A total of forty patients treated in the Tumor Hospital of Institute of Medical Science of Gansu province, China were included in the study (). The eligibility criteria for the study included: biopsy confirmed cancers of the esophagus, stomach, and colon, age less than 70 years, WHO performance status of 2 or better, a life expectancy of at least 3 months, laboratory tests of leukocytes>3.0×109/L and platelet count>100×109/L. Exclusion criteria included major organ metastases, major organ allografts, serious psychiatric diseases, and having previous systemic treatment for malignant tumors. The study was approved by the Ethical Committee of the Tumor Hospital of Institute of Medical Science of Gansu Province, China. Written consent form was obtained from each participant.
2.2. Isolation of PBL
After obtaining informed consent, a 30ml blood sample was drawn from each of the forty patients using vacuum tubes with heparin (Beijing Shuanghe Medicine Co. Ltd, Beijing, China). Peripheral blood lymphocytes (PBL) were isolated by a previously described method (Bellik et al. 2005
). Briefly, PBL were isolated from patients with malignant tumor using a lymphocyte separation medium (LSM) (Shanghai Sangon Biological Engineering Technology and Service Co. Ltd, Shanghai, China) and density gradient centrifugation. The samples were then washed three times with phosphate buffered saline (PBS). All blood draws and PBL isolation experiments were finished within 1.5 hours. The PBL were then re-suspended in RPMI 1640 medium (Sigma, St Louis, MO, USA) with HEPES supplemented with 10% fetal bovine medium, 2 mM L-glutamine, 200 IU of penicillin per ml, 150 mg of streptomycin per ml, and 50 mg of gentamycin per ml to a concentration of 107
cells per ml. PBL from each patient were divided into three groups of equal number for subsequent irradiation treatment including sham, X-ray and 12
2.3. Irradiation using heavy ion beams and X-ray
A carbon ion beam of 100 MeV/u was supplied by the Heavy Ion Research Facility in Lanzhou (HIRFL) at the Institute of Modern Physics, Chinese Academy of Sciences (IMP-CAS). The irradiation of cells was conducted at the therapy terminal of the HIRFL, which has a vertical beam line. Due to the energy degradation by the vacuum window, air gap, and Petri dish cover and medium, the energy of the ion beam on cell samples was calculated to be 89.63 MeV/u, corresponding to LET of 28.3 keV/...m and the dose rate was adjusted to be about 0.5Gy/min.
Low-LET irradiation was performed using SIEMENS Primus high energy electron linear accelerator operated at 6MV and at a source-to-surface distance of 100 cm, and the dose rate was approximately 0.5Gy/min. The dose used for each type of irradiation (carbon-ion or X rays) was 0.05Gy. All irradiations were performed once for each dish of cells at room temperature on the same day. The radiation dose selection was based on our previous results from animal data (Xie et al. 2007
; Zhang et al. 2006
2.4. Phenotype analysis
Twenty-four hours after radiation exposure, cells were obtained from PBL cultures for phenotype analysis using appropriate monoclonal antibodies, including CD3-Tc, CD3-FITC, CD4-FITC, CD8-PE, CD16-Tc, and CD56-PE (CALTAG, USA). T cells were detected by CD3, CD4 and CD8 antibodies, whereas NK cells were detected by CD3, CD16, CD56 antibodies. One million PBL were washed once in PBS containing 1% bovine serum albumin (BSA) and resuspended in 100ul of PBS buffer. The cells were incubated with various conjugated monoclonal antibodies for 20 min at 4° C, washed twice in PBS, and resuspended in 400ul of PBS. A flow cytometric analysis was performed on a FACSCalibur flow cytometry (CoulterEPICS XL, USA), and the data were analyzed using the SYSTEM statistical software. Forward and side scatter parameters were used to gate live cells (Han et al. 2005
2.5. Isolation of RNA
Total RNA was extracted using Trizol reagent (Invitrogen, USA) from irradiated and unirradiated cells 24 hours after irradiation according to the manufacturer’s instructions. The quality of total RNA was determined by the Bioanalyzer (Petro 2005
2.6. Preparation of cDNA
Five micrograms of total RNA from each sample was reversed transcribed to cDNA using PrimeScript™ reverse transcriptase kit (TaKaRa, Japan) according to the manufacturer’s protocol.
2.7. Analysis of mRNA expression by real time quantitative RT-PCR
Real-time quantitative RT–PCR analysis was performed using the Rotor-Gene RG-3000 Real-Time Sequence Detection System (Corbett Research, Australia). Reactions were carried out according to the manufacturer’s protocol of the SYBR Premix Ex Taq™ kit (Perfect Real Time) (TaKaRa, Japan). Oligonucleotides were used as primers and the predicted sizes of amplified PCR products are listed in . Using SYBR Premix Ex Taq™ kit (TaKaRa, Japan), the experiment was carried out in a final volume of 10μl of reaction mixture consisting of 5μl of SYBR Premix Ex Taq™, 0.4μl of the primers and 1μl cDNA according to the manufacturer’s instructions. The reaction mixture was then loaded into glass capillary tubes and subjected to denaturation at 95°C for 10 min, followed by 40 rounds of amplification at 95°C for 10 seconds for denaturation, 53°C for annealing, and 75°C for extension, with a temperature slope of 20°C per second, performed in the Rotor-Gene. The transcript amount for differentially expressed genes was estimated from the respective standard curves and normalized to the β-actin transcript amount which was determined in corresponding samples.
2.8. Cytokine protein production
Supernatants of PBL were harvested and assayed using enzyme-linked immunosorbent assays (ELISA) (ADL Co., USA) to quantify levels of IL-2, IFN-γ and TNF-α according to the manufacturer’s instructions.
2.9. Statistical analysis
Statistical analyses were performed using SPSS software (version 13.0). Each value was expressed as mean ± SD. An ANOVA analysis of variance was used to determine the level of any statistically significant differences between irradiated and unirradiated groups. A p-level of 0.05 was selected as a criterion for a statistically significant test.