脐血和成人外周血CD4^+CD25^+调节性T细胞的分离及功能特征的比较

The comparison of CD4^+ CD25^+ regulatory T cells in isolation and functional characterization between cord blood and adult peripheral blood

目的分离成人外周血、脐血CD4+CD25+调节性T细胞,并检测其功能,以了解脐血CD4+CD25+T细胞的特性。方法应用免疫磁珠分选法从健康成人外周血、脐血淋巴细胞中分离纯化CD4+CD25+、CD4+CD25-T细胞。应用流式细胞术检测分离纯度,胎盘蓝染色检测细胞存活率;RT-PCR技术检测CD4+CD25+、CD4+CD25-T细胞中Foxp3的mRNA的表达;体外增殖实验检测其对CD4+CD25-T细胞的免疫抑制作用。结果 MACS分离的CD4+CD25+T细胞、CD4+CD25-T细胞纯度达(92.7±1.6)%、(90.3±1.2)%,细胞存活率达(95.3±2.1)%;体外经抗CD3、CD28单克隆抗体刺激培养的脐血及成人外周血CD4+CD25+T细胞同时得到扩增,而且高表达Foxp3;CD4+CD25+T细胞能有效的抑制效应T细胞的增殖,当效靶比为1︰1时,其抑制效应T细胞的作用最强,成人外周血抑制率为(81.36±1.61)%、脐血抑制率为(90.74±2.43)%。结论脐血和成人外周血CD4+CD25+T细胞是一群具有免疫抑制功能的调节性T细胞,与成人外周血相比,培养后的脐血CD4+CD25+T细胞比成人外周血有更强的免疫抑制功能。

Objective：To study CD4＋ CD25＋ regulatory T cells functional characteristics from the cord blood and adult peripheral blood,and to have an insight into the property of CD4＋ CD25＋ T cells from cord blood.Methods： CD4^＋CD25^＋ and CD4＋CD25-T cell were isolated from cord blood of term infants and adult peripheral blood by auto MACS.The purity and the survival rate of sorted cells were determined by flow cytometry and trypan blue exclusion respectively.The expression of Foxp3 mRNA in CD4^＋CD25^＋ and CD4＋CD25-T cells were test by RT-PCR.The immuno puppressive property of CD4^＋CD25^＋T cells on proliferation of CD4＋CD25-T cells was also assessed by cell proliferation assay in vitro.Results： The purity of CD4^＋CD25^＋T cells separated by MACS reached（92.7±1.6）% with the survival rate of（95.3±2.1） %.CD4^＋CD25^＋ T cells of the cord blood and adult peripheral blood were amplified by CD3,CD28 antibodies with high expression of the Foxp3 gene.When the ratio of CD4＋ CD25＋ T cells and effector T cell were 1︰1,Treg cells could suppress the proliferation of effector T cells effectively.The suppressive rate was（81.36±1.61）% in adult peripheral blood and（90.74±2.43）% in the cord blood.Conclusion： CD4＋ CD25＋ regulatory T cells of the cord blood and adult peripheral blood were a subpopulation of immunoregulatory T cells with suppressive function,and inhibitory effect of cultured CD4＋ CD25＋ regulatory T cells was stronger in the cord blood than adult peripheral blood.