Notch1信号调控调节性T细胞在川崎病中的作用  

Effects of Notch1 signaling on regulatory T cells and coronary artery lesions in childhood Kawasaki disease

作　　者：郭育鑫 杨莉 王国兵[2] 温鹏强[2] 齐中香[3] 徐明国[4] 刘琮[4] 李成荣[3] Guo Yuxin;Yang Li;Wang Guobing;Wen Pengqiang;Qi Zhongxiang;Xu Mingguo;Liu Cong;Li Chengrong(Department of Pharmacy,Shenzhen Children's Hospital,Guangdong 518038,China;Shenzhen Institute of Pediatrics,Shenzhen Children's Hospital,Guangdong 518038,China;Department of Rheumatology,Shenzhen Children's Hospital,Guangdong 518038,China;Department of Cardiology,Shenzhen Children's Hospital,Guangdong 518038,China)

机构地区：[1]广东省深圳市儿童医院药剂科,深圳518038 [2]广东省深圳市儿童医院儿科研究所,深圳518038 [3]广东省深圳市儿童医院风湿免疫科,深圳518038 [4]广东省深圳市儿童医院心血管内科,深圳518038

出　　处：《中华风湿病学杂志》2022年第10期649-656,I0002,共9页Chinese Journal of Rheumatology

基　　金：国家自然科学基金(81870364,81102227);广东省深圳市科技计划(JCYJ2018022-8175700-233,JCYJ20150403100317055);广东省高水平医院建设专项经费(ynkt2021-zz17)。

摘　　要：目的探讨Notch1信号对川崎病患儿调节性T细胞(Treg)的影响及在血管损伤机制中的作用。方法以2019年3月至2021年6月收治的42例川崎病患儿为研究对象,分别于急性期及输注丙种球蛋白(IVIG)治疗后取样送检,对照组为32名同年龄健康儿童。采用流式细胞术检测外周静脉血CD4^(+)CD25hiFoxp3^(+)Treg数量及叉头螺旋翼状转录因子(Foxp3)、细胞毒性T细胞相关抗原4(CTLA4)、糖皮质激素诱导的肿瘤坏死因子受体(GITR)、Notch1蛋白表达水平;免疫共沉淀-定量PCR技术鉴定CD4^(+)T细胞Foxp3基因启动子组蛋白H4乙酰化(H4Ac)及Notch1受体胞内结合域1(NICD1)、重组信号结合蛋白J(RBP-J)、p300结合水平;荧光定量PCR分析早老素1(PSEN1)、主导控制样蛋白1(MAML1)、RBP-J和Foxp3 mRNA表达;ELISA检测血浆中IL-10、TGF-β蛋白浓度。采用t检验、Pearson相关分析法进行统计分析。结果①与健康对照组相比,急性期川崎病患儿CD4^(+)CD25hiFoxp3^(+)Treg比例显著降低[(4.3±1.5)%和(7.9±2.9)%;t=6.41,P<0.001],分化及功能相关分子(Foxp3、CTLA4、GITR)表达水平和血浆IL-10、TGF-β蛋白浓度明显下降(t=6.87,P<0.001;t=4.26,P<0.001;t=7.88,P<0.001;t=8.42,P<0.001;t=13.01,P<0.001),其中合并冠状动脉损伤组(CAL)前述6项指标低于无冠状动脉损伤组(NCAL)[t=5.83,P<0.001;t=3.83,P<0.001;t=3.28,P=0.002;t=5.05,P<0.001;t=5.96,P<0.001;t=5.17,P<0.001],治疗后显著上调[t=7.13,P<0.001;t=6.10,P<0.001;t=4.31,P<0.001;t=6.55,P<0.001;t=7.40,P<0.001;t=7.84,P<0.001]。②急性期川崎病患儿Foxp3基因启动子H4Ac修饰及NICD1、p300结合水平明显低于同年龄对照组(t=10.25,P<0.001;t=6.93,P<0.001;t=6.75,P<0.001),其中CAL组Foxp3基因启动子H4Ac及NICD1、p300结合水平低于NCAL组(t=6.08,P<0.001;t=2.66,P=0.011;t=6.02,P<0.001),治疗后明显上调(t=7.72,P<0.001;t=4.16,P<0.001;t=5.76,P<0.001)。Pearson相关分析显示Foxp3基因启动子H4Ac与其mRNA水平呈正相关(r=0.47,P<0.001)。各组间Foxp3/RBP-J结合水�Objective To explore the effect of Notch1 signaling on regulatory T cells and its roles in vascular damage in patients with Kawasaki disease(KD).Methods A total of 42 children with KD were enrolled in the present study from March 2019 to June 2020,as 32 age-matched healthy children were recruited as control.The proportions of CD4^(+)CD25hiFoxp3^(+)regulatory T cells(Treg)and expressions of transcription factor forkhead box protein 3(Foxp3),cytotoxic T lymphocyte associated antigen-4(CTLA4),glucocorticoid-induced tumor necrosis factor receptor(GITR),and Notch1 protein were evaluated by flow cytometry.Chromatin immunoprecipitation was conducted to detect acetylation level of histone H4(H4Ac)associated with the promoter of Foxp3 gene and its binding abilities of Notch1 intracellular domain 1(NICD1),recombination signal binding protein for immunoglobulin kappa J region(RBP-J)and p300 in CD4^(+)T cells.Transcription levels of Foxp3,presenilin 1(PSEN1),mastermind like transcriptional coactivator 1(MAML1),and RBP-J in CD4^(+)T cells were determined by real-time polymerase chain reaction(PCR).Concentrations of interleukin(IL)-10 and transforming growth factor-β(TGF-β)in plasma and culture supernatant stimulated with Jagged1 were measured by enzyme linked immunosorbent assay.Independent-sample t-test,Pearson correlation analysis was used as the statistical method in this study.Results①The frequencies of Treg in acute KD patients decreased significantly[(4.3±1.5)%vs(7.9±2.9)%;t=6.41,P<0.001],as protein levels of Foxp3,CTLA4 and GITR and concentrations of IL-10 and TGF-βin plasma reduced remarkably in acute KD patients(t=6.87,P<0.001;t=4.26,P<0.001;t=7.88,P<0.001;t=8.42,P<0.001;t=13.01,P<0.001).All parameters afore-mentioned in patients combined with coronary artery lesions(CAL)were lower than those of patients without coronary artery lesions(NCAL)(t=5.83,P<0.001;t=3.83,P<0.001;t=3.28,P=0.002;t=5.05,P<0.001;t=5.96,P<0.001;t=5.17,P<0.001),and increased after therapy(t=7.13,P<0.001;t=6.10,P<0.001;t=4.31,P<0.001;t=6.55,

关 键 词：黏膜皮肤淋巴结综合征 受体 Notch1 T淋巴细胞 调节性 叉头转录因子类 

分 类 号：R725.4[医药卫生—儿科]