| Chapter one: Study on Hepatocarcinogenesis ObjectiveHBV related hepatocellular carcinoma(HCC) usually develops in a setting of chronic necrosis inflammation(including chronic hepatitis or cirrhosis). It is still unclear as to the mechanisms of hepatocarcinogenesis. In this study, we identified the differential expression proteins in hepatic tissues using 2-DE and MALDI-TOF MS in order to better understand hepatocarcinogenesis.Methods1. Tissue specimens and sample lysis prior to 2-DE: Hepatic tissue specimens were obtained from 18 patients with HBV related HCC undergoing partial hepatectomy. Of these specimens, 18 were cancerous tissue,12 cirrhotic tissue, 6 chronic hepatitis tissue. Serologically. Hepatic tissues were dissolved in lysis buffer, samples were centrifuged for Ih at 15,000rpm to remove particle materials. The protein concentration of sample was measured on a spectrophotometer.2. 2-DE, image acquisition and analysis: The first dimensional isoelectric focusing (IEF) was performed on precast 18cm immobilized pH 3-10 gradient (IPG) strips. Following IEF, the gel strip was equilibrated for30 min in the equilibration buffer. The second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at 16癈. Proteins were visualized by silver staining. The stained 2-DE gels were scanned on an labcan. The image were analysed using the ImageMaster 2D Elite software. The differential protein spots were detected between cancerous tissues and non-cancerous tissues.3. MALDI-TOF MS and protein identification: Differential protein spots in silver-stained 2-DE gels were excised, Each spot was then subjected to discoloration > in-gel reduction > alkylation. Enzymatic digestion was performed with TPCK- trypsin. desalting by zipTip機18. Peptide mass fingerprinting maps were obtained using ProFlex橧II MALDI-TOF MS and searched in the SWISS-PROT/TrEMBL database (http: // www.expasy.org ) with the Peptldent soft.Results1. Analysis of the differential protein spots: The good 2-DE patterns including high resolution and reproducibility were obtained. The 2-DE gels analyzed by ImageMast software showed that average 1281 + 51 spots in cancerous tissues, 1188 + 41 spots in cirrhotic tissues, 1235 + 31 spots in chronic hepatitis tissues. Differential protein spots were difined as spots in 2-DE gels whose expression upregulated signi'ficantly(expression volume of proteins whose expression levels were different by more than three-fold aftercorrection) in more than 50% certain tissue compared with control tissue. As cirrhotic vs cancerous tissue, there were 19 differential protein spots in cancerous tissues, and 16 differential protein spots in cirrhotic tissues.As chronic hepatitis vs cancerous tissue, there were 21 differential protein spots in cancerous tissues, and 17 differential protein spots in chronic hepatitis tissues. As cancerous vs non-cancerous tissue (including chronic hepatitis and cirrhotic tissue), there were 14 differential protein spots in cancerous tissues, and 12 differential protein spots in non-cancerous tissues.2. Identification of the differential protein spots: Of the forty-seven differential protein spots, 28 peptide mass fingerprints (PMF) maps were obtained by MALDI-TOF-MS. The PMF maps were searched in the SWISS-PROT/TrEMBL database by Peptldent software. 17 proteins were preliminarily identifyied, among which ten proteins (Protein CDC27Hs*, Insulin-stimulated protein kinase 1, Alpha-1- fetoprotein, Lamin Bl, Cytoskeletal 8, Gankyrin, Nucleolar phosphoprotein B23*, Cytoplasmic dynein heavy chainN c-Jun N-terminal kinase 2*> ADP/ATP carrier protein*) expression were upregulated in cancerous tissue; and seven proteins (Cyclin-dependent kinase inhibitor p12, Cyclin-dependent kinase inhibitor 1 , Antioxidant protein 2 * , Protein disulfide isomerase A2 * , C-1-tetrahydrofolate synthase * , Rho-GTPase-activating protein 4 * , Insulin-like growth factor binding protein 2) expression were downregulatedin cancerous ti...
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