The In Vitro Experimental Study Of Human T Cells Stimulated By Fusion Vaccine Of Gastric Cancer Cell-dendritic Cell Against Gastric Cancer

Objective: Gastric cancer is a malignant disease in the stomach. The traditional treatments of gastric cancer, such as surgery operation, radiotherapy and chemotherapy, are usually aggressive with extensive side effects. In recent years, the biologic therapy based on dendritic cell (DC) as an antigen presenting cell has drawn more attention for it can provide very potent stimulation to immune system. However, the gastric cancer cells are lack of specific tumor antigens. The tumor antigens obtained from gastric cancer cells are very limited, which can not stimulate immune system such as T cells in vivo. Additionally the gastric cancer cells with lack of antigens could not improve DC effectively, and meanwhile, the anti-tumor immune response inspired by the DC is poor. Thus it is necessary to improve its ability to response to stimulation of tumor antigens and the reactions of anti-tumor immune system. Generally speaking, it is difficult to collect DC from peripheral blood in gastric cancer patients to prepare DC vaccine. Nevertheless, the DC can be easily separated from peripheral blood mononuclear cells of healthy blood donors, which avoid collecting blood from cancer patients. In this study, the dendritic cells derived from peripheral blood mononuclear cells (PBMCs) were obtained and fused with gastric cancer cell line, SGC7901.The purpose is to study the proliferation of autogenetic and allogenic T cells stimulated by this DC vaccine and in vitro killing efficacy of the DC vaccine against SGC7901 cells.Methods: The culture and identification of dendritic cells were performed using white coat (leukocytic cream of peripheral blood). The matured dendritic cells were induced by adding cytokines such as GM-CSF, IL-4 and TNF-αin vitro, and identified and collected successfully. The dendritic cells were then fused with gastric cells SGC7901 and the fusion cells were screened out DC-tumor vaccine by HAT/HT selective culture system. The proliferation of autogeneic and allogenic T cells caused by DC-tumor vaccine and the anti-tumor effects were tested by MTT method in vitro.Results: A total number of (3.87±0.31)×107 mature dendritic cells were averagely obtained from one blood unit (200ml whole blood) and the cells showed typical morphological character of DC. Meanwhile, the expression levels of DC-associated surface molecules such as CD83, CD1a, CD86 and HLA-DR were (48.54±2.12)%, (50.59±5.06)%, (63.04±4.42)% and (89.12±3.99)% individually. The fusion cells as DC-tumor vaccine were fused successfully and examined by light microscope, and showed status of suspension cells with transparent cytoplasm, sharpness of border and membrane integrity. The fusion cells were screened out by HAT and HT selective methods after cultured for 2 weeks and 1 week respectively, and then obtained successfully. The production levels of proliferation of autogeneic and allogenic T cells stimulated by fusion cells were closely related to the ratio of DC to T cells, and showed high proliferation activity of autogeneic or allogenic T cells as the increasing ratio of dendritic cells to T cells as well. However, the proliferation activity of autogeneic or allogenic T cells was similar at the same ratio of dendritic cells to T cells. In addition, the anti-tumor effects were similar in vitro with autogeneic or allogenic T cells induced by the fusion cells vaccine.Conclusions: The dendritic cells from peripheral blood of healthy donors were obtained after induction by cytokine including GM-CSF, IL-4 and TNF-α. Gastric cells were used to fuse with dendritic cells by polyethylene glycol (PEG), and the fusion cells were screened out and isolated with HAT/HT selective medium. The growth of fusion cells was suspension property with the character similar to dendritic cells. The number of fusion cells was less than the number of parent cells. The proliferation of autogeneic and allogenic T cells stimulated by fusion cells was closely related to the ratio of dendritic cells to T cells. However, the proliferation activity of T cells was similar at the same ratio of dendritic cells to autogeneic or allogenic T cells. The killing activity of autogeneic or allogenic cytotoxic T lymphocytes stimulated by dendritic fusion cell vaccine was much higher than that of normal T cells, and showed with the similar results of killing tumor effect of both T cells.