Patients and groups
Sixty-one patients with HCC who had undergone hepatectomies between October 2009 and April 2010 at the Liver Cancer Institute, Zhongshan Hospital of Fudan University (Shanghai, China) were enrolled. We randomized the patients between 18-65 years old, American Society of Anesthesiologists (ASA) scores I-II, with normal leukocyte and lymphocyte counts, who had an imaging diagnosis of HCC with the intention of curative surgery and without distant metastasis or any prior anti-cancer treatments into two groups, that is, the G + E group and G group. We then excluded patients who were not pathologically diagnosed as HCC or who were given a blood transfusion during or after surgery. We finally chose 61 out of 70 patients, 31 in the G + E group and 30 in the G group. Fourteen cases received right lobectomy, 11 cases left hemihepatectomy and 36 cases segmentectomy. The study was approved by the Research Ethics Committee of Zhongshan Hospital, and informed consent was obtained from all patients.
Anesthesia and analgesia
All patients were free of preoperative medication and had fasted for over 8 h. Epidural puncture for patients in the G + E group was carried out at T8 and T9 into the epidural space, with patients in a left-lateral position. After successful puncture, epidural catheters were placed toward the head for 3-4 cm. We fixed the catheters and gave 2% lidocaine (3 mL) through the catheters. After 5 min, upon confirming no spinal analgesia and that the epidural block was successful, we gave the patients 0.375% bupivacaine liquor (8 mL), then 4 mL at each 50-min interval.
Induction and maintenance of general anesthesia: All patients were induced with 5 μg/kg fentanyl, 2.0 mg/kg propofol and 0.9 mg/kg rocuronium and then incubated for approximately 90 s. Anesthesia was maintained with sevoflurane (1.5%-3.5%) based on a bispectral index monitor.
Postoperative analgesia: Patients in the G + E group used postoperative patient-controlled epidural analgesia, made up of 0.125% bupivacaine + morphine (30 μg/mL). The patients in the G group used postoperative patient-controlled intravenous analgesia containing morphine (0.4 mg/mL) administered via a jugular vein catheter. Patient-controlled analgesia was administered via an ambulatory infusion pump (AutoMed, Acemedical AM3400) and lasted for about 48 h. All patients were followed up at the end of analgesia using a visual analogue scale (VAS) for evaluation of their pain levels, and the corresponding drug dosage used was recorded.
Blood samples (10 mL) were obtained from all patients in the recumbent position with a 20-gauge needle for a clean veni-puncture of an antecubital vein at three time points: early morning on the operation day (d0), and the second (d2) and seventh day (d7) after the operation. The samples were collected into tubes containing 0.2 mL sodium heparin. Peripheral blood mononuclear cells (PBMCs) were prepared using a Ficoll density gradient, for flow cytometry and real-time polymerase-chain-reaction analyses. Plasma was obtained after centrifugation and stored at -80 °C for measurement of cytokines.
Preparation for flow cytometry analysis
For analyses of Th1, Th2 and Th17, PBMCs were suspended at a density of 2 × 106 cells/mL in complete culture medium (RPMI 1640 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mmol glutamine and 10% heat-inactivated fetal calf serum). The cell suspension was transferred to individual wells of 24-well plates. Cultures were stimulated with phorbol myristate acetate (50 ng/mL), ionomycin (1 μmol) and Brefeldin A (10 μg/mL, all from Sigma-Aldrich, St. Louis, MO, United States) for 4 h. The cultures were incubated at 37 °C, in a 5% CO2 environment. After 4 h of culture, the contents of the well were transferred to 1.5 mL sterile centrifuge tubes. To analyze Tregs, PBMCs were aliquoted into tubes for further staining.
Surface and intracellular staining
For Th1, Th2 and Th17 analyses, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 at 4 °C for 20 min. For Treg analysis, the cells were incubated with FITC-conjugated anti-human CD4 and allophycocyanin (APC)-conjugated anti-human CD25 monoclonal antibodies (mAbs). After surface staining, the cells were stained with R-phycoerythrin (PE)-conjugated anti-human IFN-γ, APC-conjugated anti-human interleukin (IL)-4 and IL-17 mAbs for detection of Th1, Th2 and Th17, respectively; or PE-conjugated anti-human FOXP3 mAb for Treg detection after fixation and permeabilization, according to the manufacturer’s instructions. Isotype controls were given to enable correct compensation and confirm antibody specificity. All antibodies were purchased from eBioscience (San Diego, CA, United States). Stained cells were analyzed using a FACScan cytometer equipped with CellQuest software (BD Bioscience, San Jose, CA, United States).
Real-time reverse transcription-polymerase chain reaction analysis
Total RNA was extracted with TRIzol regent (Invitrogen, Carlsbad, CA, United States) according to the manufac-turer’s instructions. First-strand cDNA was synthesized using random hexamer primers and RNase H-reverse transcriptase (Fermentas, Glen Burnie, MD, United States). TaqMan primers and probes for human IFN-γ, IL-4, IL-17 and FoxP3 were purchased from TAKARA Bio Inc. (Osaka, Japan), and samples were analyzed utilizing the Opticon Monitor 3 System (Bio-Rad, Hercules, CA, United States, formerly MJ Research). The following primer sets were used: IFN-γ: F: 5’-TGCAGGACCCATATGTAAAAGA-3’, R: 5’-TCAAAATGCCTAAGAAAAG-3’; IL-4: F: 5’-TACAGCCACCATGAGAAGGACA-3’, R: 5’-GCCAGGCCCCAGAGGTT-3’; IL-17: F: 5’-CGCTGATGGGAACGTGGACTAC-3’, R: 5’-GGTGGACAATCGGGGTGACA-3’; FoxP3: F: 5’-CGCTGATGGGAACGTGGACTAC-3', R: 5’-GGTGGACAATCGGGGTGACA-3’; and β-actin: F: 5’-CAACTGGGACGACATGGAGAAAAT-3’, R: 5’-CCAGAGGCGTACAGGGATAGCAC-3’. For each sample, the mRNA expression level was normalized to the level of β-actin.
Enzyme-linked immunosorbent assay analysis
The plasma levels of IFN-γ, IL-4, IL-17, IL-10 and transforming growth factor-β1 (TGF-β1) were measured by enzyme-linked immunosorbent assay (ELISA), following the manufacturer’s instructions (R and D Systems, Minneapolis, MN, United States). Intra-assay and inter-assay coefficients of variation for all ELISAs were < 5% and < 10%, respectively. All samples were measured using three independent experiments, in duplicate.
Statistical analyses were performed with SPSS 16.0 software (SPSS, Chicago, IL, United States). Values are expressed as the mean ± SE of the mean. Student’s t tests were used to compare quantitative variables, and Fisher’s exact tests were used for categorical variables. Two-tailed P < 0.05 was judged to be significant.