| 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 pregnancyChapter1The 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. |
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