| AIM: To establish the two-dimensional electrophoresis (2-DE) mapsfrom peripheral blood mononuclear cells (PBMC) in patients withhepatitis B virus (HBV) related hepatocellular carcinoma (HCC), livercirrhosis (LC), chronic hepatitis B (CHB) or healthy adults, and identifythe differential expression proteins. The method of western-blot analysiswas used to determine the differential expressional levels of the partialproteins. The immunohistochemistry method was used to detect theexpressions of differential proteins in hepatic tissue specimens. Reliableperipheral blood biomarkers for early diagnosis, treatment, mechanism ofcancerization and evaluation of HCC prognosis are needed.METHODS:(1) Peripheral blood mononuclear cells (PBMC) in patients withHBV related HCC, LC, CHB or healthy adults were gained by densitygradient centrifugation.The total proteins of PBMC were dissolved inupgraded cell disruption buffer and measured on a spectrophotometer byspecial 2-DE kits.(2) The total proteins from PBMC in patients with HBV relatedHCC (n=18), LC(n=13),CHB(n=12) or healthyadults(n=10) wereseperated by means of immobilized pH gradient-based 2-DE. Firstly, the first dimensional isoelectric focusing (IEF) was performed on precast 24cm immobilized pH 3-10NL gradient (IPG) strips. Following IEF, the gelstrip was equilibrated for 30 min in the equilibration buffer. Secondly ,thesecond dimensional sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed. Proteins were visualized byblue silver staining and were scanned on a labcan. The differentialexpression proteins were analyzed by using PDQuest analysis softwarethrough identifying spot, adjusting background, standardizing andmatching, establishing matchset gel. Finally, the partial differentialexpression proteins were identified by peptide mass fingerprint (PMF)based on matrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF-MS) and searched in database.(3) The method of Western-blot analysis was used to determine thedifferential expressional levels of the partial proteins.(4) Immunohistochemistry method was used to detect theexpressions of differential protein in hepatic tissue specimens in patientswith HBV related HCC, LC, CHB or control.RESULTS:(1) The regression equation of standard curve in total protein fromPBMC is Y=-0.0032X+0.37, R~2=0.998.(2) The well-resolved and reproducible 2-DE patterns of PBMCwere obtained in patients with HBV related HCC, LC, CHB or healthy adults. The mean numbers of protein spots for HCC, HCC, LC, CHB orhealthy adults were 1189±45, 1016±41, 1096±42 and 1123±39, withan average matching rate of 91.6 %, 90.2%, 90.7% and 92.8%respectively. There was a good reproducibility of spot position in 2-DEmaps in intergroup, For HCC, HCC, LC, CHB or healthy adults the anaverage of deviations in IEF direction was 1.21±0.14mm, 1.06±0.12mm,1.09±0.15mm and 0.99±0.12mm, respectively, and the average ofdeviations in SDS-PAGE direction was 2.22±0.26mm,2.15±0.2mm,1.93±0.20mm and 1.98±0.24mm, respectively.(3) We compared 2-DE patterns of PBMC in patients with HBVrelated HCC with those of patients with LC, CHB or healthy adults. Weobtained 72±7, 63±6 and 65±5 up-regulated significantly differentialprotein spots respectively, and 34±5, 24±4 and 27±4 down-regulatedsignificantly differential protein spots respectively. Twenty-eightdifferential proteins which were different by more than three-fold aftercorrection and expressed in more than 50% certain gel were tested byMALDI-TOF-MS, and 19 differential proteins were identified. In the 19proteins, 15 differential proteins were up-regulated in PBMC in patientswith HCC, including heat-shock protein 27, member RAS oncogenefamily (RAB14), Actin, Alpha 1 antitrypsin, RNA-binding proteinregulatory subunit and so on. By contrast, the levels of proteins such asPDX2, PDX6, HSPA8 and MnSOD were significantly reduced in HCC patients. Some of these proteins participated protein synthesis anddegradation, or chaperone, or protection and detoxification, orcytoskeleton, while some of those were involved in metabolism or signaltransduction.(4) The method of Western-blot analysis was used to determine thedifferential expressional levels of MnSOD and HSP27 and the resultswere identical with the proteome analysis. When the PBMC in patientswith HBV related HCC was compared with those of the patients with LC,CHB, gastric cancer or healthy adults, the expressional levels of HSP27were up-regulated significantly (P＜0.01), while the expressional levelsof MnSOD were down-regulated significantly (P＜0.01).(5) The result of HSP27 in hepatic tissue specimen byimmunohistochemistry method showed a significant difference betweenHCC patients and control (P＜0.01). By contrast, the expressional levelsof HSP27 in hepatic tissue specimen of LC (P=0.15) and CHB (P=0.69)were very low as that in control hepatic tissue specimen.CONCLUSIONS:(1) The PBMC proteome research system has been successfullyestablished.(2) A well-resolved, reproducible 2-DE patterns of PBMC in patientswith HBV related HCC, LC, CHB and healthy adults are established.(3) Nineteen differential proteins are characterized. 15 differential proteins were up-regulated in PBMC in patients with HCC, and 4 weresignificantly reduced. Some of those may be reliable peripheral bloodbiomarkers for early diagnosis, treatment, mechanism of cancerizationand evaluation of HCC prognosis.(4) There are almost the same expressional levels of HSP27 ofPBMC in patients with HBV related HCC, LC, CHB and healthy adultsas those in hepatic tissue specimen. It deserves to be further studied.
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