Author(s)

Ming-Jie Mai^1#*, Hao Lu^2#, Ji-Yue Liu^2#, Jing-Yun Hu², Shi-Kai Wu², Ming-Xue Cai², Sheng-Jie Liao³, Xiao-Hui Li³, Cheng-Feng Huang³, Hao Wang^2*

Affiliation(s)

¹Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangzhou, 510080, China
²Department of Anesthesia, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China
³Department of Cardiovascular Surgery, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China

Corresponding Author

Ming-Jie Mai, Hao Wang

ABSTRACT

Objective: In vitro co-culture of CD4+CD25+Tregs with Rapamycin or Cyclosporine A was observed on the proliferation of CD4+CD25+Tregs and expression of Foxp3 and TGF-β, in order to analyze the mechanism by which Rapamycin promotes TGF-β to secrete and induce in vitro differentiation and proliferation of regulatory T cells (Tregs). Methods: The mononuclear cell was isolated from a C57BL/6 mouse spleen obtained under sterile conditions; CD4+CD25+Tregs were sorted by immunomagnetic beads, which were divided into the blank control group, Rapamycin group, and Cyclosporine A group to conduct 96 hours of co-culture. CD4+CD25+Tregs were detected with a up-flow cytometry. The level of expression and secretion of FoxP3+ and TGF-β mRNA of CD4+CD25+Tregs treated by Rapamycin and Cyclosporin A were detected and analyzed with reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The expression of Smad proteins, which is the important activated molecules of the TGF-β signal pathway, was analyzed by Western blot, and observed the effects on the proliferation of CD4+CD25+FoxP3+Tregs. TGF-β neutralizing antibodies were used to further confirm the significance of Rapamycin on the promotion of differentiation and proliferation of CD4+CD25+FoxP3+Tregs. Results: Compared with that in the control group, the proportion of CD4+CD25+Tregs, decreasing significantly in CD4+T cells (3.72% vs 7.42%, p<0.01) in the Cyclosporine group, and obviously increased (11.47% vs 7.42%, p<0.01) in the Rapamycin group; the expression of T cells and Foxp3 mRNA of Rapamycin group was significantly higher than that in the Cyclosporine group and control group (all P<0.01); the expression of Foxp3 mRNA was lower markedly in the Cyclosporine A group compared with that in the control group (P<0.05). Conclusions: Rapamycin can promote the proliferation and growth of CD4+CD25+Tregs by in vitro culture, while Cyclosporin A can inhibit proliferation and growth of CD4+CD25+Tregs in vitro culture; Foxp3 and TGF-β are related positively to CD4+CD25+Tregs by the in vitro 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

CITE THIS PAPER

Ming-Jie Mai, Hao Lu, Ji-Yue Liu, Jing-Yun Hu, Shi-Kai Wu, Ming-Xue Cai, Sheng-Jie Liao, Xiao-Hui Li, Cheng-Feng Huang, Hao Wang. An experimental research of the effects of rapamycin on the in vitro proliferation and growth of tregs in mice by promoting TGF-Β secretion. Acta Translational Medicine. 2018, 1(1): 35-46.

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