Recombinant human interleukin 4

Human peripheral blood mononuclear cells (PBMNCs) were obtained from healthy volunteers (provided by Zhejiang Blood Center, Zhejiang, China), and SFs were isolated from non-tumoral gastric walls of the patients who underwent surgery in our hospital. Informed consent was obtained from all volunteers and patients. PBMNCs were isolated using a human Lymphoprep solution (Axis-shield PoC AS, Oslo, Norway), and cultured in a 10-cm Petri dish and incubated for 24 h to allow them to adhere to the dish’s surface. Adherent cells were induced to form immature DCs, supplemented with 120 ng/mL recombinant human granulocyte macrophage colony-stimulating factor (PeproTech, Offenbach, Germany), and 60 ng/mL recombinant human interleukin-4 (PeproTech) to decrease contamination by macrophages for 5 days. Nonadherent cells were harvested and used as human lymphocytes. SFs were prepared by transferring the gastric tissue to a T25 flask and cutting into 1mm³ pieces. Incubate the chopped material with 5 ml of trypsin EDTA for 5 min at 37 °C, and then the trypsin was Inactivated by adding 1 ml FBS. The cell pellet was obtained by centrifuging of suspension at 200 g speed for 10 min, which was transferred to a T25 flask containing DMEM medium with 20% FBS and 2 × Penicillin/Streptomycin) after resuspend using DMEM.

The experimental procedures were approved by the Ethics Committee of Dalian Medical University and written consent forms were obtained from each volunteer participant. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from freshly drawn peripheral blood (50 mL). Cells were allowed to adhere in plastic cell culture flask for 2 h at 37 °C in the incubator. Adherent cells were induced into DCs using 500 U/mL of recombinant human interleukin-4 (PeproTech) and 1000 U/mL of recombinant human granulocyte-macrophage colony stimulating factor (PeproTech) for 4 days, before treated with 1000 U/mL of tumor necrosis factor alpha (PeproTech) to generate mature DCs. Cells were then treated with DMSO (control), curcumin (25 μM), or apigenin (30 μM). Non-adherent cells were collected for cytokine-induced killer (CIK) cells using serum-free medium (Lonza, X-VIVO 15) supplemented with 1000 U/mL IFN-γ (Chemo Wanbang, China) for 24 h. Then cells were treated with 100 ng/mL of anti-CD3 antibody and 1000 U/mL of recombinant human IL-2 (Sanyao, China). The medium was replenished every 3 days. At day 7, CIK cells were harvested and separated into equal parts for co-culture with previously treated DCs at a ratio of 3:1 for another 7 days. CIK and DC-CIK cells were used in co-culture assays with A375 cells according to methods described above.