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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、p STAT4、p STAT5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ITLR4m RNA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-γm RNA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KDCAL+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p STAT5、p STAT4、T-bet和My D88表达明显上调(P<0.05),且KD-CAL+亚组前述各项均高于KDCAL-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。
Objective To investigate the changes of histone acetylation of IFN-γ in children with acute Kawasaki disease (KD) and its role in the pathogenesis of KD. Methods KD children diagnosed and hospitalized in Shenzhen Children’s Hospital from February 2015 to June 2016 were KD group and KD-IVIG group after IVIG treatment. The KD group was divided into coronary artery injury (KD-CAL +) subgroup and non-coronary artery injury (KD-CAL-) subgroup. Blood samples were taken before and after IVIG treatment. Healthy children in the same period were selected as control group. The levels of histone H3 acetylation and the binding of histone deacetylase HDAC1 / 2 and histone deacetylase (IFN-|Ã) in peripheral blood CD4 + T cells were detected by chromatin immunoprecipitation. The levels of CD4 + The expression of IFN-γ, IL-2Rα / β, IL-12Rβ1 / IL-12Rβ1 and CD4 + T cells were detected by real-time fluorescence quantitative PCR. 2, IL-18Rα / β and ITLR4m RNA were detected by enzyme-linked immunosorbent assay (ELISA). The concentrations of IL-12, IL-2 and IL-18 in peripheral blood were measured by ELISA. Results There were 38 children (20 males) with 1 KD, aged 1 to 5.2 years; KD-CAL + subgroup of 16 patients and KD-CAL-subgroup of 22 patients. Control group of 32 patients (17 males), aged 1.2 to 4.9 years. KD group and control group age and gender differences were not statistically significant. The proportion of Th1 cells, the expression of IFN-γmRNA and protein and the level of histone acetylation in KD children with acute KD were significantly higher than those in control group (P <0.05), and KDCAL + subgroups were higher than KD-CAL-subgroups (P < 0.05), after IVIG treatment significantly recovered (P <0.05). (P <0.05), the level of HDAC1 / 2 binding was significantly decreased (P <0.05), and the level of p300 / HDAC1 / 2 was significantly higher in CD4 + T cells than in CD4 + T cells There was a positive correlation between histone H3 acetylation level and control group (r = 0.52; P <0.05). The levels of acetylation of IFN-γ were recovered after IVIG treatment (P <0.05). The p300 binding level and p300 / HDAC1 / 2 ratio in KD-CAL + subgroup were higher than that in KD-CAL-subgroup (P <0.05), while the HDAC1 / 2 binding level was lower than the latter (P <0.05). The plasma levels of IL-2, IL-12 and IL-18 in children with acute KD were significantly increased (P <0.05). The levels of IL-2Rα / β, IL- β, TLR4 and its downstream signaling molecules p STAT5, p STAT4, T-bet and My D88 were significantly increased (P <0.05), and KD-CAL + subgroup was higher than KDCAL-subgroup After IVIG treatment, they all recovered to varying degrees (P <0.05). Conclusion Over-acetylation of IFN-γ histone may be one of the important factors leading to immune dysfunction in children with acute KD.