Abstract
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。
Keywords: 系统性硬化症, 细胞因子, 全基因组甲基化
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
系统性硬化症(systemic sclerosis,SSc)是一种病因不明的高度异质性的自身免疫病,其主要特征是微血管功能障碍、慢性炎症、免疫异常以及皮肤和内脏器官进行性纤维化。SSc患病率约为1/10 000,在风湿病中病死率最高[1]。肺间质病变(interstitial lung disease,ILD)和肺动脉高压是导致SSc死亡的主要并发症[1,2]。SSc根据皮肤受累程度,分为弥漫性系统性硬化症(diffuse cutaneous scleroderma,dcSSc)和局限性系统性硬化症(limited cutaneous scleroderma,lcSSc)两种主要亚型。dcSSc皮肤受损广泛,常累及四肢、躯干和腹壁,病情进展快,发病5年内容易出现脏器衰竭。lcSSc皮肤受损局限于肢体末端或面部,或仅累及手指(指端硬化),病情进展缓慢[3]。SSc发病机制复杂,多种细胞因子参与纤维化的发生发展,如血小板源性生长因子、内皮素1、白介素(interleukin, IL)-6和IL-13等,但目前由于SSc血清细胞因子受疾病病程、治疗药物、样本量和检测方法的影响,其表达变化在各研究中报道不一致[4,5,6]。本研究采用Luminex MAGPIX检测系统对SSc血清中27种细胞因子进行高通量检测,并将其表达量与SSc亚型、临床指标进行相关性分析。
在前期研究中,本课题组利用高通量芯片技术对SSc 外周血单个核细胞进行全基因组DNA甲基化分析[7]。本研究中,进一步将细胞因子的表达与全基因组DNA甲基化进行整合分析,以探讨细胞因子的调控机制。
1. 资料与方法
1.1. 研究对象
收集中南大学湘雅医院2013年12月至2016年6月入院的SSc患者30例,28例汉族,1例苗族,1例瑶族。男性11例,女性19例。年龄为13~64岁,平均年龄(44.23±12.86)岁,病程1个月至9年。根据SSc患者肺部影像学高分辨CT表现诊断患者是否合并ILD,分为SSc合并ILD组[SSc ILD(+)组]及SSc不合并ILD组[SSc ILD(-)组],各15例;根据皮肤受累程度,分为dcSSc组20例和lcSSc组10例;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20例及SSc Scl-70(-)组10例。80例正常对照(normal controls,NCs)均为汉族人,男性30例,女性50例,年龄为21~80岁,平均年龄(38.58±13.73)岁。所有SSc患者均符合1980年美国风湿病学会制定的分类标准[8],该标准包括以下条件:(1)主要条件:近端皮肤硬化,即手指及掌指(跖趾)关节近端皮肤增厚、紧绷和肿胀,可累及整个肢体、面部、颈部和躯干(胸、腹部)。(2)次要条件:①指硬化:上述皮肤改变仅限手指。②指尖凹陷性瘢痕或指垫消失:由于缺血导致指尖凹陷性瘢痕或指垫消失。③双肺基底部纤维化:在立位胸部X线片上,可见条状或结节状致密影,以双肺底为著,也可呈弥漫斑点或蜂窝状肺,但应除外原发性肺病所引起的这种改变。符合上述主要条件或次要条件2条及2条以上者,可诊断为SSc。NCs来自同时期来中南大学湘雅医院体检的健康志愿者。本研究通过中南大学伦理委员会审批(批准号:201303293), 所有研究对象均签署知情同意书。
1.2. 方法
1.2.1 细胞因子芯片分析 本实验收集30例SSc 患者及80例正常对照组血清,采用Luminex MAGPIX检测系统(MAGPIX,Luminex公司,美国)和Bio-Plex Pro Human Cytokine 27-plex Assay试剂盒 (Bio-Plex,Bio-Rad公司,美国), 分析血清样本中的27种细胞因子: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)。使用xPONENT 4.2 软件 (Luminex公司,美国)绘制标准曲线并计算细胞因子的浓度(ng/L)。
1.2.2 全基因组DNA甲基化分析 全基因组DNA甲基化分析方法详见本课题组已发表文章[7]。本实验提取30例SSc 患者及80例NCs外周血单个核细胞DNA,利用Illumina 450K甲基化芯片(Infi-nium,Illumina公司,美国)检测全基因组485 000个CpG位点。实验步骤如下:使用EZ DNA 甲基化试剂盒(Zymo Research Corp,Zymo Research公司,美国)将1 μg外周血单核细胞DNA进行转化、染色和扫描。利用R 3.5.1软件包 (Bell Laboratories实验室,美国)进行数据提取和归一化处理。归一化处理后的数据使用SAM软件(斯坦福大学,美国)进行分析,P <0.05被认为是有差异的甲基化位点。全基因组DNA甲基化程度(β值)用0~1之间的数值表示,0代表完全未甲基化,1代表完全甲基化。基因转录本可分为几个功能区,分别为TSS200(转录起始位点到上游200 nt)、TSS1500(转录起始位点到上游200 nt至1 500 nt)、5'非翻译区(5'UTR)、第1外显子区、基因主体和3'非翻译区(3'UTR)。
1.3. 统计学分析
使用Perseus 1.5.5.0软件(Max Planck Institute of Biochemistry,德国)和Graphpad Prism 7软件(GraphPad Software公司,美国)进行统计分析,正态分布的计量资料两两比较采用两样本t检验,非正态分布资料组间比较采用Mann-Whitney U检验,细胞因子与红细胞沉降率(erythrocyte sedimentation rate,ESR)或C反应蛋白(C-reactive protein,CRP)的相关性分析采用Spearman相关性分析。细胞因子表达值数据用中位数(四分位距)[ Median(IQR)]表示,全基因组DNA甲基化程度用均值(Mean)表示, P<0.05认为差异具有统计学意义。
2. 结果
2.1. 细胞因子在SSc中的表达变化
首先对NCs组和SSc组细胞因子表达谱进行比较,在27种细胞因子中,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,IL-5在SSc中表达降低,差异有统计学意义(P<0.05);进一步比较dcSSc组和lcSSc组细胞因子表达谱变化,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(表1)。
1.
血清细胞因子表达谱在SSc、dcSSc和lcSSc组中的变化/(ng/L), 中位数(四分位距)
Altered serum cytokine expression profiles in SSc, dsSSc and lcSSc groups/(ng/L), Median (IQR)
| Cytokine | NCs (n=80) | SSc (n=30) |
P value (NC vs. SSc) |
lcSSc (n=10) | dcSSc (n=20) |
P value (lcSSc vs. dcSSc) |
| SSc, systemic sclerosis; dcSSc, diffuse cutaneous scleroderma; lcSSc, limited cutaneous scleroderma; NCs, normal controls; BASIC FGF, basic fiber growth factor; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; IL-1ra, interleukin-1 receptor antagonist; IP-10, interferon-gamma induced protein 10; MCP-1, monocyte chemotactic protein 1; TNF-α, tumor necrosis factor α; RANTES, regulated on activation in normal T-cell expressed and secreted; VEGF, vascular endothelial growth factor; MIP-1α, macrophage inflammatory protein-1α; MIP-1β, macrophage inflammatory protein 1β; PDGF-BB, platelet-derived growth factor BB. | ||||||
| BASIC FGF | 64.27 (42.37) | 88.51 (46.58) | 0.002 | 94.07 (49.47) | 86.70 (48.08) | >0.05 |
| Eotaxin | 145.90 (89.30) | 178.80 (155.70) | 0.039 | 139.40 (127.30) | 188.60 (156.60) | >0.05 |
| G-CSF | 75.87 (58.45) | 113.90 (104.84) | <0.001 | 122.40 (149.89) | 108.00 (82.50) | >0.05 |
| GM-CSF | 30.75 (57.51) | 53.17 (124.65) | 0.039 | 106.50 (96.09) | 42.76 (134.16) | >0.05 |
| IFN-γ | 65.50 (75.52) | 198.60 (149.60) | <0.001 | 259.20 (224.60) | 194.90 (116.90) | >0.05 |
| IL-5 | 8.76 (23.13) | 3.28 (4.27) | 0.001 | 3.28 (10.23) | 4.63 (4.57) | >0.05 |
| IL-1β | 1.62 (27.08) | 6.58 (6.64) | <0.001 | 7.69 (5.82) | 5.46 (10.36) | >0.05 |
| IL-1ra | 143.70 (177.36) | 481.60 (613.10) | <0.001 | 541.40 (514.60) | 359.50 (399.10) | >0.05 |
| IL-6 | 21.58 (32.72) | 35.92 (31.39) | 0.002 | 34.49 (40.10) | 33.86 (21.01) | >0.05 |
| IP-10 | 766.00 (523.40) | 1 307.00 (2 506.40) | 0.002 | 2 096.00 (2 885.00) | 1 094.00 (2 081.60) | >0.05 |
| MCP-1 | 118.30 (73.10) | 229.60 (319.40) | <0.001 | 180.30 (284.18) | 261.00 (548.40) | >0.05 |
| TNF-α | 43.17 (33.35) | 62.64 (43.74) | <0.001 | 63.43 (61.72) | 55.81 (39.60) | >0.05 |
| RANTES | 23 404.00 (27 482.00) | 52 632.00 (75 694.00) | 0.019 | 42 802.00 (80 383.00) | 71 752.00 (75 270.00) | >0.05 |
| IL-2 | 37.29 (54.92) | 5.04 (30.77) | 0.053 | 4.48 (36.44) | 5.04 (29.21) | >0.05 |
| VEGF | 153.10 (139.00) | 204.00 (278.90) | 0.052 | 304.80 (338.20) | 150.30 (285.10) | >0.05 |
| IL-10 | 19.43 (30.78) | 19.84 (15.76) | >0.05 | 23.91 (36.54) | 18.92 (12.44) | >0.05 |
| IL-15 | 13.88 (46.35) | 2.03 (20.51) | >0.05 | 2.03 (7.33) | 2.03 (42.40) | >0.05 |
| IL-17A | 92.95 (108.01) | 127.20 (64.19) | >0.05 | 119.00 (76.45) | 143.50 (97.83) | >0.05 |
| MIP-1α | 7.46 (23.51) | 6.76 (4.90) | >0.05 | 7.56 (5.43) | 6.66 (4.48) | >0.05 |
| IL-4 | 7.30 (17.02) | 5.92 (3.51) | >0.05 | 6.39 (6.85) | 5.98 (4.23) | >0.05 |
| IL-7 | 23.91 (19.28) | 19.26 (15.27) | >0.05 | 20.67 (29.32) | 19.79 (13.86) | >0.05 |
| IL-8 | 48.05 (55.05) | 41.05 (32.61) | >0.05 | 36.59 (54.53) | 42.06 (31.75) | >0.05 |
| IL-9 | 35.19 (71.11) | 36.04 (32.79) | >0.05 | 43.78 (52.64) | 35.57 (26.95) | >0.05 |
| IL-13 | 21.49 (23.94) | 17.51 (43.08) | >0.05 | 21.25 (88.46) | 16.15 (15.30) | >0.05 |
| MIP-1β | 317.80 (280.30) | 290.60 (260.70) | >0.05 | 254.30 (273.70) | 324.60 (231.60) | >0.05 |
| IL-12P70 | 45.26 (25.25) | 45.89 (45.64) | >0.05 | 57.69 (43.41) | 41.08 (43.54) | >0.05 |
| PDGF-BB | 4 623.00 (2 680.00) | 4 701.00 (2 770.00) | >0.05 | 3 911.00 (2 521.00) | 5 598.00 (3 036.00) | >0.05 |
2.2. 细胞因子表达谱在SSc ILD(+)、SSc Scl-70(+)组中的变化
与SSc ILD(-)组相比较,IL-15在SSc ILD(+)组表达增高,差异有统计学意义(P<0.05)。与SSc Scl-70(-)组相比较,IP-10在SSc Scl-70(+)组表达降低,差异有统计学意义(P<0.05,表2)。
2.
血清细胞因子表达谱在SSc ILD(+)、SSc Scl-70(+)组中的变化/(ng/L), 中位数(四分位距)
Altered serum cytokine expression profiles in SSc ILD(+)、SSc Scl-70(+) groups/(ng/L), Median (IQR)
| Cytokine | SSc ILD(-) (n=15) |
SSc ILD(+) (n=15) |
P value [ILD(-) vs. ILD(+)] |
SSc Scl-70(-) (n=10) |
SSc Scl-70(+) (n=20) |
P value [Scl-70(-) vs. Scl-70(+)] |
| ILD, interstitial lung disease; other abbreviations as in Table 1. | ||||||
| BASIC FGF | 88.51 (42.70) | 99.08 (51.46) | >0.05 | 77.49 (40.47) | 89.70 (50.58) | >0.05 |
| Eotaxin | 172.10 (140.60) | 185.40 (195.20) | >0.05 | 158.40 (402.00) | 188.60 (179.00) | >0.05 |
| G-CSF | 122.40 (128.54) | 112.00 (97.04) | >0.05 | 105.40 (230.59) | 115.90 (89.78) | >0.05 |
| GM-CSF | 52.97 (86.94) | 121.60 (136.55) | >0.05 | 49.65 (143.35) | 53.17 (122.49) | >0.05 |
| IFN-γ | 205.90 (131.00) | 184.00 (188.80) | >0.05 | 238.10 (943.10) | 187.70 (114.80) | >0.05 |
| IL-12P70 | 36.59 (44.73) | 47.48 (34.56) | >0.05 | 50.77 (186.11) | 45.89 (46.69) | >0.05 |
| IL-1β | 6.30 (5.34) | 9.23 (23.73) | >0.05 | 6.58 (40.19) | 7.05 (6.57) | >0.05 |
| IL-1ra | 460.40 (508.50) | 502.90 (792.10) | >0.05 | 500.90 (6421.90) | 456.30 (524.80) | >0.05 |
| IL-6 | 35.74 (16.38) | 36.27 (119.28) | >0.05 | 27.34 (170.27) | 36.36 (33.49) | >0.05 |
| IP-10 | 1 418.00 (2 969.90) | 1 182.00 (2 306.60) | >0.05 | 1 878.00 (2 964.00) | 1 030.00 (2 196.60) | 0.039 |
| MCP-1 | 180.40 (179.90) | 323.10 (571.20) | >0.05 | 175.00 (216.12) | 296.90 (499.10) | >0.05 |
| TNF-α | 71.00 (39.17) | 52.80 (118.41) | >0.05 | 51.18 (315.81) | 69.48 (41.94) | >0.05 |
| VEGF | 193.70 (210.20) | 298.10 (388.80) | >0.05 | 275.20 (428.40) | 176.30 (232.30) | >0.05 |
| IL-13 | 24.73 (83.62) | 15.12 (36.66) | >0.05 | 18.94 (76.56) | 17.51 (55.58) | >0.05 |
| IL-7 | 18.19 (18.71) | 19.61 (12.01) | >0.05 | 18.02 (57.74) | 21.20 (15.01) | >0.05 |
| RANTES | 45 504.00 (74 609.00) | 59 759.00 (77 217.00) | >0.05 | 78 450.00 (76 739.00) | 38 953.00 (75 650.00) | >0.05 |
| IL-10 | 18.28 (11.81) | 23.40 (13.45) | >0.05 | 14.85 (156.50) | 21.64 (14.77) | >0.05 |
| IL-15 | 2.03 (0.05) | 18.20 (172.97) | 0.024 | 2.03 (63.31) | 2.03 (25.19) | >0.05 |
| IL-17A | 122.30 (66.79) | 145.20 (112.53) | >0.05 | 123.90 (43.10) | 134.50 (73.82) | >0.05 |
| IL-2 | 2.84 (16.57) | 5.95 (34.50) | >0.05 | 4.36 (180.43) | 5.08 (29.21) | >0.05 |
| MIP-1α | 6.76 (3.18) | 6.46 (20.36) | >0.05 | 6.51 (6.51) | 7.74 (4.39) | >0.05 |
| IL-4 | 6.23 (4.63) | 5.72 (3.04) | >0.05 | 6.05 (10.81) | 5.85 (3.51) | >0.05 |
| IL-5 | 3.21 (3.90) | 3.34 (4.84) | >0.05 | 4.09 (10.88) | 3.21 (3.21) | >0.05 |
| IL-8 | 37.45 (21.89) | 49.71 (47.20) | >0.05 | 40.31 (58.24) | 41.05 (28.72) | >0.05 |
| IL-9 | 36.22 (35.10) | 35.86 (30.29) | >0.05 | 33.01 (30.67) | 38.56 (29.77) | >0.05 |
| MIP-1β | 260.70 (285.40) | 310.60 (420.10) | >0.05 | 312.30 (293.10) | 290.60 (243.80) | >0.05 |
| PDGF-BB | 4 359.00 (3 072.00) | 5 043.00 (3 128.00) | >0.05 | 4 827.00 (3 340.00) | 4 701.00 (2 958.00) | >0.05 |
2.3. SSc血清细胞因子表达谱与ESR、CRP的相关性分析
血清27种细胞因子与ESR相关性分析发现,17种细胞因子(BASIC FGF、eotaxin、G-CSF、GM-CSF、IFN-γ、IL-10、IL-12P70、IL-1β、IL-1ra、IL-2、VEGF、TNF-α、IL-13、IL-4、IL-5、IL-7和IL-9)与ESR负相关,IL-6与ESR正相关,其余细胞因子与ESR无相关性;血清27种细胞因子与CRP相关性分析发现,IL-13和PDGF-BB与CRP负相关;MCP-1和MIP-1β与CRP正相关,且相关性较大(表3), 其余细胞因子与CRP无相关性。
3.
SSc血清细胞因子表达谱与ESR、CRP的相关性
The correlation of serum cytokine expression profile with ESR, CRP in SSc
| Cytokine | ESR | CRP | ||
| r | P | r | P | |
| ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; other abbreviations as in Table 1. | ||||
| BASIC FGF | -0.09 | 0.009 | 0.12 | >0.05 |
| Eotaxin | -0.12 | 0.013 | 0.15 | >0.05 |
| G-CSF | -0.11 | 0.004 | 0.19 | >0.05 |
| GM-CSF | -0.04 | 0.012 | 0.05 | >0.05 |
| IFN-γ | -0.07 | 0.010 | 0.10 | >0.05 |
| IL-10 | -0.09 | 0.002 | 0.25 | >0.05 |
| IL-12P70 | -0.19 | <0.001 | 0.16 | >0.05 |
| IL-15 | 0.09 | >0.05 | 0.08 | >0.05 |
| IL-17A | 0.05 | >0.05 | 0.14 | >0.05 |
| IL-1β | -0.06 | 0.008 | 0.23 | >0.05 |
| IL-1ra | -0.02 | 0.011 | 0.10 | >0.05 |
| IL-2 | -0.31 | <0.001 | 0.07 | >0.05 |
| IL-6 | 0.04 | 0.017 | 0.30 | >0.05 |
| IP-10 | -0.02 | >0.05 | 0.09 | >0.05 |
| MCP-1 | 0.09 | >0.05 | 0.49 | 0.043 |
| MIP-1α | -0.05 | >0.05 | 0.38 | >0.05 |
| TNF-α | -0.06 | 0.011 | 0.16 | >0.05 |
| VEGF | -0.13 | 0.024 | 0.17 | >0.05 |
| IL-13 | -0.35 | <0.001 | -0.07 | 0.050 |
| IL-4 | -0.2 | 0.005 | 0.04 | >0.05 |
| IL-5 | -0.07 | 0.034 | 0.06 | >0.05 |
| IL-7 | -0.04 | 0.006 | 0.17 | >0.05 |
| IL-8 | 0.09 | >0.05 | 0.32 | >0.05 |
| IL-9 | -0.21 | 0.001 | 0.30 | >0.05 |
| MIP-1β | -0.02 | >0.05 | 0.41 | 0.007 |
| PDGF-BB | -0.04 | >0.05 | -0.01 | 0.042 |
| RANTES | 0.17 | >0.05 | 0.02 | >0.05 |
2.4. 细胞因子的DNA甲基化表达调控
进一步分析上述SSc中差异表达的细胞因子的DNA甲基化位点变化,发现位于IL-10 TSS1500区的cg17744604、IL-12P70 TSS200区的cg06111286、IL-1β TSS200 区的cg07935264、IL-1ra TSS1500区的cg01467417、IL-1ra 5'UTR区 的cg03989987和VEGF TSS200区的cg21099624 均呈低甲基化(表4)。
4.
细胞因子甲基化表达的改变
Altered methylation expression of cytokines
| Cytokine | Gene | Methylation sites | NCs β (Mean) | SSc β (Mean) | Δβ | P value | Gene locus | Chromosomal location |
| SSc, systemic sclerosis; NCs, normal controls; IL, interleukin; IL-1ra, interleukin-1 receptor antagonist; VEGF, vascular endothelial growth factor. | ||||||||
| IL-10 | IL10 | cg17744604 | 0.403 | 0.297 | -0.106 | <0.001 | TSS1500 | 1 |
| IL-12P70 | IL12B | cg06111286 | 0.316 | 0.250 | -0.066 | 0.001 | TSS200 | 5 |
| IL-1β | IL1B | cg07935264 | 0.195 | 0.150 | -0.045 | <0.001 | TSS200 | 2 |
| IL-1ra | IL1RN | cg01467417 | 0.127 | 0.108 | -0.019 | 0.014 | TSS1500 | 2 |
| IL-1ra | IL1RN | cg03989987 | 0.301 | 0.238 | -0.063 | 0.006 | 5'UTR | 2 |
| VEGF | VEGFA | cg21099624 | 0.097 | 0.089 | -0.008 | 0.011 | TSS200 | 6 |
3. 讨论
细胞因子是自身免疫和炎症性疾病中的关键炎症介质,在风湿病中,细胞因子不仅在免疫细胞中具有重要调控作用,在非免疫细胞如成纤维细胞、内皮细胞、成骨细胞和破骨细胞等中也均起着重要作用。细胞因子生物学研究的进步为我们了解SSc的发病机制提供了重要帮助[9]。早在1992年,Needleman等[10]发现了IL-2、IL-4和IL-6在SSc中表达增高。Hasegawa等[11]于1997年发现了IL-4、IL-10和IL-13在SSc中表达增高,IL-13水平与ESR、CRP相关。2011年,该团队利用流式微珠阵列细胞因子检测试剂盒分析了10种细胞因子在31例日本SSc患者中的表达水平,发现IP-10、膜结合免疫球蛋白、MCP-1显著性表达增高,IL-6和IL-8表达增高,但差异无统计学意义。IL-2、IL-4、IL-10、TNF-α和IFN-γ在多数样本中未检测到[12]。Dantas等[6]采用酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测了8种细胞因子的表达,发现IL-17A在SSc中增高,但差异无统计学意义,IL-2、 IL-4、IL-10、 IL-6和TNF未检测到。本研究采用Luminex MAGPIX分析了SSc患者血清27种细胞因子表达谱,发现在SSc患者血清中,12种细胞因子在SSc中表达明显升高,IL-5在SSc中表达降低。Luminex MAGPIX检测方法较ELISA更为敏感,我们能检测到所有SSc患者及正常对照组中细胞因子的表达变化,且大部分细胞因子表达水平变化与文献[5]报道相符。本研究还发现细胞因子的表达水平与SSc临床特征相关,有9种细胞因子在dcSSc组中的表达较在lcSSc组中的表达更高,IL-15在SSc ILD(+)组增高,IP-10在SSc Scl-70(+)组表达降低。IL-15是一种多功能的细胞因子,在调节免疫、血管病变和结缔组织分化中具有重要作用。研究报道SSc血清中的IL-15水平增高,且与肺功能受损相关,在早期SSc中,IL-15与SSc纤维化和肺血管疾病及肺血管病变相关[13]。在抗黑色素瘤分化相关基因-5(melanoma differentiation related gene-5,MDA5)抗体阳性的无肌病性皮肌炎合并ILD患者中,血清IL-15水平在死亡患者中显著高于未死亡患者,提示IL-15在抗MDA5抗体阳性的无肌病性皮肌炎肺间质病变中发挥着重要作用[14],结合本研究结果,提示IL-15可能是SSc ILD(+)的重要生物标志物。IP-10是由多种细胞分泌的炎症趋化因子,可通过募集淋巴细胞调节免疫反应并抑制血管新生。IP-10在SSc ILD(+)患者中增高,且与心血管累及程度、肺动脉高压和ESR上升正相关[15]。本研究发现,IP-10与SSc Scl-70(+)存在相关性,但其作用和意义还需进一步研究。ESR和CRP属于非特异性的炎症指标,IL-6、MCP-1和MIP-1β均属于促炎症细胞因子,IL-6是急性炎症反应细胞因子,在类风湿关节炎等多种炎症性疾病中发挥重要作用。MCP-1和MIP-1β属于促炎症趋化因子,广泛调节多种免疫细胞的功能,如巨噬细胞、树突细胞和中性粒细胞等[16]。本研究发现,IL-6、MCP-1和MIP-1β与ESR或CRP相关,提示此类细胞因子可能与SSc疾病活动度相关,可能成为判断SSc疗效的潜在标志物。
本研究结合全基因组甲基化表达谱,分析了细胞因子的DNA甲基化变化,发现IL-10、IL-12P70、IL-1β、IL-1ra和VEGF的转录起始区均存在DNA低甲基化,提示其可能受DNA甲基化调控。由于多数细胞因子在体外不稳定,如前文所述多个研究中利用ELISA方法无法检测到细胞因子的表达。细胞因子DNA甲基化水平与其mRNA表达存在一定相关性,且在体外可长期保存,血标本或尿液均易检测,所需标本量少。如果通过检测细胞因子DNA甲基化水平替代常规血清细胞因子检测,将对SSc的诊断、分类、并发症的预防和预后判断具有更重要的意义[17]。
本研究利用高通量分析方法检测了SSc血清27种细胞因子的表达,并与SSc亚型、临床指标进行相关性分析,最后探讨了细胞因子的表达调控机制。本研究发现13种细胞因子在SSc血清中存在表达变化,IL-15与SSc ILD相关,IP-10与SSc 抗Scl-70抗体相关,IL-6、MCP-1和MIP-1β与ESR、CRP正相关,部分细胞因子受DNA甲基化调控。细胞因子在SSc血管新生、免疫炎症和纤维化中均发挥着重要作用,其表达受多种免疫细胞影响,单一细胞因子可能不足以反映疾病的活动性或并发症。在今后的研究中,联合使用多种细胞因子进行疗效和预后判断对临床将具有更重要的指导意义[18,19,20]。
Funding Statement
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）
Supported by the National Natural Science Foundation of China(81671622); Supported by the National Natural Science Foundation of China(81771765); Hunan Provincial Natural Science Fundation(2018JJ3823)
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