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系统性硬化症血清细胞因子表达谱变化及调控机制
引用本文:朱红林,杜倩,谌威霖,左晓霞,李全贞,刘思佳. 系统性硬化症血清细胞因子表达谱变化及调控机制[J]. 北京大学学报(医学版), 2019, 51(4): 716-722. DOI: 10.19723/j.issn.1671-167X.2019.04.021
作者姓名:朱红林  杜倩  谌威霖  左晓霞  李全贞  刘思佳
作者单位:中南大学湘雅医院风湿免疫科,长沙,410008;中南大学湘雅医院风湿免疫科,长沙,410008;中南大学湘雅医院风湿免疫科,长沙,410008;中南大学湘雅医院风湿免疫科,长沙,410008;中南大学湘雅医院风湿免疫科,长沙,410008;中南大学湘雅医院风湿免疫科,长沙,410008
基金项目:国家自然科学基金(81671622);国家自然科学基金(81771765);湖南省自然科学基金(2018JJ3823)
摘    要:目的:分析系统性硬化症(systemic sclerosis,SSc)血清细胞因子表达谱,探讨其可能的调控机制。方法:收集30例SSc 患者及80例正常对照组血清和外周血单个核细胞DNA,按照SSc有无合并肺间质病变(interstitial lung disease, ILD)分为SSc合并ILD组及SSc不合并ILD组,根据皮肤受累程度,分为弥漫性系统性硬化症(diffuse cutaneous scleroderma,dcSSc)组和局限性系统性硬化症(limited cutaneous scleroderma,lcSSc)组,根据SSc患者血清中是否存在抗拓扑异构酶-1抗体(即抗Scl-70抗体), 分为SSc Scl-70(+)组及SSc Scl-70(-)组。使用Luminex MAGPIX检测系统和Bio-Plex Pro Human Cytokine 27-plex Assay试剂盒检测血清中的27种细胞因子:白细胞介素(interleukin,IL)1β(IL-1β)、IL-1受体拮抗剂(interleukin-1 receptor antagonist,IL-1ra)、IL-2、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12P70、IL-13、IL-15、IL-17、碱性纤维生长因子(basic fiber growth factor,BASIC FGF)、嗜酸性粒细胞趋化因子(eotaxin)、粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)、干扰素γ(interferon-γ,IFN-γ)、人干扰素诱导蛋白10(interferon-gamma induced protein 10,IP-10)、单核细胞趋化蛋白-1(monocyte chemotactic protein 1, MCP-1)、人巨噬细胞炎性蛋白1α(macrophage inflammatory protein-1α,MIP-1α)、人巨噬细胞炎性蛋白1β(macrophage inflammatory protein 1β,MIP-1β)、人血小板衍生生长因子BB (platelet-derived growth factor BB,PDGF-BB)、调节激活正常T细胞表达和分泌细胞因子(regulated on activation in normal T-cell expressed and secreted,RANTES)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)。利用Illumina 450K甲基化芯片检测单个核细胞DNA全基因组甲基化位点变化。结果:与正常对照组相比较,12种细胞因子(BASIC FGF、eotaxin、G-CSF、GM-CSF、IFN-γ、IL-1β、IL-1RA、IL-6、IP-10、MCP-1、TNF-α和RANTES)在SSc中表达明显升高(P<0.05), IL-5在SSc中表达降低(P<0.05),其余细胞因子表达差异无统计学意义。与lcSSc组比较, 9种细胞因子(eotaxin、IL-5、MCP-1、IL-2、RANTES、IL17A、IL-8、MIP-1β和PDGF-BB)在dcSSc组增高,但差异无统计学意义。与SSc不合并ILD组相比较,IL-15 在SSc合并ILD组增高[18.2(172.97) ng/L vs. 2.03(0.05) ng/L,P<0.05];与SSc Scl-70(-)组相比较,IP-10在SSc Scl-70(+)组表达降低[1 030(2 196.6) ng/L vs. 1 878(2 964) ng/L,P<0.05]。分析血清细胞因子与红细胞沉降率(erythrocyte sedimentation rate,ESR)、C反应蛋白(C-reactive protein,CRP)的相关性发现,IL-6与ESR正相关(r= 0.04, P= 0.017);MCP-1(r =0.49, P =0.043)和MIP-1β(r =0.41, P =0.007)与CRP正相关。分析细胞因子甲基化位点变化,发现IL-10 TSS1500区的cg17744604、IL-12P70TSS200区的cg06111286、IL-1β TSS200区的cg07935264、IL-1 ra TSS1500区的cg01467417、IL-1 ra 5'非翻译区的cg03989987和VEGF TSS200区的cg21099624 均呈低甲基化。结论:SSc患者血清存在多种细胞因子变化,且细胞因子变化与皮肤受损程度、肺纤维化有关,多种细胞因子表达受甲基化调控。

关 键 词:系统性硬化症  细胞因子  全基因组甲基化
收稿时间:2019-01-11

Altered serum cytokine expression profile in systemic sclerosis and its regulatory mechanisms
Hong-lin ZHU,Qian DU,Wei-lin CHEN,Xiao-xia ZUO,Quan-zhen LI,Si-jia LIU. Altered serum cytokine expression profile in systemic sclerosis and its regulatory mechanisms[J]. Journal of Peking University. Health sciences, 2019, 51(4): 716-722. DOI: 10.19723/j.issn.1671-167X.2019.04.021
Authors:Hong-lin ZHU  Qian DU  Wei-lin CHEN  Xiao-xia ZUO  Quan-zhen LI  Si-jia LIU
Affiliation:Department of Rheumatology and Immunology, Xiangya Hospital, Central South University, Changsha 410008, China
Abstract:Objective: To analyze the expression profile of serum cytokines in patients with systemic sclerosis (SSc) and explore its possible regulatory mechanisms.Methods: Serum and DNA of peripheral blood mononuclear cells were collected from 30 SSc patients and 80 normal controls (NCs). According to the presence or absence of interstitial lung disease (ILD) in SSc, the patients were divided into SSc with ILD group and SSc without ILD group. According to the degree of skin involvement, the patients were divided into diffuse systemic scleroderma (dcSSc) group and limited systemic scleroderma (lcSSc) group. According to the presence of anti-topoisomerase-1 antibody (anti-Scl-70 antibody) in the serum of patients with SSc, they were divided into SSc Scl-70 (+) group and SSc Scl-70 (-) group. 27 cytokines in serum were detected by Luminex MAGPIX detection system and Bio-Plex Pro Human Cytokine 27-plex Assay kit: interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12P70, IL-13, IL-15, IL-17, basic fiber growth factor (BASIC FGF), eotaxin, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN-γ), interferon-gamma induced protein 10(IP-10), monocyte chemotactic protein 1(MCP-1), macrophage inflammatory protein-1α(MIP-1α), macrophage inflammatory protein 1β(MIP-1β), platelet-derived growth factor BB (PDGF-BB), regulated on activation in normal T-cell expressed and secreted (RANTES), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor(VEGF). Methylation sites were detected by Illumina 450K methylation chip.Results: Compared with NCs group, the expression of 12 cytokines (BASIC FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-6, IP-10, MCP-1, TNF-α and RANTES) in the SSc group significantly increased (P<0.05), IL-5 was decreased expression in the SSc group (P<0.05), there was no signi-ficant difference in the expressions of the other 14 cytokines. Compared with lcSSc group, 9 cytokines (eotaxin, IL-5, MCP-1, IL-2, RANTES, IL17A, IL-8, MIP-1β and PDGF-BB) increased in dcSSc group, but there was no significant difference. Compared with SSc without ILD group, IL-15 increased in SSC with ILD group [18.2(172.97) ng/L vs. 2.03(0.05) ng/L, P<0.05]. Compared with SSc Scl-70 (-) group, the expression of IP-10 decreased in SSc Scl-70 (+) group [1 030 (2 196.6) ng/L vs. 1 878 (2 964) ng/L, P<0.05]. The correlation analysis of serum cytokines with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) showed that IL-6 was positively correlated with ESR (r =0.04, P= 0.017), MCP-1 (r= 0.49, P= 0.043) and MIP-1β (r= 0.41, P= 0.007) positively correlated with CRP. By analyzing the changes of methylation sites of cytokines, it was found that cg17744604 in IL-10 TSS1500 region, cg06111286 in IL-12P70 TSS200 region, cg07935264 in IL-1 β TSS200 region, cg01467417 in IL-1ra TSS1500 region, cg03989987 in IL-1ra 5'UTR region and cg21099624 in VEGF TSS200 region were all hypomethylated.Conclusion: There were different cytokines expression profiles in the serum of SSc patients, and the altered cytokines were correlected with the degree of skin damage and pulmonary fibrosis. Many cytokines were regulated by methylation.
Keywords:Systemic sclerosis  Cytokine  Genome-wide methylation  
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