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中国循证儿科杂志

中国循证儿科杂志 ›› 2019, Vol. 14 ›› Issue (5): 365-370.DOI: 10.3969/j.issn.1673-5501.2019.05.009

• 论著 • 上一篇    下一篇

急性期川崎病IL-4基因组蛋白甲基化的改变及意义

赖恒1, 王国兵2, 温鹏强2, 梅洁花3, 俞灵盈2, 徐明国3, 刘琮3, 李成荣2   

  1. 1 遵义医科大学珠海校区 珠海,519041;
    2 遵义医科大学深圳市儿童医院儿科研究所 深圳,518038;
    3 遵义医科大学深圳市儿童医院心血管内科 深圳,518038
  • 收稿日期:2019-08-28 出版日期:2019-10-25
  • 通讯作者: 王国兵, E-mail:rogasan@163.com
  • 基金资助:
    1 深圳市医学科研基金:201401053;2 深圳市科技计划项目:JCYJ20180228175700233;3 国家自然科学基金:81102227

Changes and significances of histone methylation of interleukin-4 gene in acute pediatric Kawasaki disease

LAI Heng1, WANG Guo-bing2, WEN Peng-qiang2, MEI Jie-hua3, YU Ling-ying2, XU Ming-guo3, LIU Cong3, LI Cheng-rong2   

  1. 1 Zhuhai Campus, Zunyi Medical University, Zhuhai 519041, China;
    2 Institute of Pediatrics, Shenzhen Children's Hospital, Zunyi Medical University, Shenzhen 518038, China;
    3 Department of Cardiology, Shenzhen Children's Hospital, Zunyi Medical University, Shenzhen 518038, China
  • Received:2019-08-28 Online:2019-10-25
  • Contact: WANG Guo-bing, E-mail: rogasan@163.com

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摘要： 目的 探讨急性期川崎病(KD)患儿IL-4基因组蛋白甲基化改变及其在KD Th2细胞异常机制中的作用。方法 KD患儿急性期及IVIG治疗4～5 d后取血备检,同年龄健康儿童为对照组。采用流式细胞术检测外周血Th2比例及CD4+ T细胞IL-4、pSTAT6和GATA3蛋白水平;染色质免疫共沉淀分析外周血CD4+ T细胞IL-4基因CNS1、HSⅡ、HSVa组蛋白甲基化H3K4me3及GATA3、MLL1结合水平;荧光定量PCR分析CD4+ T细胞IL-4、IL-5、IL-13、IL-4Rα、IL-2Rγ、SOCS5 mRNA水平;双抗体夹心ELISA检测血浆细胞因子IL-4、IL-5和IL-13蛋白浓度。结果 KD组42例,对照组36例。① KD患儿急性期Th2细胞比例,功能相关分子(IL-4、IL-5和IL-13)mRNA表达,血浆蛋白水平,IL-4基因CNS1、HSⅡ、HSVa位点组蛋白甲基化H3K4me3修饰水平均明显增加(P<0.05),且冠状动脉损伤组(CAL)前述指标均高于无冠状动脉损伤组(NCAL)(P<0.05),经IVIG治疗后明显降低(P<0.05); ②KD患儿急性期外周血CD4+ T细胞转录因子GATA-3、组蛋白甲基化酶MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平显著增高(P<0.05),且MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平与IL-4 mRNA表达显著正相关(r分别为0.42、0.33和0.39, P均<0.05),经IVIG治疗后均表现不同水平的恢复(P<0.05)。其中CAL组MLL1、GATA-3与IL-4基因CNS1、HSⅡ、HSVa结合水平明显高于NCAL组 (P<0.05); ③KD患儿急性期血浆IL-4浓度和CD4+ T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA-3、SOCS5表达显著增高(P<0.05),其中CAL组血浆IL-4浓度和CD4+T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA3表达均高于NCAL组、SOCS5表达低于NCAL组(P<0.05),经IVIG治疗后均表现不同水平的下调(P<0.05)。结论 IL-4基因组蛋白甲基化修饰异常可能是导致KD患儿急性期Th2细胞异常的始动因素之一。

关键词: Ⅱ型辅助性T细胞, H3K4me3, 白细胞介素-4, 川崎病, 免疫

Abstract: Objective To explore the changes of histone methylation of IL-4 gene and its roles in dysfunction of Th2 cells during acute pediatric Kawasaki disease (KD).Methods Forty-two children with KD were recruited in present study, while thirty-six age-matched healthy volunteer were as controls. Flow cytometry was performed to determine the frequencies of Th2 cells and levels of IL-4, phosphorylated STAT6 (pSTAT6) and GATA3 protein. Chromatin immunoprecipitation (ChIP) and quantitative PCR were used to evaluate trimethylation of histone H3 at lysine 4 at CNS1, HSⅡ and HSVa of IL-4 gene and binding levels of GATA3 and MLL1 with loci afore-mentioned in CD4+ T cells. Transcription levels of IL-4, IL-5, IL-13, IL-4Rα, IL-2Rγ and SOCS5 mRNA in CD4+ T cells were quantitated by real-time PCR. Plasma concentrations of IL-4, IL-5 and IL-13 were analyzed by enzyme-linked immunosorbent assay.Results (1) In comparison with healthy controls, the frequencies of Th2 cells, mRNA levels and plasma concentrations of Th2 cytokines (IL-4, IL-5 and IL-13) ,and methylation levels of H3K4me3 associated with IL-4 loci (CNS1, HSⅡ and HSVa) increased significantly in patients with acute KD(P<0.05). The items mentioned afore in patients with coronary artery lesions (CAL) were found to be higher than those without coronary artery lesions (NCAL), and restored to some extent after IVIG therapy (P<0.05). (2) ChIP results showed binding abilities of GATA-3 and MLL1 proteins with IL-4 gene loci (CNS1, HSⅡ and HSVa), were up-regulated remarkably in CD4+ T cells from patients with acute KD (P<0.05), and down-regulated after IVIG treatment(P<0.05). There are positive correlations between binding abilities of MLL1 protein with IL-4 loci (CNS1, HSⅡ and HSVa) and transcription level of IL-4 mRNA detected during acute KD (r=0.42, 0.33, 0.39, P<0.05). In parallel, binding abilities of GATA-3 and MLL1 with the three IL-4 loci in CAL group were higher than those in NCAL group (P<0.05). ⑶ During acute phase of KD, plasma levels of IL-4 protein, and expression levels of IL-4 signaling downstream molecules (IL-4Rα, IL-2Rγ、pSTAT6 and GATA-3) and negative regulator SOCS5 increased remarkably (P<0.05), and all the items decreased after IVIG treatment (P<0.05). Furthermore, the five items aforementioned in CAL group were higher than those in NCAL group, while the last items in CAL group was lower than that in NCAL group(P<0.05).Conclusion Excessive histone methylation of H3K4me3 with IL-4 gene might be one of initiative factors resulting in aberration of Th2 cells in KD.

Key words: H3K4me3, IL-4, Immune, Kawasaki disease, MLL1