Construction Of MSC Highly Express IFN-γ And Curative Effect Analysis Of That MSC In Dextran Sulfate Sodium (DSS) Induced Inflammatory Bowel Disease

Mesenchymal stem cells (MSC) are multipotential nonhematopoietic progenitor cells capable of differentiating into multiple mesenchymal tissue cells. Except ability of differentiation, MSCs are able to suppress T- and B-lymphocyte activation and proliferation and may also affect dendritic cell maturation. Umbilical cord derived MSCs because of many advantages, such as their primitive, higher proliferation rate and differentiation capacity, noninvasive accessibility, lack of ethical controversy surrounding them, and reduced risk of viral contamination; So MSCs derived from umbilical cors are highly suitable for further development of novel cell-based therapeutic potential. Various studies have demonstrated that MSCs are strongly immunosuppressive in vitro and in vivo and have great potential for treating various diseases, especially those related to tissue damage involving immune reactions. Interestingly, Various studies have found that IFN-y influence the ability of MSC, for instance enhance the inhibitory effects of the proliferation of T cells, increase the expression of chemokine receptors and intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, increase the expression of IDO and then influence the differentiation of monocytes into M2 immunosuppressive macrophages (CD14(+)/CD206(+)), less susceptible to NK killing, less antagonism of transplant to hosts in Allotransplantation. Hypothesis, IFN-y gene-modified MSC (IFN-y-MSC) may have more potently immunosuppressive ability. So in this study, we structure MSC highly express IFN-y and the curative effect of IFN-y-MSC in dextran sulfate sodium (DSS) induced inflammatory bowel disease model.In this paper, we first succeed reconstructed plasmid pcDNA3.1-IFNγ and transfected to Passage 5 MSCs. Then we detected the expression of IFN-y and the phenotypic surface antigens and it shows that MSC transfected with plasmid pcDNA3.1-IFNγ did not only highly express IFN-γ but also positivity for CD44, CD90, CD105 (SH2), CD73 (SH3) and negativity for immunophenotype of bone marrow-derived MSCs such as CD11, CD14, CD34 and CD31. Those results showed that we successfully constructed MSC highly express IFN-γ. We detected the vitality, apoptosis, proliferation and cell cycle of IFN-y-MSCs and the result is that the action of transfection did not change the basis characters of MSCs.In order to determine whether the action of transfection affect the immunosuppressive effects on the proliferation of T lymphocytes in response to mitogenic stimulation in vitro, purified peripheral blood mononuclear cells (PBMCs) were obtained from healthy subjects and were stimulated with phytohaemagglutinin (PHA,2.5ug/ml) and co-cultured with different managed groups of MSC, un-stimulated cells were used as their negative control. Cells were finally plated in quintuplet into flat-bottom 96-well plates in a final volume of 200ul/well, containing MSC/PBMC at the ratio of 1:10 or 1:60. Cells were harvested, and cells proliferation was measured using [3H] thymidine incorporation 3 days later. The result is that when MSC/PBMC at the ratio of 1:10, IFN-y-MSCs have distinct immunosuppressive effects.Secondly, we analyzed the effects of immune cells and curative effect of IFN-γ-MSCs in dextran sulfate sodium (DSS) induced inflammatory bowel disease model. According to existing reports, colitis was induced by administration of dextran sulfate sodium (DSS) (3% W/V) in drinking water to C57BL/6 for 7 consecutive days. DSS challenge induced UC model that was well characterized morphologically and biochemically. DSS produced shrinkage of colon length and accompanied by mucosal edema and bloody stool. Histologically, DSS produced sub-mucosal erosions, ulceration, inflammatory cell infiltration and crypt abscess. To bowel disease model induced by DSS, when at the day 2 of inducing process, we injected difference handled MSCs through tail vein to cure colitis, Systemic infusion with MSCs transfected with pcDNA3.1-IFNy protected mice against colitis-related ease of body weight, increase of colon length, decrease in disease activity index(DAI) and improve structure of small intestine tissues. We also found that IFN-y-MSC induce Treg and increase the quantity of Th2 in Spleen and obviously suppress the expression of inflammatory cytokines from colon such as TNF-a, IL-6 and LL-1β.Taken together, this study successfully constructed MSC highly express IFN-y, discovered IFN-y-MSC can obvious suppress T cells and have a higher capacity to improve the symptom in colitis induced by DSS. This study provides a theoretical basis for the further use of genetically modified MSC and makes it more suitable for the treatment of related diseases.