Effects Of U0126 In Processes Of ImDC Induce The Naive CD4~+T Cells To Differentiate Treg Cells In Vitro

Object:To obtain the immature dendritic cells derived from human peripheral blood, and then cocultured it with allogeneic CD4+T cells, to block ERK1/2 signal pathway through U0126,for study the effect of ERK1/2 signal pathway in the processes of immature dendritic cells induce the allogeneic CD4+T cells to differentiate regulatory T cells, and explore the probable molecular mechanisms about immune tolerance.Methods: 50ml elbow vein blood in a healthy adult male volunteer was draw, The peripheral blood mononuclear cells (PBMC) were isolated from the elbow vein blood with gradient centrifugation,PBMC were cultured in 2 hours,then were induced and amplified by GM-CSFand IL-4 for 7 days,PBMC were transformed into imDC.After induced, the immature dendritic cells were identified by cell morphology, cell surface marker and cell functions individually. To separate allogenic PBMC from infant cord vein blood by the foregoing same way,and separate CD4+T cells from PBMC by immunological magnetic beads.Then, to coculture the imDC and the CD4+T cells in the ratio of 1:10,the cocultured cells were divided into five groups: 1. Group Blank: CD4+T cells were cultured only,and the RPMI-1640 at equal capacity was added to cell culture medium;2. Group mixed contrast:the suspension wasn't added the inhibitor (U0126);3.Group low-concentration U0126: U0126 solutrope whose concentration was 10μmol/l was added to cell culture medium;4.Group middle -concentration U0126: U0126 solutrope whose concentration was 30μmol/l was added to cell culture medium;5.Group high-concentration U0126 :U0126 solutrope whose concentration was 50μmol/l was added to cell culture medium; After coculture for 5days, to detect the conversion ratio of regulator T cells by flow cytometry. Results:(1)The identification of the immature dendritic cells: in the morphology alteration aspect,after cultured for 7 days, the PBMC was suspended growth, burr processes can be seen on the surface of cells under the light microscope, the volume of cells enlarged.(2)The regulatory T cells transformtion ratio were detected by FCM: After cocultured,to detect the transformtion ratio of the regulatory T cells by FCM,the result as follows: Group blank:3.25±0.89; Group mixed contrast : 21.80±0.40;Group low-concentration U0126: 22.58±0.52; Group middle -concentration U0126:40.81±0.81;Group high-concentration U0126:41.58±0.81;Through One-way ANOVA analysis,the regulatory T cells transformtion ratio of the middle -concentration U0126 group was little difference in contrast to the high-concentration U0126 group ( P >0.05),and the low-concentration U0126 group was little difference in contrast to the mixed contrast group(P>0.05), There was a significance difference from each other between other groups(P<0.05).The results indicate that the transduction pathway was blocked better,while the concentration of the U0126 was 30μmol/l.while increased the concentration,but the transformtion ratio of the regulatory T cells didn't declined, and while decreased the concentration of the U0126,there was little difference in contrast to the transformtion ratio of the mixed contrast group.Conclusion:(1)Through applied rhGM-CSF+rhIL-4 combinedly inducing PBMC to obtain imDC, we constructed a kind of ripe and high effective culture method of imDC in vitro.(2)The immature dendritic cells derived from the human peripheral blood can induce the naive CD4+ T lymphocyte to differentiate the regulatory T cells.(3) U0126(ERK1/2 signal pathway specific blocking agent) can partial promote the naive CD4+ T lymphocyte transform into the regulatory T cells,the result shows that the blocking agent played an important function in the immune tolerance inducing.