CAJ | 학술논문

目的：探讨多环芳烃（ polycyclic aromatic hydrocarbons，PAHs）对新生儿调节性T细胞（ Treg）的影响。方法收集7例新生儿脐带血，分离CD4＋CD25＋T细胞，体外CD3、CD28及IL-2诱导培养3 d，设加PAHs组及对照组。流式细胞术检测Foxp3、信号传导转录激活因子3（ STAT3）、STAT5的表达及Treg细胞抑制功能，结合重亚硫酸盐的焦磷酸测序法检测Foxp3基因启动子区和Treg特异性甲基化区域（ TSDR）甲基化水平，RT-PCR检测DNA甲基化酶（ DNMT）1、DNMT3a、DNMT3b、IL-4 mRNA表达水平，ELISA检测培养后上清液IL-4蛋白水平。结果（1） PAHs抑制Foxp3表达及Treg细胞抑制功能；（2）PAHs抑制STAT5表达（P＜0．05），上调STAT3表达（P＜0．05）；（3）PAHs增加Foxp3基因启动子区和TSDR区甲基化；（4）PAHs上调DNMT1、DNMT3a、DNMT3b表达（P＜0．05）；（5）PAHs诱导CD4＋CD25＋T细胞产生IL-4，IL-4与STAT5表达呈负相关，与STAT3表达呈正相关。结论 PAHs抑制新生儿Treg细胞数量及功能，其机制可能与诱导IL-4产生、导致STAT5／STAT3表达异常、Foxp3基因过度甲基化等有关。
목적：탐토다배방경（ polycyclic aromatic hydrocarbons，PAHs）대신생인조절성T세포（ Treg）적영향。방법수집7례신생인제대혈，분리CD4＋CD25＋T세포，체외CD3、CD28급IL-2유도배양3 d，설가PAHs조급대조조。류식세포술검측Foxp3、신호전도전록격활인자3（ STAT3）、STAT5적표체급Treg세포억제공능，결합중아류산염적초린산측서법검측Foxp3기인계동자구화Treg특이성갑기화구역（ TSDR）갑기화수평，RT-PCR검측DNA갑기화매（ DNMT）1、DNMT3a、DNMT3b、IL-4 mRNA표체수평，ELISA검측배양후상청액IL-4단백수평。결과（1） PAHs억제Foxp3표체급Treg세포억제공능；（2）PAHs억제STAT5표체（P＜0．05），상조STAT3표체（P＜0．05）；（3）PAHs증가Foxp3기인계동자구화TSDR구갑기화；（4）PAHs상조DNMT1、DNMT3a、DNMT3b표체（P＜0．05）；（5）PAHs유도CD4＋CD25＋T세포산생IL-4，IL-4여STAT5표체정부상관，여STAT3표체정정상관。결론 PAHs억제신생인Treg세포수량급공능，기궤제가능여유도IL-4산생、도치STAT5／STAT3표체이상、Foxp3기인과도갑기화등유관。
Objective To investigate the effects of polycyclic aromatic hydrocarbons ( PAHs) on regulatory T ( Treg) cells in newborns.Methods Blood samples were taken from the umbilical cord of sev-en newborn babies.CD4+CD25+T cells were isolated and treated with or without 300 nmol/L of phenan-threne for 72 hours in the presence of 100 IU/ml of IL-2.The expression of forkhead box P3 (Foxp3), sig-nal transducer and activator of transcription 3 (STAT3) and STAT5 were analyzed by 8-color flow cytometry. RT-PCR was performed to detect the expression of DNA ( cytosine-5 )-methyltransferase 1 ( DNMT1 ) , DNMT3a, DNMT3b and IL-4 at mRNA level.Pyrosequencing in combination with bisulphite sequencing was used to evaluate the methylation within the promoter and the Treg-specific demethylated region ( TSDR) of Foxp3 locus.Treg cells were cultured for 7 hours with autologous Tresp at Tresp/Treg ratios of 1 ∶1, 2 ∶1, 4 ∶1 and 8 ∶1 and stimulated with anti-CD3/CD28 beads and IL-2 for the evaluation of the immunosuppres-sive activities of Treg cells.Results (1) PAHs inhibited the expression of Foxp3 and the function of Treg cells collected from newborns.(2) PAHs significantly decreased the expression of STAT5 and Foxp3, but increased the expression of STAT3 (P<0.05).(3)PAHs enhanced the methylation of the promoter and the TSDR within Foxp3 gene.(4) The transcription levels of DNMT1, DNMT3a and DNMT3b in PAHs treated group were significantly higher than those of the control group (P<0.05).(5) More IL-4 was secreted by PAHs treated CD4+CD25+T cells, indicating that IL-4 was negatively correlated with STAT5, but positively correlated with STAT3.Conclusion PAHs decreased the number and inhibited the function of Treg cells in newborns.The possible mechanism might be related to the abnormal expression of STAT3 and STAT5 in-duced by IL-4 as well as the methylation within Foxp3 gene.