Experimental Study Of Rapamycin And Ciclosporin On The Proliferation Of Human Regulatory T Cells

[Objective] Isolation and amplification of human CD4+CD25+regulatory T cells in vitro, and to identify immunosuppressive function; To observe the influence of rapamycin and ciclosporin on the proliferation of human regulatory T cells, then explore its possible mechanism.[Methods]Part1:Isolation and amplification of CD4+CD25+regulatory T cells (Treg cells) in vitro from human peripheral blood, and to identify their immunosuppressive function:Get50-100ml fresh human peripheral blood, Treg cells were isolated by application of density gradient centrifugation and immunomagnetic beads separation system, The purity and viability of enriched cells were measured by flow cytometry and Trypan blue dyeing. Then on part of Treg cells were labled by carboxyfluorescein diacetate succinimidyl ester(CFSE), with the other part of Treg cells were incubated in RPMI-1640special media which was added in anti-CD3/CD28coated beads and high concentration recombinant human IL-2. After7days, the labled Treg cells were analysised by flow cytometry to identify the amplification of them. The other unlabled Treg cells were incubated for another7days and counted by optical microscope regularly. Tregs’ suppressive function was evaluated by MLR(mixed lymphocyte culture) using co-culture of CFSE labled CD4+CD25-T cells and proliferated Tregs. CD4+CD25T cells and Treg cells were co-cultured in1:1for7days, then labled cells were analysised by flow cytometry in order to identify Treg cells’immunosuppressive function.Part2:The influence of rapamycin and ciclosporin on the proliferation of human regulatory T cells:isolate and identify the purity of Treg cells, then according to cells number, divide Treg cells into control group, rapamycin group and cyclosporine group co-culture with normal media,100nmol/L rapamycin added and410nmol/L cyclosporine added, respectively. After7day, cells of three groups were analysised by flow cytometry to identify Treg cells’proliferation state among groups. The results were analyzed using SPSS13.0statistieal analysis software.[Results] Part1:We isolated15adult blood by application of density gradient centrifugation and immunomagnetic beads separation system. One sample was polluted,6samples’CD4/CD25purity were80%below, The purity of8Treg cell samples were (97±0.8)%, Foxp3expression rate in cells were (99±0.5)%. Treg cells could increase normally in vitro, the percentage of primary cells were (37.0±3.5)%by flow cytometer, which were cultured for7days. After14days’ culture, by optical microscope the number of Treg cells were150times than before; After7days’ MLR(CD4+CD25+T cells:CD4+CD25-T cells=1:1), the percentage of primary CD4+CD25-T cells were79.4±6.9%on average, statistical analysis indicates that CD4+CD25+Treg cells can significantly inhibit the proliferation of CD4+CD25-T cells(P<0.05);Part2:The percentage of primary Treg cells in rapamycin group、cyclosporine group and blank group were (8.8±3.2)%,(48.9±7.0)%and(33.2±4.3)%respectively. Two experimental group respectively compared with blank group, statistical analysis indicated that Rapamycin can significantly promote the proliferation of CD4+CD25+T cells(P<0.05), inversely cyclosporine inhibits the proliferation of CD4+CD25+T cells(P<0.05).(Conclusion]1. CD4+CD25+Treg cells were isolated by application of density gradient centrifugation and immunomagnetic beads separation system is one ideal method which has proved high expression of CD4/CD25/Foxp3in cells;2. Rapamycin promote the proliferation and growth of CD4+CD25+Treg cells in vitro, while cyclosporine inhibit the proliferation and growth of CD4+CD25+Treg cells. These data has indicated that different immunosuppressive drugs(rapamycin or cyclosporine) might have different roles in tolerance induction. Maybe, rapamycin could promoted the induction of allo-antigen tolerance, but cyclosporine hampered the progression.