Study On Immune Adjustment Of Mice Caused By PC-3 Cell Transferred MIL-18 And MGM-CSF Gene In Vivo And In Vitro

【Abstract】　Objectives To prepare the PC-3 pancreatic cancer cell which transfected with IL-18 and GM-CSF gene. Methods PC-3 cells were transfected with IL-18 and GM-CSF or the control vector by liposome transfection. The expression of those genes was measured by Western blot analysis. The concentration of IL-18 and GM-CSF in the supernatant was measured by ELISA assay. Results　The expression of IL-18 and GM-CSF was identified by Western blot assay. The concentration of GM-CSF was 35.28±2.52 pg/ml in the group with cell number of 6×104/ml as well as 17.35±2.41 pg/ml in the group with cell number of 3×104/ml at the time of 24 hours after pCDNA3.1-GM-CSF transfection. The concentration of GM-CSF was 534.88±54.83 pg/ml in the group with cell number of 6×104/ml as well as 170.50±48.26 pg/ml in the group with cell number of 3×104/ml at the time of 24 hours after pCDNA3.1-IL-18 transfection. Conclusions 　The vector expressing IL-18 and GM-CSF had been transferred to PC-3 cell. The cell could be used in further research. Part TwoStudy on the cytotoxicity of macrophage and PBMC to pancreatic cancer cell line PC-3transferred with GM-CSF and IL-18【Abstract】 Objectives To study the killing potential of macrophage and peripheral blood mononuclear cell (PBMC) to pancreatic cell line PC-3 transfected with IL-18 and GM-CSF. Methods PC-3 cells were labeled by CalceinAM. After ConA injection to mouse abdomen, the macrophages were separated and then cultured in vitro. PBMC was acquired from the blood of peripheral blood of nude mice. The transfected IL-18 and GM-CSF potential oftumor immune regulation was evaluated by macrophage killing test. Results The cytotoxicity mediated by macrophage and PBMC on PC-3 transferred with IL-18 and GM-CSF were significantly higher than those cells transfected with control vector and the untransfeced (p< 0.001). The cytotoxicities of macrophage and PBMC coculture with PC-3 at the ratio of 20:1 were significantly higher than those of 10:1. Conclusions GM-CSF could enhance the ability to excrete TNF-αand IL-18 by macrophages. IL-18 could enhance the ability to excrete TNF-αa by macrophages and PBMC. IL-18 and GM-CSF could enhance the killing potential of macrophage and PBMC to PC-3 cell line. Therefore, it was suggested that IL-18 and GM-CSF might be used as immune therapy of pancreatic cancer.Part ThreeStudy on the subcutaneous tumor established by the PC-3 cell transferred with IL-18 and GM-CSF【Abstract】 Objectives To investigate the effect of IL-18 and GM-CSF as well as their interact relationship by the subcutaneous tumor established by the PC-3 cell transferred with IL-18 and GM-CSF Methods Human pancreatic carcinoma strain PC-3 cells transferred with pCDNA3.1, pCDNA3.1-IL-18, pCDNA3.1-GM-CSF and the mixture of the later two were inoculated to 8 nude mice subcutaneous on their right back for each group. Two weeks after the inoculation, TNF-αin the peripheral blood was measured by ELISA assay. All the mice were killed five weeks later. The weight of the mice was measured. Tumors were excised and the size and weight were measured, too. Tumor cell cycle kinetics was analyzed by microscope and by flow cytometry method (FCM). The apoptotic index (AI) was detected by terminal-deoxynucleotide transferase mediated d-UTP nick end labeling (TUNEL) through the analysis the result of immunohistochemical staining. Results The concentration of TNF-αin the peripheral blood of the group used the PC-3 cells transferred with pCDNA3.1-GM-CSF and the mixture group was significantly high(P< 0.01).Compareto the control, the group used the PC-3 cells transferred with pCDNA3.1-GM-CSF was high (P< 0.05). The percent of S phase, Proliferation Index, were lower in the IL-18 groups and mixture group than in the control groups and the GM-CSF group (p< 0.05). The extent of local inflammation and incrassation of blood vessel membrane were more seriously in the GM-CSF group and mixture group. The novel vessel beside the tum...