| 1. Through the effect of human plasma exosomes-like vesicles or recombinant Wnt molecules on CD4+ CD25+ CD127low regulatory T cells (Treg), observed the survival of Treg cells in vitro and the changes of the Wnt signaling pathway.2. Through the expansion of Treg cells by allogeneic human peripheral blood B lymphocytes in vitro, observed the changes of Treg cells in the number, phenotype and function after the expansion, established an effective way of the expansion of Treg cells in vitro, and compared with the expansion method by regulatory dendritic cells (rDC).1.CD4+CD25+CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) using Magnetic cell sorting, Wnt molecules and human plasma exosomes-like vesicles isolated from several healthy donors were cocultured with the Treg cells respectively, with interleukin 2 (IL-2,50U/ml). Analyzed the changes of apoptosis related genes what Wnt signaling pathway involved in after 12h by RT-PCR, detected the changes of phosphorylatedp-catenin level in Wnt signaling pathway and the apoptosis of co-culture cells in every group after 14 days by flow cytometry. The untreated Treg cells were the control group.2. The purified Treg cells handled by B-lymphocyte isolated from allogeneic peripheral blood with IL-2 (100U/ml) and CD28 monoclonal antibody (500ng/ml), detected the changes of phenotype of Treg cells in the 14 days co-culture. On the 14th day, the expanded Treg cells were cultured with CD4+ effector T cells which were stimulated strongly (addition of 500ng/ml CD3 monoclonal antibody and 300U/ml IL-2), detected the inhibition of the proliferation of CD4+ effector T cells by flow cytometry on the 5th day. The CD4+ effector T cells stimulated strongly only were established as the control group. 1. Analyzed the Frizzled receptors (FRZ) and LDL receptor related protein 5 (LRP5) and LRP6 gene expression on Treg cells by RT-PCR, the results showed that the Treg cells expressed FRZ2,3,4, LRP6 genes. Detected the protein expression of Wnt3a and Wnt5a in Plasma Exosomes by Western Blotting, both appeared the bands. Detection of apoptosis-related genes Bcl-xL, Bcl-2, c-Myc expression after 12h co-culture by RT-PCR showed that anti-apoptosis gene Bel-2 increased significantly in Treg cells cultured with plasma exosomes-like vesicles or the Wnt molecules, the expression of Bcl-xL and c-Myc had no significant change. On the 14th day, the survival rate of Treg cells which added exosomes-like vesicles or the Wnt molecules were higher than the control group by detecting the apoptosis with flow cytometry.2. After 14 days of coculturation with B cells combined with IL-2 and CD28 monoclonal antibody, the positive rate of Foxp3 and CD4/CD25 of the Treg cells were both high, and the absolute number of Treg cells were amplified 20 times. The expanded Treg cells cultured with the CD4+ T cells which were stimulated by CD3 monoclonal antibody and IL-2, the results detected by flow cytometry using CFSE technology after 5 days showed that the expanded Treg cells can effectively inhibit the proliferation of CD4+ effector T cells.1. We can initially judge that the Wnt signaling pathway can influence the survival status of Treg cells, plasma Exosomes-like vesicles were able to prolong the survival time of the Treg cells, in which the interaction between Wnt molecules carried by Exosomes-like vesicles and the Frizzled receptors on Treg cells can activate the Wnt signaling pathway of the Treg cells, which played a certain role in improving the survival rate of the Treg cells. The plasma Exosomes-like vesicles can affect the survival of Treg cells in vitro through the Wnt singaling pathway.2. Allogeneic B lymphocytes can effectively amplify human peripheral blood Treg cells in vitro, the expansion efficiency can be up to 20 times compared to the initial, and the expanded Treg cells can inhibit the proliferation of CD4+ effector T cell significantly. |
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