Transient Transfection Of Human Interleukin-10 In HEK293 Cells And Its Immunological Effects

Interleukin-10 was originally characterized as a T-cell growth and differentiation factor.Because of its ability to inhibits the production of IL -2﹑IFN-γ﹑ TNF-αand granulocyte-macrophage colony-stimulating factor (GM-CSF),it was named cytokine synthesis inhibitory factor. Interleukin-10 can impair the antigen-presenting properties of Dendritic Cells(DCs) by reducing the expression of antigen-presenting and costimulatory molecules and by interfering with the maturation of monocytes to DCs .As a result of these biologic properties, IL-10 is classified as a Th2 type cytokine with potent anti-inflammatory and immunosuppressive activities. Accordingly, its use has been advocated and is currently under clinical evaluation in human beings for the therapy of autoimmune diseases and to induce tolerance after transplantation.In this study, we constructed the recombinant of eukaryotic expression of hIL-10, using molecular biological techniques; then infected human Embryonic Kidney Cell Line HEK293 by the way of transient transfection, aiming at studying the immunological efforts mediated by HEK293 cells.Objective:In this study, we set up to construct the recombinant of eukaryotic expression of hIL-10, and transfer it into human Embryonic Kidney Cell Line HEK293 to explore the mechanism of its immunosuppressive activity.Methods:(1) cDNA fragment encoding hIL-10 gene was amplified from human peripheral blood mononuclear cells by RT-PCR.(2)The PCR production was cloned to pMD18-T vector via T4 DNA ligase enzymes. PMD18-T-hIL-10,the cloning recombinant,was constructed.(3)Then pMD18-T-hIL-10 was cleaved by restriction enzymes HindⅢ,EcoRI and hIL-10 gene fragment was obtained. Finally hIL-10 gene was cloned to pcDNA3.0 which is also cleaved by restriction enzymes Hind III,EcoRI. The eukaryotic expression recombinant of pcDNA3.0-hIL-10 was constructed and confirmed by restriction enzyme mapping and sequencing. (4)In order to examine the immunosuppressive activities of the constructed plasmid at the cellular level,HEK293 cells transferred by pcDNA3.0-hIL-10 using liposomes . (5)The expression of IL-10 mRNA in HEK293 cells was identified with reverse transcription polymerase chain reaction (RT-PCR) at 24h,48h and 72h after transfection and its quality of expression was observed with luminance of electrophoresis strips and DNA sequencing.(6) Culturing mice spleen and thymus cells with the supernatant which collected from transfected HEK293 culture medium at 24h,48h and72h to measure the effects on the proliferation of spleen and thymus cells,respectively. (7)Culturing mice spleen cell with the supernatant collected from transfected HEK293 and then after a period of culture, collecting the supernatant to detect the cytokineTNF-α, IL-2 and IFN-γ.(8)The expression of VEGF,TGF-? and FasL mRNA in HEK293 cells were identified with reverse transcription polymerase chain reaction (RT-PCR) at 24h,48h and 72h after transfection.Results: (1) We got the cDNA containing the complete sequence of hIL-10 from human peripheral blood mononuclear cells by RT- PCR technique. (2) The recombinant clone vector pMD18-T-hIL-10 was confirmed by restrict digestions of EcoRI and HindⅢ. (3) The recombinant eukaryotic expression vector pcDNA3.0-hIL-10 was confirmed by restrict digestions of EcoRI and HindⅢand further confirmed by DNA sequencing.(4) The supernatant collected from transfected HEK293 at different time points restrains the proliferation of spleen and thymus cells,especially at 48h after transfection.(5) In the supernatant stimulated by spleen cells, TNF-α, IL-2 and IFN-γwere decreased in pcDNA3.0-hIL-10 transfected group ,particularly at 48h at the dilution of 1:2,1:4 and 1:8(p<0.05).And TNF-αwas decreased evidently at 24h at the dilution of 1:8(p<0.05),IL-2 was decreased evidently at 72h at the dilution of 1:2,1:4 and 1:8(p<0.05).The above were controlled by the free vector-transfected group. (6) VEGF mRNA in transfected HEK293 was expressed in the recombinant, while there were no significant difference between the free vector-transfected group and control group.Conclusions:(1)The eukaryotic expression recombinant of pcDNA3.0-hIL-10 was constructed. (2) pcDNA3.0-hIL-10 was transfected into HEK293 cells,successfully. (3) hIL-10 can inhibit the proliferation of T and B cells and reduce the production of IL-2,IF N-γ,and TNF-α. (4) hIL-10 can stimulate the expression of VEGF mRNA in HEK293 cells.