Proteomic Study On Pathogenic Mechanism Of Pregnancy Induced Hypertension And Pancreatic Cancer

Nowadays, proteomics is an essential discipline as a current intersect of analytical chemistry and life science, proteomics develops rapidly in recent years. It observes all the components in whole proteomic level changes of the proteins caused by multi-genes and reveals the life essence more precisely than genomics. Proteomics has great potentials in medical and drug targets. Now proteomics is widely used as a tool for researching the cause and mechanism of various diseases, many great progresses have been made in finding disease biomarkers.Contents and advances of this dissertation described as follows.In the first chapter, advances in 2D and multidimensional separation techniques were reviewed in detail. Fundamentals of multidimensional separation techniques were introduced and the advantages and shortcomings of existing technical modes were discussed.Aims and advances of this dissertation were described.Chapter 2 described the total protein expression profile analysis of pregnancy induced hypertension (PIH) patients tissue. Chapter 3 described differential protein expression profile analysis between normal and PIH tissue. Chapter 4 described differential protein expression profile analysis between BeWo cell under normal and hypoxia conditions. Chapter 5 described differential protein identification between normal and pancreatic tumor tissue. And chapter 6 described some results of research, including fractionating complex protein samples from rat liver and French human liver prior to 2-DE using RPLC and differential protein expression profile analysis between normal and using RNAi technology to knock out 14-3-3Ï„protein.The main advances of this study can be summarized as follows. This study is focused on the fundamental of pathogenic mechanism of PIH and pancreatic cancer. A relative complete proteomic profile has been established, providing a new method for disease early diagnostic biomarker and treatment. 2D technology and the optimized experimental conditions for pancreatic cancer were established for this study use. The differential expressed proteins were found as the eventually disease biomarker and drug target for pancreatic cancer. Experimental findings of discovering disease biomarker and cell signal transduction pathways provide very useful information for the further study, In addition, RNAi to interfere and knock out 14-3-3Ï„protein to have functional validation have been studied.1. This is the first part of the dissertation. In this part, the proteomics study and resulted advances for PIH were described. A protein expression profile of PIH patient was established for the first time. By using proteomic technique of 2D-gel electrophoresis and image analysis software, the differential protein expression profile of tissue between the normal pregnant patient and PIH patient was compared. The differential proteins were identified via in-gel digestion followed MALDI-TOF-TOF-MS and database search; those especially essential proteins had been verified with Western blot. In addition, laser capture micro-dissection (LCM) technique was used to cut the sample tissue and then combined with hyphenation of ICAT with LC-MS/MS, for the comparision of the differential expressed protein profile between the normal pregnant patient and PIH patient. A molecular mechanism of PIH pathogenesis was proposed on the basis of the data resulted from both approaches.1.1 Total protein expression profile analysis of tissue of pregnancy induced hypertension (PIH) patientsPIH is a complex and serious condition of pregnancy, resulting in maternal and fetal complications even death if it is untreated or treated improperly. Up to now, there is no clinical measure to prevent this disease and treat it properly. Trophoblast cells in human placenta which can be separated from heterogeneous cells and collected by laser capture micro-dissection (LCM), plays an important role in early pregnancy. In this work, trophoblast cells in human placenta are considered as the functional protein in PIH. After total proteins have been extracted and identified by 1D-LC-MS/MS, 962 proteins of human trophoblast cell in PIH were analyzed by LTQ-Orbitrap. The identified proteins are classified according to molecular function, biological process and cellular component. Proteins of interest include14-3-3, which participate in signal transduction, heat shock protein 27 and annexinâ…¡andâ…¤in stress response like oxidation.A relatively complete protein data file of PIH was established, providing abundant experimental information for the further study of PIH pathogenesis mechanism, and laying a foundation of the further analysis of the low-abundance proteins.1.2 Differential protein expression profile analysis between normal and PIH tissueOn the basis of establishment of a relatively complete protein expression profile of PIH, a differential protein profile between PIH tissue and normal trophoblast cells was further established, via using 2D-gel, 2D-HPLC techniques for the separation of the whole extracted proteins. Afterwards, the differential proteins were identified via in-gel digestion combined with MALDI-TOF-TOF-MS, and then made a search with the database. 13 differential expressed proteins have been identified, especially essential proteins among these were verified with the Western blot for the validation of function. According to the MS identification and preliminary classification and functional search, it was found that 3 proteins in PIH were down-regulated and 10 proteins were up-regulated, they were signal transduction protein, molecular chaperon, cell skeleton protein and proteases which take part in material transportation and metabolism. All these proteins were related to the cause of preeclampsia.Another approach to the differential protein research was to cut the PIH tissue by LCM technique then mark the differential proteins by ICAT and identify them with 2D-LC-MS hyphenation technique. Total 28 differential proteins were identified. Among them, 15 proteins were up-regulated and13 were down-regulated. They are transferrin, laminin and collagen. It is noticeable that the proteins detected from the two different approaches are so different, meaning that two approaches should be complementary one another.1.3 Differential protein expression profile analysis of BeWo cells under normal conditions and hypoxia conditions Syncytiotrophoblast formation is affected by a number of pathological conditions and the suppressed syncytiotrophoblast formation due to hypoxia may play a role in the pathogenesis of preeclampsia. However, the molecular mechanism of hypoxia-inhibited trophoblast syncytialization is poorly understood. To determine the effect of hypoxia on trophoblast syncytialization, a proteomic analysis was performed in the human cytotrophoblast cell line BeWo using two-dimensional electrophoresis and MALDI-TOF-TOF-MS. Hypoxia induced marked inhibition of BeWo cell fusion and differentiation. The proteomic profiling was established under hypoxia in BeWo cell syncytialization. The results showed that twenty proteins were significantly up- or down-regulated under hypoxia, compared with cells under normoxia. In response to hypoxia, three antioxidants, peroxiredoxin 1, peroxiredoxin 2 and 1-Cys peroxiredoxin, were down-regulated, two proteins involved in glycolysis pathway (malate dehydrogenase and enolase) were up-regulated. The expression of two members of the annexin family (annexin A2 and annexin A5) was increased. We also found a decreased expression of 14-3-3Ï„protein in hypoxia treated cells. The expression of two cytoskeleton components (keratin 1 and b-actin) was found to be down-regulated. In addition, galectin-3 was up-regulated. These proteins have been implicated in regulating cellular oxidative stress, glycolysis, signal transduction, protein folding and degradation, cell mobility and cytoskeletal structure formation. Western blot analysis revealed that the levels of peroxiredoxin 1 and14-3-3Ï„were decreased, whereas the levels of annexin A5 and annexin A2 were increased in BeWo cells under hypoxia. These findings provided new insights into the molecular mechanisms in mediating cellular response to hypoxia in trophoblast syncytialization. 2. Differential protein identification between normal tissue and pancreatic tumor tissueThis part of work is mainly focused on the establishment of a method for pancreatic tissue total protein extraction and optimization of the working conditions for 2D gel, to achieve a high resolution and reproducibility. Twelve cancer samples and six normal tissue samples were analyzed. Total 30 differential expressed proteins were discovered. Among them, 24 proteins were identified by MALDI-TOF-TOF-MS, 15 proteins were up-regulated in pancreatic cancer and 9 proteins were down-regulated. Those proteins are various proteases and precursor proteins which take part in the life progress. They are trypsin precursor, coenzyme A dehydrogen protease; S100 calcium binding protein family like Calgizzarin; heat shock protein family; cell skeleton protein family; Annexin A5, correlated with membrane transportation; some other proteins related to cell and tissue and some unknown function protein. These proteins are related to the pancreatic cancer, and eventually become disease biomarker and drug targets.3. Differential protein expression profile analysis for the normal tissue and the RNA interference technology to knock out 14-3-3Ï„protein; fractionating complex protein samples from rat liver and French human liver prior to 2-DE using RPLCThis part of work is focused on RNA interference technology to knock out 14-3-3Ï„protein expression. Compared with before and after knocked out, differential expressed proteins were identified. The role of 14-3-3Ï„protein was validated in the formation of BeWo cells syncytialization under normoxia and hypoxia.An approach for fractionating complex protein samples from rat liver prior to 2-DE using RPLC was described. Experimental findings indicated that RPLC pre-fractionation provided an efficient approach to the low abundance proteins enrichment. This method is complementary to 2D-gel and can provide the new possibility for high throughput proteomic research. The proposed method has been applied to the the study of profiling mode of French Human Liver.