三甲基化组蛋白H3赖氨酸4对调节性T淋巴细胞foxp3基因表达的影响

Influence of Tri-Methylated Histone H3 at Lysine 4 on Regulatory T Cells foxp3 Expression

目的探讨天然调节性T淋巴细胞(nTreg)的三甲基化组蛋白H3赖氨酸4(H3K4me3)对foxp3基因的影响,寻找维持调节性T淋巴细胞(Treg)表型长期稳定的方法。方法使用免疫磁珠细胞(MACS)分选法从健康人外周血中分离出CD4+CD25+Treg。对CD4+CD25+Treg分别进行0 d、3 d、7 d培养,其中0 d为阴性对照组,3 d为阳性组1,7 d为阳性组2,采用流式细胞仪(FCM)对0 d、3 d、7 d nTreg膜表面标志CD25的变化进行检测,并分别对培养0 d、3 d、7 d的nTreg细胞通过染色质免疫共沉淀(ChIP-PCR)法检测foxp3基因启动子区H3K4me3及DNA水平变化。结果 1.使用细胞计数Kit-8(CCK-8)法确定植物血凝素(PHA)质量浓度在10 mg.L-1时为最佳的药物质量浓度,对Treg细胞的增殖作用最为显著。nTreg细胞培养0 d、3 d、7 d的表型变化呈递减趋势。随着培养时间(0 d、3 d、7 d)的延长,与foxp3基因启动子区结合的H3K4me3表达逐渐减少。2.采用ChIP-PCR法对培养0 d、3 d、7 d的nTreg细胞检测与H3K4me3结合的foxp3基因启动子区DNA水平变化。DNA凝胶电泳图显示,0 d、3 d在204 bp处可见条带(灰度值分别为2.31、0.91),7 d有模糊条带或无条带。三者比较差异有统计学意义(P<0.05)。结论 PHA不能维持Treg细胞的表型稳定,其机制与H3K4me3减少有关。

Abstract： Objective To investigate the effects of tri -methylated histone H3 at lysine 4 （H3K4me3）on foxp3 in naturally occurring regulatory T ceils（nTreg） in vitro and search a new method for maintaining a long - term stable Treg cells phenotype. Methods CD4＋CD25＋Treg cells were isolated with magnetic cell sorting （MACS） from human peripheral blood of healthy individuals.CD4＋CD25＋Treg cells were cultured for 0 day,3 days, and 7 days separately and divided into control group （0 d）, positive group 1 （ 3 d）, positive group 2 （7 d）. Superna- tant CD25 levels of the 3 groups cells were detected with flow cytometry （FCM）, correspondingly, after 0 day,3 days, and 7 days for eulturing, changes of DNA and H3K4me3 of foxp3 gene promoter regions in nTreg cells were examined respectively with polymerase chain reaction （PCR） - based - ChIP assay. Results 1. Phytohemagglutinin （PHA） of 10 mg. L -1 was optimal concentration that led to the most obvious expansion of Treg ceils in vitro. After 0 day ,3 days, and 7 days for culturing ,results of FCM were declined dramatically. 2. After 0 day ,3 days, and 7 days for culturing,change of DNA with H3K4me3 of foxp3 gene promoter locus in nTreg cells were respectively detected by PCR - based - ChIP assay. DNA gel electrophoresis showsed that 0 day and 3 days had a full - resolution band at 204 bp （ gray value respectively were 2.31,0.91 ） ; in comparison with 0 day and 3 days,7 days were obviously dim or not band. Three groups had distinct difference（P 〈0. 05 ）. Conclusion PHA cannot maintain stable phenotype of Treg cells,its mechanism is related to reduction of H3K4me3.