The Effect Of AG490 In Processes Of ImDC Induce CD4~+T Cells To Differentiate Treg Cells

Object: The culturation of immature dendrtic cell from human peripheral blood in vitro ,observe the effects of AG490 on tranformation from CD4+ T lymphocytes to CD4+ CD25+T lymphocytes in vitro by imDC transduction. And ascertain the effection that imDCs impact on the activity of JAK-STAT signal transduction pathway in the trnaformation from the CD4+ T cell to regulatoion T lymphocytes.Accordly,which determine its molecular mechanism.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 were induced and amplified by GM-CSF(final concentration: 100ng/ml)and IL-4(final concentration: 50ng/ml) for 7 days.After induced, the immature dendritic cells were identified by cell morphology, cell surface marker and cell functions individually. Separate allogenic PBMC from human peripheral blood by the foregoing same way,and separate CD4+T cells from PBMC by immunological magnetic beads.Then, for 5days,the cocultured cells were divided into five groups: 1. Group blank contrast:the suspension wasn't added the inhibitor (AG490);to coculture the imDC and the CD4??ells in the ratio of1: 10 were divided into four groups: 2.Group Blank: CD4+T cell cultured only. After coculture, to detect the conversion ratio of regulator T cells by flow cytometry. 3. Group low-concentration AG490: AG490 solutrope whose concentration was 10μmol/l was added to cell culture medium 4. Group middle -concentration AG490: AG490 solutrope whose concentration was 30μmol/l was added to cell culture medium; 5.Group high-concentration AG490 :AG490 solutrope whose concentration was 50μmol/l was added to cell culture medium;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, cell membrane was stained green fluorescence under the fluorescence microscope;After induced, the cell surface molecule was detected by FCM,and the result of molecule on imDC: CD1a:higher express,CD80:lowser expressCD83:lowser express;(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: 1.74±0.18%,Group contrast : 22.23±0.77%,Group low-concentration AG490: 22.31±0.82%, Group middle -concentration AG490 : 41.39±1.88% ; Group high-concentration AG490 : 42.32±1.18%.Through One-way ANOVA analysis,the regulatory T cells transformtion ratio of the middle -concentration AG490 group was little difference in contrast to the high-concentration AG490 group(P>0.05),and the low-concentrat??AG490 group was little difference in contrast to the control group(P>0.05), There was a significance difference from each other between other groups(P<0.05).Conclusion:(1)The immature dendritic cells derived from the human peripheral blood can differentiate induced the allogeneic CD4+ T lymphocyte to regulatory T cells.(2) JAK2/3 pathway inhitor AG490 can improve the conversion rate of the regulatory T cells partly.The imDC induced immune torelance by differentiating CD4+.T cell to Treg,that use the pathway of JAK-STATs.