目的探讨Th17细胞在川崎病(Kawasaki disease,KD)免疫发病机制中的作用。方法急性期KD患儿60例,正常同年龄对照组32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。采用荧光定量PCR(real—timePCR)检测CD4^+T细胞IL-17A/F、转录因子ROR-γt、Foxp3及PBMCIL-6、TGF—β、IL-23p19、IL-27p28、IL-27EBI3、IFN-γ等mRNA表达;酶联免疫吸附试验检测血浆中IL-6、TGF—β、IL-23、IL-27、IFN-γ的蛋白浓度;流式细胞术检测外周血CD4^+CD25^+调节性T细胞(Tr)的比例。结果急性期KD患儿CD4^+T细胞高表达IL-17A及IL-17F(P〈0.01),IVIG治疗后明显降低(P〈0.01);急性期KD患儿IL-17A/IL-17F与红细胞沉降率(ESR)、C反应蛋白(CRP)、白细胞数目(WBC)呈正相关(IL-17A:0.70,0.85,0.80,P〈0.01;IL一17F:0.63,0.65,0.69,P〈0.01);急性期KD患儿Th17细胞转录因子ROR-γt及前炎症细胞因子IL-6转录水平明显高于对照组(P〈0.01),IVIG治疗后显著降低(P〈0.01);TGF—β、IL-23p19、IL-27p28、IL-27EBI3mRNA水平及血浆蛋白浓度与对照组比较差异无统计学意义(P〉0.05);血浆IFN-γ浓度显著升高,mRNA水平无变化;急性期KD患儿CD4^+CD25^+Tr细胞比例明显低于正常对照组(P〈0.01),其转录因子Foxp3表达亦明显降低(P〈0.01)。结论急性期KD患儿Th17细胞过度活化可能参与了KD免疫发病机制。
Objective To investigate the changes and the role of Th17 cells in immunological pathogenesis of Kawasaki Disease (KD). Methods Sixty children with KD and thirty-two age-matched healthy children were studied. Real-time PCR were used to evaluate the mRNA levels of IL-17A/F, ROR-γt and Foxp3 in CD4-positive cells, or IL-6, TGF-β, IL-23p19, IL-27p28, IL-27EBI3, IFN-γ in peripheral blood mononuclear cells. Protein levels of cytokines in plasma were measured by ELISA. The proportion of CD4^+CD25^+ regulatory T (Tr) cells was analyzed by flow cytometry. Results Compared with healthy controls, transcription levels of IL-17A/F were significantly up-regulated during acute phase of KD (P 〈 0.01 ), and down-regulated after treated with intravenous immunological globulin(IVIG) therapy. Significant positive correlation between IL-17A/F and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and white blood cells(WBC) were observed during acute phase of Kawasaki disease( IL-17A: 0.70, 0.85, 0.80, P 〈 0.01 ; IL-17F : 0.63, 0.65, 0.69, P 〈 0.01 ). Transcription levels of ROR-γt in CD4 ^+ T cells and proinflammatory cytokine IL-6 in PBMC, and protein level of IL-6 in plasma were significantly increased during acute phase of KD( P 〈 0.01 ), and decreased to some extents after IVIG therapy. No differences of TGF-β, IL-23p19, IL-27p28 and IL-27EBI3 mRNA and protein levels in plasma were detected among all groups( P 〉 0.05 ). The protein level of IFN-γ in plasma was significantly elevated during acute phase of KD(P 〈 0.01 ). No change of IFN-γ, mRNA level was observed( P 〉 0.05 ). The proportion of CD4 ^+ CD25 ^+ Tr cells in peripheral blood from patients with KD was significantly lower than that of healthy subjects ( P 〈 0.01 ), and increase significantly after treatment of IVIG therapy. Transcription level of Foxp3 was significantly down-regulated in acute phase of KD ( P 〈 0.01 ), and up-regulated to some extent after IVIG the