| The main contributions of this dissertation are: a proteome profile of the nuclei in the liver of C57 mouse was built up by using density gradient centrifugation for the nuclei isolation and 2DE/MALDI MS for the protein identification, 1193 nonredundant proteins were identified, and they are great possible to be localized in nuclei. The data can act as a reference in the research work of liver disease; and then, the expressional proteome profile of the metastasis HCC nude mice model LCI-D20 were analyzed, 995 proteins were found. Some of them participate pathways related with HCC and HCC metastasis, for example the MAPK pathway. These data would offer great information for the HCC metastasis; At last, the different expressed nuclear proteome is analysed for human hepatocellular carcinoma cell lines with different metastasis potential (Hep3B, MHCC97-L and MHCC97-H) by combining sub-proteomic and comparative proteomic technologies, and a group of nuclear proteins were found to be related with HCC metastasis, such as the hnRNP proteins and ASF2 protein.Hepatocellular Carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. Metastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients. Like other cancers, the development and migration of HCC is a multifactor and multistage pathogenesis. Because of the limitations in conventional techniques, researchers have been allowed to focus on a few biological target molecules at a time, and little has been known regarding to the specific alterations responsible for the development or progression of this aggressive malignancy. Recent technological advancements in proteomics allow us to study global patterns of protein content and activity and how these change during development or in response to disease, it has boosted our understanding of systems-level cellular behavior and mechanism of disease. In addition, proteomics benefits the identification of new drug targets and the development of new diagnostic markers in clinical research.This work is based on the prior work of Institutes of Biomedical Sciences of Fudan University, and the Liver Cancer Institute of Zhongshan Hospital, Fudan University.Firstly, the nuclear proteome in the normal C57 mouse liver were studied in our work, great data have been obtained for comprehensive understand of the physiological process of normal liver and other liver disease. And then global proteomic profile of the human HCC metastasis nude mice model LCI-D20 were built up. At last sub-proteomic studies on HCC cell lines with different metastasis potential were carried out in detail in this work. Some nuclear protein targets have been found out, and the expression and intracellular location of 3 hnRNP proteins were validated.This dissertation consists of 5 parts and the contents are summarized as follows:In the first chapter, the status quo of proteomics and the newest technical development in proteomics such as 2DE, bio-mass spectrometry, chromatography, protein chip, Yeast two-hybrid system, tandem affinity purification, and so on, were summarized. A review of liver proteomics study was presented.Work in the second chapter is focus on the nuclear proteome of the normal liver of C57 mouse. Firstly, purified nuclei were separated by several density gradient centrifugations. Verified by electron microscope and western blot, the purity of nuclei were up to 90. Nuclear protein were separated with 2-DE. In the first dimension, IPG strips with different pH range (pH 3-10, NL and pH 4-7, L) were used. Recast gels with the concentration of 12.5% were used in the second dimension. The gels were stained by comassia brilliant blue and silver stain. All the protein spots were excised, digested, and then identified by MS. There are about 1200 proteins successfully identified in this work.Proteins in the liver tissue were extracted by both one-step-extraction and sequential extraction. Fractions were separated with 2-DE and Top-down technologies. After MS analysis, 995 nonredundant proteins were identified successfully; including many HCC and HCC metastasis related proteins, such as AFP, ras-protein, Heat shock protein 27KD, MAPK protein, et al. Those proteins participate many processes during the HCC metastasis, such as metabolism, cell motility and invasion, and signal transduction. Different protein extraction method and separation technologies were also evaluated on the basis of experimental data. We have built up a proteome database of LCI-D20 in the first time, and this database should have a great value for the understanding of HCC metastasis.In the last chapter, different expressed nuclear proteomes of three HCC cell lines with different metastasis potential were studied through the combination of organelle purification and proteomics technologies. Firstly, the method of muclei purification was built up, and then the nuclear protein of the 3 cell lines, Hep3B (HCC without metastasis), MHCC97L (HCC lower metastasis potential) and HCCLM3 (HCC higher metastasis potential), were extracted. After 2DE, 142 differentially expressed protein spots in three celllines were in-gel digested manually and identified by tandem mass spectrometry. A total of 91 proteins were identified definitely, including proteins associated with nuclear matrix, DNA/RNA binding, translation, mRNA alternative splicing, etc.. Some of the proteome results were verified by western blot and immunohistochemistry. Our study provided the global information of proteomic alteration during HCC metastasis, and it also provided the clues for further understanding of the mechanism of HCC metastasis.
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