The Effect Of ZVAD-fmk In Processes Of Human Immature Dendritic Cell Induce The Differentiation-of T Cell

Objectives: we obtained the immature dendritic cells from human peripheral blood, and then mixed-cultured it with CD4+ T cell which from human umbilical cord blood, while blocked Caspase signal pathway with zVAD-fmk during them cultured. The purpose of the study is hoped to find the effect of apoptosis signal pathway in the processes of immature dendritic cell induce the regulatory T cells, and explore the probable molecular mechanisms.Method: In the study, we get 50ml human peripheral vein blood from a health adult, then we separated the semi-attaching-wall mononuclear cells by lymphocyte separating and gradient centrifugation. The peripheral blood mononuclear cells (PBMC) was cultured in 2 hours then was induced and amplified by rhGM-CSF and IL-4 for 7 days. After that PBMC was transformed into imDC. Meanwhile, we separated allogenic PBMC from human umbilical cord blood by the same way, and then we separated CD4+T cells from PBMC by immunological magnetic beads. We mixed-cultured the imDC and CD4+T cells for 5 days. The mixed-cultured cells were divided into five groups: 1. Blank group: CD4+T cell cultured with equal volume of RPMI-1640 only. 2. Control group: the mix-cultured suspension??n't added the obstructor. 2. low-concentrantion group: the mix-cultured suspension was added the Caspase obstructor (zVAD-fmk) in 10μmol/L. 2. Middle-concentration group: the mix-cultured suspension was added zVAD-fmk in 20μmol/L. 3. High-concentration group: the mix-cultured suspension was added zVAD-fmk in 30μmol/L. After the mixed-culture, we detected the conversion ratio of CD4+CD25+T cell (regulator T cell) by flow cytometry (FCM).Results: (1). after 7 days cultured the PBMC was suspended growth, burr processes can be see on the surface of cells under the light microscope, the volume of cells enlarged. (2). The T cell conversion rate was detected by FCM after mixed-cultured: blank group: 1.78±0.11, control group: 22.23±0.77, low-concentrantion group: 21.63±0.82, middle-concentration group: 12.24±0.54, high-concenttration group: 12.20±0.96. Detecting data were done by statistics analysis, Except the comparing between the control group and low-concentration group, the high-concentration group and middle-concentration group, there were significance difference between other groups (P<0.05). Compare with the control group the T cell conversion rate of experiment group was lower (P<0.05).Conclusion:1.Through applied rhGM-CSF+rhIL-4 combinedly inducing PBMC to obtain DC in different growth phase, we constructed a kind of culture method of imDC in vitro.2. imDC can induce allogenic human umbilical cord blood originated initial CD4+T cell transformed into Treg cell.3. The peculiar obstructor zVAD-fmk of Caspase signal pathway can partly inhibit the transforming of Treg, it shows that Caspase signal pathway played an important role in the inducing of immune tolerance.