Volume 1, Issue.1, Pp35-46, 2018

AN EXPERIMENTAL RESEARCH OF THEEFFECTS OF RAPAMYCIN ON THE IN VITROPROLIFERATION AND GROWTH OF TREGS INMICE BY PROMOTING TGF-Β SECRETION

Abstract : Objective: In vitro co-culture of CD4+CD25+Tregs with Rapamycinor Cyclosporine A was observed on the proliferation of CD4+CD25+Tregs andexpression of Foxp3 and TGF-β, in order to analyze the mechanism by whichRapamycin promotes TGF-β to secrete and induce in vitro differentiation andproliferation of regulatory T cells (Tregs). Methods: The mononuclear cell wasisolated from a C57BL/6 mouse spleen obtained under the sterile conditions;CD4+CD25+Tregs were sorted by immunomagnetic beads, which were dividedinto the blank control group, Rapamycin group, and Cyclosporine A group toconduct 96 hours of co-culture. CD4+CD25+Tregs were detected with a up-flowcytometry. The level of expression and secretion of FoxP3+ and TGF-βmRNA ofCD4+CD25+Tregs treated by Rapamycin and Cyclosporin A were detected andanalyzed with reverse transcription-polymerase chain reaction (RT-PCR) andenzyme linked immunosorbent assay (ELISA). The expression of Smad proteins,which is the important activated molecules of the TGF-β signal pathway, wasanalyzed by Western blot,and observed the effects on the proliferation ofCD4+CD25+FoxP3+Tregs. TGF-β neutralizing antibodies were used to furtherconfirm the significance of Rapamycin on the promotion of differentiation andproliferation of CD4+CD25+FoxP3+Tregs. Results: Compared with that in thecontrol group, the proportion of CD4+CD25+Tregs, decreasing significantly inCD4+T cells (3.72% vs 7.42%, p<0.01)in the Cyclosporine group, and obviouslyincreased (11.47% vs 7.42%, p<0.01) in the Rapamycin group; the expression ofT cells and Foxp3 mRNA of Rapamycin group was significantly higher than thatin the Cyclosporine group and control group (all P<0.01); the expression of Foxp3mRNA was lower markedly in the Cyclosporine A group compared with that inthe control group (P<0.05). Conclusions: Rapamycin can promote theproliferation and growth of CD4+CD25+Tregs by in vitro culture, whileCyclosporin A can inhibit proliferation and growth of CD4+CD25+Tregs in vitroculture; Foxp3 and TGF-β are related positively to CD4+CD25+Tregs by the invitro experiment. Rapamycin can promote the proliferation of CD4+CD25+FoxP3+Tregs by inducing the secretion and expression of TGF-β.

Keywords： Regulatory T cells; Mouse; Rapamycin; Cyclosporine A; TGF-β;Foxp3