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Proteomic Study on Pathogenic Mechanism of Pregnancy Induced Hypertension and Pancreatic Cancer

Author: JinHong
Tutor: YangPiYuan
School: Fudan University
Course: Analytical Chemistry
Keywords: Pregnancy-induced hypertension syndrome Pancreatic Cancer Proteomics Two-dimensional gel electrophoresis Biological Mass Spectrometry Liquid chromatography MS Laser microdissection RNAi interference 14-3-3τ
CLC: R735.9
Type: PhD thesis
Year: 2007
Downloads: 226
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Abstract

Proteomics as interdisciplinary point of today's analytical chemistry and life sciences in recent years has been rapid development. The nature of the unique advantages of Proteome Research has observed multiple protein components caused by multiple genes events overall change than genomic school closer to the phenomenon of life. It has a powerful potential value in the study of medical and drug targeting, has been widely used to study the occurrence of various diseases, the development of the law, and made significant progress in the search for molecular markers of disease. This dissertation includes the following sections: 1. Pregnancy induced hypertension tissue expression profiles full spectrum analysis; 2. Differential proteomics study of pregnancy induced hypertension and normal tissues: 3.BeWo cells in normal and lack oxygen under the conditions of the differential proteome analysis; 4., pancreatic cancer and paraneoplastic identification of differentially expressed protein 5. research papers: interference by RNAi silencing of 14-3-3τ research, and in rat liver and liver the protein of RPLC/2-DE separation of identification. The papers made major progress: the papers from the proteomics level depth in the pathogenesis of a common multiple of pregnancy-induced hypertension and pancreatic cancer two basic research, so far relatively complete proteome information; as the early diagnosis and treatment of illness to explore new avenues. Paper the initial establishment and optimization of two-dimensional gel electrophoresis of pancreatic tissue, found that the incidence of pancreatic cancer differentially expressed proteins, these proteins found in pancreatic cancer protein biomarkers and drug treatment may become the target protein. The results of this paper to provide an experimental basis of disease marker discovery and cell signaling molecules passing avenues for further research. The paper conducted a study of the RNAi interference silent 14-3-3τ; first verified rat liver for the study of feasibility of RPLC/2-DE separation system, and applied to the the Frenchman liver samples Isolation and Identification get better experimental results, it is an effective complement to existing research methods and technology roadmap. A paper for the first time established a pregnancy-induced hypertension patients proteome expression profiling, and the use of research methods in two-dimensional gel electrophoresis and image analysis software, such as proteomics approach, comparing normal pregnancy and preeclampsia and placental tissue protein group differences in the expression; gel digestion, MALDI-TOF-TOF-MS and database search to identify differentially expressed proteins, and especially important differences protein with Westernblot method validation. In this thesis, on the other hand, using laser microdissection cutting tissue in combination with ICAT isotope quantitatively associated with LC-MS/MS technology to compare the differences in normal pregnancy and preeclampsia placenta tissue proteome expression. Combined with the data obtained by these two methods, the paper presents the molecular mechanisms of the pathogenesis of preeclampsia. 1, pregnancy-induced hypertension tissue expression profiles of full-spectrum analysis of pregnancy-induced hypertension (referred to as PIH) is a common and serious impact on maternal and child safety diseases, its prevention and treatment is extremely important. Cause of pregnancy-induced hypertension is still not clear, is still based on its predilection factors, as well as pathophysiological changes characteristics taken antispasmodic, antihypertensive, diuretic, and the timely termination of pregnancy, the principles of treatment, the lack of effects of drugs and vaccine. PIH pathogenic mechanism with the theories and methods of proteomics research, looking for a target protein can be used for the diagnosis or treatment of important significance. This thesis is to establish the technology program, which includes a comprehensive extraction and separation of PIH placental tissue protein the laser microdissection PIH placental tissue, total protein extraction, one-dimensional SDS-PAGE and chromatography - mass spectrometry and The shot-gun technology on the two-dimensional separation of the total protein. And total protein PIH placental tissue expression profiling of biological mass spectrometry identification of 962 non-redundant proteins were identified, including prolactin and other pregnancy-specific protein, cytoskeletal proteins, heat shock proteins, and cells cycle, apoptosis, and signal transduction proteins, by far the most complete PIH placental tissue protein profiling, providing a wealth of experimental data for further study of the pathogenesis of pregnancy-induced hypertension; further analysis detected low abundance proteins laid the foundation. 2, pregnancy induced hypertension and normal tissues of the differences in protein expression profiling research to establish a more complete pregnancy hypertension placental tissue protein profiling on the basis of the paper continues to apply the two-dimensional gel electrophoresis, two-dimensional high-performance liquid separation and other technology to establish the difference between the spectrum of pregnancy-induced hypertension and normal placental trophoblast cells between the proteome map. Identification of differentially expressed proteins and in-gel digestion, MALDI-TOF-TOF-MS and database search identified 13 differentially expressed proteins, and the significance of differences in protein validated by Western blot method, based on mass spectrometry identification results, preliminary classification and function to retrieve the protein in preeclampsia placenta 3 down, 10 up-regulated, including signal transduction proteins, molecular chaperones, cytoskeletal proteins involved in intracellular material transport and energy metabolism enzyme class, these proteins are associated with preeclampsia. Another parallel route PIH placental tissue laser microdissection ICAT isotope labeling differentially expressed proteins, with a satisfied upgrade two-dimensional liquid chromatography mass spectrometry identification of proteins were identified 28 differentially expressed proteins, which upregulation of 15 proteins, 13 proteins downward; these proteins transferrin layer of fibrin, collagen. The comparative analysis of differentially expressed proteins obtained by the two methods, the majority of these proteins are not the same, indicating that the two methods complement. The BeWo cells under normoxic and hypoxic conditions differential proteome analysis order further research PIH \expression between proteome map between the the abundance difference; proteome analysis PIH trophoblastic expression spectrum and hypoxia the proteomic expression spectrum similar and the differences between trophoblastic. The application of mass spectrometry techniques to identify those trophoblastic hypoxia induced PIH mechanism key proteome component, to determine whether inhibition of protein. The classic methods of molecular biology applied to verify the role of these proteins in PIH. The study compared the anoxic conditions and under normal oxygen conditions, BeWo cells differentiate into syncytiotrophoblast cell proteome expression differences and found some differences in the expression levels of protein. These proteins are involved in the regulation of cellular oxidative stress, cell cycle, and signal transduction, and contribute to the further understanding of hypoxia led to the relationship of these protein expression changes with preeclampsia. The hypoxia BeWo cells form syncytiotrophoblast 20 protein expression changes in cellular processes, including antioxidant proteins involved in the glycolytic enzymes, molecular chaperones, signal transduction proteins and cytoskeletal proteins such as. 20 protein, in addition the individual protein have been reported with trophoblastic hypoxia related to changes in this paper for the first time they hypoxia impact syncytiotrophoblast formation. These results provide a new perspective for the study of trophoblast hypoxia-reactive protein, and a better understanding of hypoxia affect syncytiotrophoblast formation of molecular mechanisms. Normal pancreatic tissue and cancer adjacent tissues differentially expressed protein identification papers in this part of the work of the establishment and optimization of two-dimensional gel electrophoresis of pancreatic tissue extract protein, with good resolution and repeatability, application two-dimensional gel electrophoresis of the 12 pairs of pancreatic cancer and paraneoplastic tissue samples, three benign pancreatic disease samples, three normal pancreatic tissue samples for analysis, and found 30 differentially expressed proteins. With MALDI-TOF-MS/MS technical identification of differentially expressed proteins, 24 proteins were identified, 15 proteins were up-regulated in pancreatic carcinoma 9 protein downregulation. Various enzymes involved in the life processes and their precursors, such as trypsin precursor, acetyl coenzyme A dehydrogenase; S100 calcium-binding proteins, such as Calgizzarin (S100A11); cytoskeletal proteins heat shock protein family: family; Annexin A5; membrane transporter related tissue as well as some unknown function protein. The occurrence of these proteins with pancreatic cancer related target proteins, can be used as protein markers of pancreatic cancer and drug treatment. Three study of 14-3-3τ and rat liver and Law liver by reversed-phase liquid chromatography combined with two-dimensional gel electrophoresis separation route in this chapter work interference silencing by RNAi RNA interference (RNA interference, RNAi) silent 14-3-3τ expression of silence before and after the two-dimensional gel electrophoresis to identify differentially expressed proteins, to study the role of 14-3-3τ in the the syncytiotrophoblast formation of hypoxia inhibit syncytiotrophoblast formation of. Reversed phase high performance liquid chromatography / two-dimensional gel electrophoresis (RPLC/2-DE) established for liver tissue protein separation, the initial establishment of a low-abundance protein identification mode, so that the method with two-dimensional gel electrophoresis methods complement each other supplement, offers new possibilities for high-throughput proteomics research.

Proteomic Study on Pathogenic Mechanism of Pregnancy Induced Hypertension and Pancreatic Cancer

Abstract

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