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中国循证儿科杂志

中国循证儿科杂志 ›› 2016, Vol. 11 ›› Issue (6): 410-415.

• 论著 • 上一篇    下一篇

急性期川崎病γ干扰素组蛋白乙酰化改变及意义初探

梅洁花1,3，崔冬1,3，王勤2，温鹏强1，徐明国1，唐根1，刘琮1，李成荣1，王国兵1   

  1. 1 遵义医学院深圳市儿童医院儿科研究所 深圳，518038；2 南方医科大学附属深圳市妇幼保健院中心实验室 深圳，518028；3 并列第一作者
  • 收稿日期:2016-10-10 修回日期:2016-12-07 出版日期:2016-12-25 发布日期:2016-12-25
  • 通讯作者: 王国兵

Changes and significances of histone 3 acetylation of IFN-γ gene during acute phase of Kawasaki disease

MEI Jie-hua1,3, CUI Dong1,3, WANG Qin2, WEN Peng-qiang1 , XU Ming-guo1 , TANG Gen1 , LIU Cong1 , LI Cheng-rong1 , WANG Guo-bing1   

  1. 1 Shenzhen Institute of Pediatrics, Shenzhen Children's Hospital, Zunyi Medical College, Shenzhen 518038, China; 2 Lab Centre, Shenzhen Maternity & Child Healthcare Hospital, Southern Medical University, Shenzhen 518028, China; 3 has equal contribution
  • Received:2016-10-10 Revised:2016-12-07 Online:2016-12-25 Published:2016-12-25
  • Contact: WANG Guo-bing

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摘要：

目的 探讨急性期川崎病(KD)患儿IFN-γ组蛋白乙酰化改变及其在KD免疫发病机制中的作用。 方法 以2015年2月至2016年6月深圳市儿童医院诊断并住院治疗的KD患儿为KD组，经IVIG治疗后为KD-IVIG组，KD组依冠状动脉有无损伤分为冠状动脉损伤（KD-CAL+）亚组和无冠状动脉损伤（KD-CAL－）亚组，于IVIG治疗前、后取血备检；同期体检的健康儿童为对照组。采用染色质免疫共沉淀法检测外周血CD4+ T细胞IFN-γ组蛋白H3乙酰化水平及组蛋白乙酰化酶p300和去乙酰化酶HDAC1/2结合水平；流式细胞术检测外周血CD4+IFN-γ+细胞(Th1)比例及IFN-γ、pSTAT4、pSTAT5和T-bet蛋白表达水平；实时荧光定量PCR检测CD4+ T细胞IFN-γ、IL-2Rα/β、IL-12Rβ1/2、IL-18Rα/β和ITLR4 mRNA表达；ELISA检测外周血浆中IL-12、IL-2和IL-18浓度。 结果 ①KD患儿38例（男20例），年龄1～5.2岁；KD-CAL+亚组16例，KD-CAL－亚组22例。对照组32例（男17例），年龄1.2～4.9岁。KD组和对照组年龄、性别差异无统计学意义。②急性期KD患儿Th1细胞比例、IFN-γ mRNA和蛋白表达及其组蛋白乙酰化水平显著高于对照组(P<0.05)，且KD-CAL+亚组高于KD-CAL－亚组(P<0.05)，经IVIG治疗后明显恢复(P<0.05)。③急性期KD患儿CD4+ T细胞IFN-γ组蛋白乙酰化酶p300结合水平显著增加(P<0.05)，HDAC1/2结合水平明显降低(P<0.05)，p300/HDAC1/2明显高于对照组且与IFN-γ组蛋白H3乙酰化水平呈正相关 (r=0.52; P<0.05)，经IVIG治疗后均有所恢复(P<0.05)。其中KD-CAL+亚组p300结合水平及p300/HDAC1/2比值均高于KD-CAL-亚组 (P<0.05)，而HDAC1/2结合水平则低于后者(P<0.05)。④急性期KD患儿血浆IL-2、IL-12和IL-18浓度显著增高（P<0.05），CD4+ T细胞表面受体IL-2Rα/β、IL-12Rβ1/2、IL-18Rα/β和TLR4及其下游信号分子pSTAT5、pSTAT4、T-bet和MyD88表达明显上调（P<0.05），且KD-CAL+亚组前述各项均高于KD-CAL-亚组（P<0.05），经IVIG治疗后均有不同程度恢复（P<0.05）。 结论 IFN-γ组蛋白过度乙酰化可能是导致急性期KD患儿免疫功能紊乱的重要因素之一。

Abstract:

Objective To investigate the changes and significances of histone 3 acetylation of IFN-γ gene in patients with acute Kawasaki disease(KD). Methods Children with KD were enrolled in Shenzhen Children's Hospital from February 2015 to June 2016, and sub-grouped as with or without coronary artery lesions (CAL). Age-matched healthy children attending routine physical examination were recruited as controls during the same period. Chromatin immunoprecipitation was performed to determine acetylation level of histone 3 of IFN-γ gene, and binding level of histone acetyltransferase p300 and deacetylase HDAC1/2 with IFN-γ gene. The proportion of CD4+IFN-γ+ cells (Th1) and protein levels of IFN-γ, pSTAT4, pSTAT5 and T-bet were analyzed by flow cytometry. Quantitative real-time PCR was used to evaluate the levels of IFN-γ, IL-2Rα/β, IL-12Rβ1/2, IL-18Rα/β and ITLR4 mRNA in CD4+ T cells. Plasma concentrations of IL-12, IL-2 and IL-18 were measured by enzyme-linked immunosorbent assay. Results ① Thirty-eight children with KD, including 16 children with CAL (KD-CAL+) and 22 children without CAL(KD-CAL－), aged from 1 to 5.2 years with a mean of 2.9 years were recruited. The KD group consisted 20 males and 18 females, and 32 age-matched healthy children with 17 males were recruited as controls. No difference of age or sex was found between the two groups(P>0.05). ② The proportion of Th1, levels of IFN-γ protein and mRNA, and acetylation levels of histone 3 of IFN-γ gene increased remarkably during acute KD(P<0.05), and restored after IVIG therapy(P<0.05). Meanwhile, all the four items aforementioned in KD-CAL+ subgroup were higher than those in KD-CAL－ subgroup (P<0.05). ③ During acute KD, binding level of p300 with IFN-γ gene was up-regulated significantly whereas binding level of HDAC1/2 with IFN-γ gene was down-regulated(P<0.05), resulting in the higher ratio of p300/HDAC1/2 that was positive correlated with the acetylation level of IFN-γ gene in patients in acute KD(r=0.52, P<0.05). Moreover, binding level of p300 with IFN-γ gene and the ratio of p300/HDAC1/2 in KD-CAL+ subgroup were higher than those in KD-CAL－ subgroup (P<0.05), while a lower binding level of HDAC1/2 with IFN-γ gene was found in KD-CAL+ subgroup (P<0.05). All the three items mentioned above restored remarkably after IVIG treatment(P<0.05). ④ In comparison with healthy controls, plasma concentration of inflammatory cytokines (IL-2, IL-12 and IL-18), levels of their receptors(IL-2Rα/β, IL-12Rβ1/2, IL-18Rα/β and TLR4) and downstream molecules (pSTAT5, pSTAT4, T-bet and MyD88) were elevated pronouncedly during acute KD(P<0.05), and restored to some extent after IVIG treatment (P<0.05). Simultaneously, all the items mentioned before in KD-CAL+ subgroup were higher than those in KD-CAL－ subgroup (P<0.05). Conclusion Hyper-acetylation of histone of IFN-γ gene might be one of the important factors contributing to immune dysfunction of KD.

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