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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。
Objective To investigate the effect of H3K4me3 on foxp3 gene in natural regulatory T lymphocytes (nTregs) and to find a way to maintain the long-term stability of regulatory T lymphocyte (Treg) phenotype. Methods CD4 + CD25 + Tregs were isolated from healthy human peripheral blood using immunomagnetic bead sorting (MACS). The CD4 + CD25 + Tregs were cultured on day 0, day 3 and day 7, respectively, in which 0 d was negative control group, 3 d was positive group, and 1 d was positive group 2, FCM (0, d, 3 d, 7 d nTreg membrane surface markers of CD25 changes were detected, and cultured 0 d, 3 d, 7 d of nTreg cells by chromatin immunoprecipitation (ChIP-PCR) assay foxp3 gene promoter region H3K4me3 and DNA level changes. Results 1. The best mass concentration of phytohemagglutinin (PHA) at the concentration of 10 mg.L-1 was determined by the cell counting kit-8 (CCK-8) method and the proliferation of Treg cells was the most significant. The phenotypic changes of nTreg cells on day 0, day 3 and day 7 showed a decreasing trend. With the extension of culture time (0 d, 3 d, 7 d), the expression of H3K4me3, which binds to the promoter of foxp3 gene, gradually decreased. The level of DNA of promoter region of foxp3 gene, which binds to H3K4me3, was detected by ChIP-PCR on day 0, day 3 and day 7 of cultured nTreg cells. DNA gel electrophoresis showed that bands were visible at 204 bp on day 0 and day 3 (gray values were 2.31 and 0.91, respectively), and fuzzy or no bands were detected on day 7. The difference between the three was statistically significant (P <0.05). Conclusion PHA can not maintain the phenotype of Treg cells, the mechanism of which is related to the decrease of H3K4me3.