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

摘要

Objective To investigate the effects of tri - methylated histone H3 at lysine 4( H3K4me3 ) on foxp3 in naturally occurring regulatory T cells( nTreg) in vitro and search a new method for maintaining a long - term stable Treg cells phenotype. Methods CD4 * CDj, * Treg cells were isolated with magnetic cell sorting ( MACS) from human peripheral blood of healthy individuals. CD4 + CD^ * 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). Supernatant CD25 levels of the 3 groups cells were detected with flow cytometry (FCM) , correspondingly, after 0 day, 3 days, and 7 days for culturing, 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~ was optimal concentration that led to the most obvious expansion of Treg cells 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.%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(H3 K4me3)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0d、3d、7d培养,其中0d为阴性对照组,3d为阳性组1,7d为阳性组2,采用流式细胞仪(FCM)对0d、3d、7 d nTreg膜表面标志CD25的变化进行检测,并分别对培养0d、3d、7d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H3 K4 me3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0d、3d、7d的表型变化呈递减趋势.随着培养时间(0d、3d、7 d)的延长,与foxp3基因启动子区结合的H3 K4 me3表达逐渐减少.2.采用ChIP-PCR法对培养0d、3d、7d的nTreg细胞检测与H3 K4 me3结合的foxp3基因启动子区DNA水平变化.DNA凝胶电泳图显示,0d、3d在204 bp处可见条带(灰度值分别为2.31、0.91),7d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H3 K4 me3减少有关.