IL-35 Gene Transfection And Its Effect On The Immune Function Of Mice

IL-35 gene transfection and its effect on the immune function of miceObjective:To investigate the feasibility of IL-35 in vivo gene transfection and its effect on the immune function of mice.Methods:(1) The IL-35 expression plasmid vector (pSecTag2-IL35) from Glasgow University was characterized. Upperstream primer and downstream primer were designed and synthesized. The IL-35 gene was amplified with polymerase chain reaction (PCR). The plasmid was aslo digested with specific restriction enzymes. The DNA fragments were determined by agarose gel electrophoresis. (2) The IL-35 expression plasmid vector or enhanced green fluorescence protein (EGFP) expression vector were transfected into 293 cells by lipofectamine. The transfection efficiency was determined by fluorescence microscope and flow cytometry. IL-35 expression was detected by ELISA. (3) In vivo gene transfer was performed through hydrodynamic tail vein injection of IL-35 expression plasmid. The proportion of CD4+T lymphocyte,CD8+T lymphocyte,CD3-CD16+NK cell,CD4+CD25+Treg in peripheral blood or spleen were determined by FCM. (4) Spleen mononuclear cells (SMCs) of BALB/c(H-2d) of injected IL-35 expression plasmid and C57BL/6(H-2b) mice were harvested through ficoll-hypaque density gradient centrifugation, one-way mixed lymphocyte reaction(MLC) was performed, of which the mitomycin(MMC) treated SMCs from BALB/c or C57BL/6 acted as the stimulators and the non-MMC treated SMCs from BALB/c injected with IL-35 acted as the responsers, after 4-day's culture the percentage of CD4+T lymphocyte,CD8+ T lymphocyte,CD4+CD25+ Treg were determined by FCM.Results:(1) PCR and retriction enzyme digestion confirmed the pSecTag2-IL35 was correct. (2) In vitro transfection results showed that green fluorescent protein (GFP) could be detected in the transfected cells 24 hours after transfection. The percentage of transfected cells was 35.30%. The IL-35 expression in culture supernatant of pSecTag2-IL35 transfectant was detected by ELISA. (3) After in vivo gene transfer, IL-35 expression increased the level of CD4+CD25+Treg in the peripheral blood and spleen of mice, reduced the level of CD4+T lymphocyte,CD3-CD16+NK cell in the peripheral blood. (4) In vivo MLC experiment showed that IL-35 up-regulated the percentage of CD4+CD25+ Treg cells, down-regulated the percentage of CD4+T lymphocytes. The reason maybe ascribed to that IL-35 transfection had promoted the level of CD4+CD25+Treg and enhanced its immunoregulatory function in mice.Conclusions:IL-35 plasmid transfection can induce differentiation and proliferation of CD4+CD25+Treg, inhibit the activation and differentiation of CD4+T lymphocyte and NK cells. IL-35 gene therapy may be able to inhibit acute transplantation rejection and induce immunological tolerance.