Induction Of The Immunomodulatory Activity Of Interleukin-35 Modified Mesenchymal Stem Cells

Objective: Mesenchymal stem cells(MSCs) of SD rats were isolated and cultured in vitro, the IL-35 expression plasmid vectors were transfected into MSCs, co-culture of MSCs and DC cell or T cells in vitro were sat up to explore the immunomodulatory activity of modified MSCsMethods:(1) MSCs were isolated by whole bone marrow adherent method and passaged by trypsin-digestion. The surface markers of the 3rd passage MSCs were detected by flow cytometry and the capacity of their adipocyte and cartilage differentiation were examined.(2) Human Ebi3 and p35 mature peptide gene were amplified by PCR from the c DNA extracted from human peripheral blood mononuclear cells. By overlap PCR, we got the IL-35 fusion gene and cloned it into the expression vector PMSCV-IRES-GFP. The plasmids were extracted and transient transfected into MSCs, some gene expression of modified MSCs were tested by Real-time PCR and the secretion of IL-10、TGF-β1、IL-12 were tested by ELISA.(3) The DC cells of Wistar rats were isolated and co-culture of MSCs and DC cell were sat up including cell-to-cell contact and transwell, 48 hours after co-culture, the DC phenotype was detected by flow cytometry and the secretion of IL-12 by DC were tested by ELISA.(4) Co-culture of MSCs and T cell were sat up including cell-to-cell contact and transwell, the CD8+T and CD4+CD25+ Treg were detected by flow cytometry, T cell proliferation were tested by MTS, The expression of IL-10 and TGF-β1 of MSCs were tested by Real-time PCR.Results:(1) MSCs were mainly spindle-shaped and vortex-like in culture. Surface marker detection showed that MSCs highly expressed CD29, CD44 and CD90, but CD31 and CD45 were negative and 3rd passage MSCs could differentiate into adipocytes and chondrocytes in vitro.(2) 630 bp human Ebi3 and 590 bp p35 genes were amplified by PCR. By overlap PCR, we got the IL-35 fusion gene and cloned it into expression vector and recombinant clone was analyzed by digestion of restriction endonuclease. The plasmids were extracted and transient transfected into MSCs cell. The expression of IL-35 of the modified MSCs significantly enhanced, and the IL-10 and TGF-β1 whichclosely related with IL-35 also enhanced.The ELISA test showed that the secretion of IL-10 and TGF-β1 of the modified MSCs increased and secretion of IL-12 reduced.(3) The DC cells were isolated by density gradient centrifugation method and were detected by flow cytometry. The mature markers of DC were in low expression. After stimulated with LPS, the expression of mature markers significantly enhanced. Whlie CD86 was still at lower levels despite the addition of LPS under the co-culture with MSCs and the inhibition effect was more obvious in MSC-IL-35 group and transwell group. The ELISA test showed that the secretion of IL-12 of the DC decreased after mixed with MSCs, and this effect was more obvious in MSC-IL-35 group.(4) The CD8+ effector T cells got effectively decrease and the CD4+CD25+ T cells were significantly increased 48 hours post co-culture of allogeneic spleen T cells and MSCs and this effect was more obvious in MSC-IL-35 group. MTS shows that MSCs inhibited the proliferation of T cells. Real-time PCR examination showed that the expression of IL-10 and TGF-β1 of MSCs significantly increased after co-culture with T cells, and this effect was more obvious in cell-to-cell contact group.Discussion: MSCs modified by IL-35 gene could suppress the DC maturation stimulated by LPS through the way of cell to cell and cytokine secretion. It could decrease the CD8+ T cells proportion and increase CD4+CD25+ regulatory T cells proportion, this effect was more stronger than normal MSCs. MSCs modified by IL-35 showed more immunomodulatory activity and might have more applications in the field of organ transplantation tolerance.