[Separation and amplification of CD4(+)CD25(+) regulatory T cells from sensitized mice].

The aim of this study was to separate and amplify CD4+CD25+Treg cells from splenocytes of sensitized mice. The percentage of CD4+CD25+Treg cells was detected by flow cytometry in sensitized and normal control mice.CD4+T,CD4+CD25+Treg and CD4+CD25-T cells were isolated from mouse splenocytes by MACS.CD4+CD25+Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2.The activity of cells was detected with 0.4% trypan blue staining.The purity of cells after sorting,the main surface marker and the level of Foxp3 were detected by flow cytometry.The results showed that CD4+CD25+Treg cell proportion was higher in sensitized mice than normal control mice(P0.05).The average purity of CD4+CD25+Treg cells was 87%.The activity of these cells was more than 97%,and the expression of Foxp3 in these cells was high.The amplification mutiples achieved 42 times after 2 weeks in vitro.The percentage of CD4+CD25+ regulatory T cells was 85.32%,and the expression of Foxp3 decreased from(76.92±1.72)% to(75.33±2.11)%(P0.05).It is concluded that the sorting of CD4+CD25+Treg cells is isolated successfully by MACS without affecting the vitality of target cells.The amplification of CD4+CD25+Treg cells is successful in vitro.Expression of surface markers and Foxp3 gene does not obviously change after amplification,so that to establish a practical method to recover and enlarge the amount of CD4+CD25+Treg cells in good condition.