小鼠调节性T细胞的CD4^+ CD25^+ CD127^(low/-)标记分析

Analysis of CD4^+ CD25^+ CD127^(low/-) Treg Cells in Mice

新的特异性细胞表面标记的发现是提高调节性T细胞(Treg)测量和分选技术的重要环节,Foxp3是公认的天然Treg的标记,却不能作为分选细胞的标记,CD127是新发现低表达于Treg细胞表面的标记。本实验测定CD4+CD25+CD127low/-和CD4+CD25+Foxp3+标记的小鼠外周血及脾脏Treg细胞的数量,并测定CD4+CD25+CD127low/-细胞内Foxp3转录因子的表达水平,分析CD4+CD25+CD127low/-标记在小鼠Treg细胞测定及分选的价值。收集BALB/c小鼠外周血及制备脾细胞悬液,分别三重荧光染色CD4、CD25、CD127及CD4、CD25、Foxp3后应用流式细胞仪检测,得出CD4+CD25+CD127low/-、CD4+CD25+Foxp3+这两种标记的小鼠外周血和脾脏Treg细胞的比例;再在CD4、CD25、Foxp3基础上加CD127染色(四重荧光染色),检测CD4+CD25+CD127low/-细胞Foxp3转录因子的表达情况。结果显示:①BALB/c小鼠外周血CD4+T细胞的分布:在总T细胞中CD4+TCD4+CD25+T细胞的中位表达水平分别为39.02%、5.35%;在CD4+T细胞中CD4+CD25+CD127low/-、CD4+CD25+Foxp3+细胞的中位表达水平分别为7.13%、3.97%;②BALB/c小鼠脾脏CD4+T细胞的分布:在总T细胞中CD4+T、CD4+CD25+T细胞的中位表达水平分别为23.49%、3.86%;在CD4+T细胞中CD4+CD25+CD127low/-、CD4+CD25+Foxp3+细胞的中位表达水平分别为12.8%、8.23%;③BALB/c小鼠外周血和脾脏CD4+CD25+CD127low/-细胞比例高于CD4+CD25+Foxp3+细胞,以脾脏更为明显(P<0.01);④小鼠外周血及脾脏CD4+CD25+CD127low/-细胞高表达Foxp3转录因子,CD4+CD25+Foxp3+细胞低表达CD127。结论:BALB/c小鼠外周血和脾脏CD4+CD25+CD127low/-较CD4+CD25+Foxp3+能定量更多的Treg细胞;CD127low/-作为CD4+CD25+Treg细胞表面的特征性标志,在细胞分选时受其他细胞的干扰最小,是定量和纯化小鼠Treg细胞的更好标记选择。

The quantitative identification and enrichment of viable regulatory T cells （Treg） requires reliable surface markers that are selectively expressed on Treg. Foxp3 is the accepted marker of natural Treg, but it cannot be used to isolate cells for functional studies. CD127 is a new surface marker expressed in Treg cells. In this study, two populations of Treg, including CD4 ^＋ CD25^＋ CD127low/- and CD4^＋ CD25^＋ Foxp3^＋ T cells, and profiles of the Foxp3 expression in CD4^＋ CD25 ^＋ CD127low/-cells were compared to evaluate which population is better. The peripheral blood cells were collected and spleen suspension of BALB/C mice were prepared, and using triple staining CD4, CD25, CD127 and CD4, CD25, Foxp3. The profiles of Treg, including CD4 ^＋ CD25 ^＋ CD127^＋^＋w/- and CD4 ^＋ CD25 ^＋ Foxp3 ^＋ were detected by flow cytometry. The quadruple staining CD4, CD25, Foxp3 and CD127 were used to determine the CD127 expression in CD4 ^＋ CD25^＋ Foxp3^＋ cells. The results showed that on T cell subset the median expression levels of CD4^＋ , CD4^＋ CD25 ^＋ were 39.02% ,5.35% in peripheral blood and 23.49% ,3.86% in spleen. On CD4 ^＋ T cell subset, the median expression level of CD4 ^＋ CD25 ^＋ CD127low/- and CD4 ^＋ CD25 ^＋ Foxp3 ^＋ T cells were 7.13% ,3.97% in peripheral blood and 12.8% ,8.23% in spleen. The ratio of CD4 ^＋ CD25 ^＋ CD127low/- T cells was higher than that of CD4 ^＋ CD25^＋ Foxp3 ^＋ cells in both peripheral blood and spleen cells （ P 〈 0.01 ）. The CD4 ^＋ CD25 ^＋ CD127low/- cells highly expressed Foxp3, while the CD4 ^＋ CD25 ^＋ Foxp3 ^＋ T cells lowly expressed CD127. It is concluded that compared with the CD4 ^＋ CD25 ^＋ Foxp3 ^＋ populations, CD4 ^＋ CD25 ^＋ CD127low/- T cells better fit thedefinition of naturally occurring regulatory T ceils in peripheral blood cells and spleen of BALB/C mice. CD127low/- is a charicteristic marker on surface of CD4 ^＋ CD25 ^＋ Treg cells, and has been confirmed to be more specific marker for quantitatively sorting Treg cells.