship among hypertensive, proteinuria and pregnancy. Most of pateints have transient hypertensive, proteinuria and it will be disappear after dilivery. Hypertensive disorders in pregnancy is harm the health of mother and baby and it is the major reason for mortality and morbidity.The classification of hypertensive disorders in pregnancy is very difference between our country and other countries. According to the standard in our countries, we can classify this disease into five different types. (1) gestational hypertension. The BP>140/90mmHg and it is the first time appearance in pregnancy. It should be normol at twelve weeks after dilivery. No proteinuria. Few of pateints maybe have obdomen uncomfortable and thrombocytopenia. This kind of disease will be diagnosised after dilivery. (2) preeclampsia.①Slight. BP≥140/90mmHg and it appears after twenty weeks gestation, significant proteinuria(>0.3g/24h or positive proteinuria). Maybe some patients have symptom of obdomen uncomfortable and headache.②severe. BP≥160/110mmHg, significant proteinuria(>2.0g/24h or positive proteinuria), creatinine>106umol/L,blood platelet<100×109/L, higher LHD, higher ALT and AST, persistent headache, visual disturbanres, epigastric pain. (3) eclampsia. Preeclampsia pateints happen to convulsion and this symptom can not explain as other reason. (4)preeclampsia superimposed upon chronic hypertension. The proteinuria and it is the first time appearance in pregnancy, or higher hypertensive, or blood platelet<100×109/L. (5) chronic hypertension complicating pregnancy. Diastolic pressure≥90mmHg happens before pregnancy or before twenty weeks gestation (except trophoblatic diseases), the symptom will not severer during pregnancy, or hypertention is the first time diagnosised after-twenty weeks gestation and it persistent to twelve weeks after dilivery.Nearly half a century, the scholar in our countries and in other countries had do a lot of research for knowledge of pathogen, pathogenetic, treatment and preventive measure. The department of gynaecology and obtetrics in our countries always heels the steps of international scholar's research and makes a lot of great results. These results provide a basement of treatment of hypertensive disorders in pregnancy.Although the cause of hypertensive disorders in pregnancy remains unknow, most of doctors agree that the pathogens of hypertensive disorders in pregnancy include: abnormal gestational trophocyte invade myometrium, immunologic mechnism, vascular endothelial cell demage, hereditary factor, trophic factors and insulin resistance etc.ObjectiveBecause of the unknow pathogenetic of hypertensive disorders in pregnancy. There is not a medicine can specially treat this disease. The clinical practice in our hospital show that heparin can treat this disease effectively.Heparin is a wide use anticoagulant. It is a kind of sulfated polysaccharides which is obtained from animals and it exist in kernel which is secreted from mastocyte. An American scholar named Mclean extracted a material from mammiferous liver and this material can prolong the clotting time. After two years, this kind of material is called heparin. After twenty years, Brinkhous and his colleague confirm that heparin can not directly inactive plasma thromboplastin component. The function of anticoagulation is produce effect though plasma cofactor. In 1968, Abildgaard called this cofactor as ATⅢ. After 1970, the mechanism of heparin was recognized by people. The mechanism is that heparin combine with AT and inactive plasma thromboplastin componentⅡa,Ⅹa,Ⅸa,ⅩⅠa andⅩⅡa. If without AT, heparin can not works very well. When heparin combine with AT, the coagulation will accelerate 1000-2000 times.MethodsThrough comparing the protein expression level between differences samples, differential expression proteins were identified, which can be used to solve various biological problems. With the development of proteomics technology, it is feasible to study whole changes of proteome in the physiological and pathological process now. 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 label protein samples with different fluorescent dyes before 2-D electrophoresis, and then 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. Since The most obvious advantage of DIGE system is integrating the advantages of both CyDye DIGE dye multiple labeling method and DeCyder difference 2-D analysis software. DeCyder software takes the advantage of the spots co-detecting algorithm, which can automatically detected fluorescence images, eliminate background, quantifiy, normalize and match spots in gel, thus, systematic errors caused by different operators can be eliminated. Based on the advantages of DIGE technology platform, great opportunities comes to us to discover the information of differential proteome contained in sample of the patients with hypertension disorder in pregnancy.A technology platform based on difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and bioinformatics analysis was established first, and then some assays on its sensitivity and reproducibility was done in our research. Through analysis by use of DeCyder difference 2-D analysis software,7 differential protein spots were found. All of them are down-regulated proteins are. Finally, a total of 6 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/TOF).The co-expression of these differential proteins indicates that they must have direct or indirect relations. Therefore, how to find out and discover the pathophysiology meaning of these relations in mechanism of heparin treatment is the key for further research. Bioinformatics is a useful tool to solve this problem. Firstly the identified differential proteins were processed by subcellular localization analysis with subcellular localization software WoLF PSORT.After subcellular localization analysis, the protein functional analysis had been done. Through analyzing protein domain and motif database, the identified proteins were roughly categorized into cellular signal transduction.However, this is not enough to know the relationship among proteins, and predicting proteins interactions were also needed. Using the "String" protein interaction databases, we had analyzed the primary interaction of the 6 kinds of proteins (direct effect) and the result showed that two groups of the proteins were of direct interaction.ResultsThrough the experimental techniques exploration, we have established a set of differential proteomics analysis techniques based on the difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and Bioinformatics. This set of techniques is successfully applied to the analysis of differential proteomics. In conclusion, The research not only provide us a novel technology system on revealing the molecular mechanism of life process, but also provide us a new chance for further studying the pathogenesis of mechanism of heparin treatment for hypertension disordered in pregnancy.conclusions1. Established DIGE technology platform which providing a new system with high reliability, high reproducibility for differential proteomics research.2. Extracted and purified proteins from patients with hypertension disordered in pregnancy successfully. Identified difference gel electrophoresis patterns of before and after heparin treatment.3. Found 7 different express proteins by differential software analysis, then identified 6 different proteins using mass spectrometry identification.4. Predicted 6 direct protein-protein interaction complexes by applying bioinformatics analysis on 6 different proteins.