| Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumorswith a very high morbidity and mortality in Southeast Asia. Particularly in China, mostpatients of HCC are HBV-positive or HCV-positive. Concerning the limitations ofefficient of several biomarkers such as AFP, it is still urgent to look for new candidatemarkers with higher sensitivity and specificity molecules to improve HCC diagnosisefficient.Nowadays, proteomics approach is considered to be a powerful technology in theglobal analysis of protein expression and has been widely used in disease proteomics,especially in cancer research fields. Just for liver cancer, to study the change of proteinstypes and volume in the HCC process dynamically and quantitatively not only can helpfurther reveal the pathogenesis of HCC, but also can identify key carcinogenesis-associated molecules, and then develop the novel detection methods to advance HCCdiagnosis.Liver tissue interstitial fluid (TIF) is the media between the body fluids (such asblood) and the intracellular fluid. The hepatocytes exchange material and informationwith other cell types and cyclic body fluids through TIF. So, we regard TIF as apromising and novel material for biomarker discovery. Meanwhile, secrete proteinsfrom tumor cells or cellular proteins released by apoptosis will exist in the TIFmicroenvironment, those proteins become a potential source of tumor markers.Comparing plasma, specific protein in TIF will not be diluted by a large number ofbiological fluids, and also won’t be interfered by other organs of protein, so, lowabundance local proteins are more enriched in TIF. We put those thought into use and analyzed differential proteins. We establishedthe TIF extraction methods of mouse liver and human adult normal liver before, andevaluated the contamination from cellular organelles proteins. Based on that research,we accurately distinguished the difference among6matched clinical HBV-HCC caseswith2D DIGE (Differential in-gel electrophoresis) technology (mainly present partly),iTRAQ (Isobaric tag for relative and absolute quantitation) technology and MassSpectrometry (MS) subtract technology. Through the three analytical methods, thedifference expression proteins were identified). There are113differential proteins,which include41up-regulation proteins and72down-regulation proteins in2D DIGEdata. The number of iTRAQ and MS subtract identified is529and728respectively andtotally there are1276proteins.We analyzed the subcellular localization of113differential proteins, includingplasma membrane, cytosol, nucleus, secreted protein and so on. GO analysis reached aconclusion that those involved in cell communication, recognition, adhesion, signaltransduction, the actin cytoskeleton assembly and transport were enriched and might beplay a role in the occurrence and development process of HCC. Only22plasmaproteins could be found in TIF (19.5%) compared with HPPP, implicating the moreinformation and higher concentration of local proteins in TIF.The expression of four candidate proteins were confirmed by Western Blot in TIF,including Chloride intracellular channel protein1(CLIC1), Transketolase (TKT),Tropomyosin alpha-3chain (TPM3), Nicotinate-nucleotide pyrophosphorylase (QPRT).There is no report about those proteins related to HCC. Besides, we firstly documentedTIF is the best material than tissue and plasma. Since serum/plasma has good maneuverability and clinical application in tumormarkers study, so Enzyme linked immunosorbent assay (ELISA) was applied tovalidate expression amount of protein S100A9and Vitamin D-binding protein (VDBP).We chose four coherent and20sera samples per group, including healthy volunteersgroup (N), HBV infected patients group (CHB), cirrhosis patients group (LC) andhepatocellular carcinoma patients group (HCC). The aera of under curse (AUC) ofthose candidates are0.955and0.898. The results indicated that the two candidates arebetter than AFP in sensitivity and specificity. It is worth to validate the results in largerclinical serum samples. |
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