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目的探讨 Toll 样受体 MyD88非依赖性途径及其调节因子在川崎病(KD)免疫发病机制中的作用。方法急性期 KD 患儿32例,正常同年龄对照组16例,KD 患儿分别于静脉丙种球蛋白(IVIG)治疗前后直接取血备检。采用逆转录-聚合酶链反应(RT-PCR)及荧光定量 PCR(RealTime PCR)检测单核/巨噬细胞(MC)Toll 样受体4及 MyD88非依赖性途径传导分子/效应分子,如含 Toll-白细胞介素-1受体结构域诱导干扰素接头分子(TRIF)、TRIF 相关接头分子(TRAM)、TANK 结合激酶1(TBK-1)、干扰素β(IFN-β)、干扰素诱导蛋白10(IP-10)、活化正常 T 细胞表达和分泌的调节因子(RANTES)、诱导性一氧化氮合成酶(iNOS)等,及负性调节因子细胞因子信号抑制因子1(SOCS-1)mRNA 表达;流式细胞术检测 MC 细胞表面共刺激分子 CD40的表达;甲基化特异性-荧光定量 PCR 分析 SOCS-1基因胞嘧啶鸟嘌呤二核苷酸(CpG)基序甲基化状态。结果 (1)急性期KD 患儿 MC MyD88非依赖性途径传导分子 TLR4、TRIF、TRAM、TBK-1和 IFN-β mRNA 表达水平显著高于正常同年龄对照组(P<0.05);(2)趋化因子 IP-10、RANTES 和 iNOS mRNA 表达水平明显增加(P<0.05);(3)急性期 KD 患儿 MC 细胞表面共刺激分子 CD40明显高于同年龄对照组[(6.19±2.25)% vs.(2.00±1.37)%,t=7.98,P<0.05],合并冠状动脉组(KD-CAL~+)CD40表达明显高于无冠状动脉组[KD-CAL~-,(9.63±2.96)% vs.(4.12±1.91)%,t=16.02,P<0.05];(4)急性期KD 患儿 MC 细胞 SOCS-1 mRNA 水平显著高于同年龄对照组[(4.31±0.83)×10~(-3) vs.(1.09±0.23)×10~(-3),t=20.43,P<0.05],KD-CAL~+组 SOCS-1 mRNA 表达明显低于 KD-CAL~-组[(5.73±1.04)×10~(-3) vs.(1.94±0.46)×10~(-3),t=14.15,P<0.05];(5)KD 患儿急性期 SOCS-1基因 CpG 序列去甲基化水平明显增高[(26.9±8.6)% vs.(5.9±1.4)%,t=13.46,P<0.05],KD-CAL~+组SOCS-1基因去甲基化水平显著低于 KD-CAL~-组[(35.1±10.3)% vs.(13.2±3.7)%,t=8.63,P<0.05]。结论急性期 KD 患儿 MyD88非依赖性途径异常活化可能是导致 KD 免疫功能紊乱的因素之一。
Objective To investigate the role of Toll-like receptor MyD88-independent pathway and its regulatory factors in the pathogenesis of KD. Methods Thirty-two children with KD in acute phase and 16 normal children in the same age group were enrolled. Blood samples were collected from patients with KD before and after intravenous gamma globulin (IVIG) treatment. Monocyte / macrophage (MC) Toll-like receptor 4 and MyD88-independent pathways were detected by reverse transcription-polymerase chain reaction (RT-PCR) and RealTime PCR Toll-Interleukin-1 receptor domain-induced interferon linker (TRIF), TRIF-related linker (TRAM), TANK-binding kinase 1 (TBK- 1), interferon- (IP-10), RANTES, iNOS and so on, as well as the negative regulator of cytokine signaling inhibitor 1 (SOCS-1) The expression of costimulatory molecule CD40 was detected by flow cytometry. The methylation status of SOCS-1 cytosine guanine dinucleotide (CpG) motif was analyzed by methylation-specific fluorescence quantitative PCR. Results (1) The mRNA expression levels of TLR4, TRIF, TRAM, TBK-1 and IFN-β in MC MyD88-independent pathways in children with KD were significantly higher than those in normal controls (P <0.05) The expression of chemokines IP-10, RANTES and iNOS mRNA were significantly increased (P <0.05). (3) The CD40 of costimulatory molecules on MCs in children with acute KD was significantly higher than that of the control group [(6.19 ± 2.25) (2.00 ± 1.37)%, t = 7.98, P <0.05]. The expression of CD40 in KD-CAL ~ + group was significantly higher than that in non-coronary artery group [KD-CAL ~ % (4.12 ± 1.91)%, t = 16.02, P <0.05]. (4) The level of SOCS-1 mRNA in MCs in KD children in acute stage was significantly higher than that in control group [(4.31 ± 0.83) The expression of SOCS-1 mRNA in KD-CAL ~ + group was significantly lower than that in KD-CAL group [(3) vs. (1.09 ± 0.23) × 10-3, t = 20.43, P < 5.73 ± 1.04) × 10 -3 (1.94 ± 0.46) × 10 -3, t = 14.15, P <0.05]. (5) The CpG sequence of SOCS-1 gene in children with acute KD The methylation level of SOCS-1 in KD-CAL ~ + group was significantly lower than that in KD group (26.9 ± 8.6% vs. 5.9 ± 1.4%, t = 13.46, P <0.05) - [(35.1 ± 10.3)% vs. (13.2 ± 3.7)%, t = 8.63, P <0.05] . Conclusion The abnormal activation of MyD88-independent pathways in children with acute KD may be one of the factors leading to KD immune dysfunction.