慢性阻塞性肺疾病患者辅助性T细胞17/调节性T细胞变化及其与肺功能的相关性研究
2016-07-25李玲春
李玲春
·论著·
慢性阻塞性肺疾病患者辅助性T细胞17/调节性T细胞变化及其与肺功能的相关性研究
李玲春
610000四川省成都市西区医院呼吸内科
【摘要】目的观察慢性阻塞性肺疾病(COPD)患者辅助性T细胞17(Th17细胞)/调节性T细胞(Treg细胞)变化,探讨其与肺功能的相关性。方法选取2013年9月—2015年9月四川省成都市西区医院收治的老年COPD稳定期患者36例作为稳定期组,COPD急性加重期患者29例作为急性加重期组;另选取同期在本院体检健康者50例作为对照组,其中吸烟者22例作为吸烟对照组,不吸烟者28例作为正常对照组。比较4组受试者外周血Th17细胞分数、Treg细胞分数、Th17细胞/Treg细胞比值,血清IL-17、IL-10水平及肺功能指标〔第1秒用力呼气容积占预计值百分比(FEV1%)、用力肺活量占预计值百分比(FVC%)及第1秒用力呼气容积与用力肺活量比值(FEV1/FVC)〕,并分析外周血Th17细胞分数、Treg细胞分数及Th17细胞/Treg细胞比值与肺功能指标的相关性。结果急性加重期组和稳定期组患者外周血Th17细胞分数、Th17细胞/Treg细胞比值及血清IL-17水平高于吸烟对照组和正常对照组,外周血Treg细胞分数及血清IL-10水平低于吸烟对照组和正常对照组(P<0.05);急性加重期组患者外周血Th17细胞分数、Th17细胞/Treg细胞比值及血清IL-17水平高于稳定期组,外周血Treg细胞分数及血清IL-10水平低于稳定期组(P<0.05);吸烟对照组受试者外周血Th17细胞分数、Th17细胞/Treg细胞比值高于正常对照组(P<0.05)。4组受试者FVC%比较,差异无统计学意义(P>0.05);急性加重期组和稳定期组患者FEV1%、FEV1/FVC低于吸烟对照组和正常对照组,急性加重期组患者FEV1%、FEV1/FVC低于稳定期组(P<0.05)。Pearson相关性分析结果显示,外周血Th17细胞分数与FEV1、FEV1%/FVC呈负相关(r值分别为-0.736、-0.694,P<0.05),Th17/Treg细胞比值与FEV1、FVC%、FEV1%/FVC呈负相关(r值分别为-0.892、-0.222、-0.748,P<0.01),而外周血Treg细胞分数与FEV1、FVC%、FEV1%/FVC呈正相关(r值分别为0.837、0.368、0.822,P<0.01)。结论COPD患者存在Th17/Treg细胞失衡,而Th17/Treg细胞失衡与吸烟、COPD病情变化及肺功能损伤等密切相关。
【关键词】肺疾病,慢性阻塞性;T淋巴细胞亚群;免疫,细胞;肺功能
李玲春.慢性阻塞性肺疾病患者辅助性T细胞17/调节性T细胞变化及其与肺功能的相关性研究[J].实用心脑肺血管病杂志,2016,24(6):38-41.[www.syxnf.net]
LI L C.Change of Th17/Treg cells and its correlation with pulmonary function of patients with chronic obstructive pulmonary disease[J].Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease,2016,24(6):38-41.
1资料与方法
1.1一般资料选取2013年9月—2015年9月四川省成都市西区医院收治的老年COPD稳定期患者36例作为稳定期组,COPD急性加重期患者29例作为急性加重期组。纳入标准:(1)符合2007年中华医学会制定的“慢性阻塞性肺病诊断指南”中的COPD诊断标准;(2)年龄≥60岁;(3)患者能配合完成肺功能检查并签署知情同意书。排除标准:(1)合并支气管哮喘;(2)合并其他肺部疾病,如支气管扩张、肺纤维化、肺结核、胸腔积液等;(3)合并心力衰竭;(4)合并自身免疫性疾病,如系统性红斑狼疮、类风湿关节炎、系统性硬化症、炎症性肠病等。另选取同期在本院体检健康者50例作为对照组,其中吸烟者22例作为吸烟对照组,不吸烟者28例作为正常对照组。稳定期组中男31例,女5例;年龄60~77岁,平均年龄(66.3±3.4)岁。急性加重期组中男26例,女3例;年龄60~79岁,平均年龄(66.7±3.9)岁。吸烟对照组中男20例,女2例;年龄60~78岁,平均年龄(67.7±3.8)岁。正常对照组中男25例,女3例;年龄60~79岁,平均年龄(67.2±3.8)岁。各组受试者性别、年龄间具有均衡性。本研究经医院伦理委员会审查批准。
1.2Th17细胞分数、Treg细胞分数及血清IL-17、IL-10水平检测方法
1.2.1主要试剂及仪器抗人IL-17、IL-10 ELISA试剂盒(美国ADL公司)、离子霉素、氟波酯、布雷杆氏菌素A(Sigma公司)、FIX & PERM试剂盒(固定/破膜剂)、异硫氰基标记的CD4、别藻蓝蛋白标记的CD25、藻红蛋白标记的Foxp3试剂盒、PE标记的IL-17A、APC标记的CD3、FITC标记的CD8(eBioscience公司)、RPMI 1640培养基(Hyclone公司)、淋巴细胞分离液、胎牛血清、流式细胞分析仪、Bio-Rad 550型酶标仪等。
1.2.2检测方法所有受试者在测定肺功能当天采集外周血5 ml,分别放入2支抗凝管中,1支抗凝管保留全血(3 ml),另1支抗凝管2 500 r/min离心10 min,吸取血清并置于-20 ℃冰箱保存待检;取100 μl全血并加入2 ml溶血剂,37 ℃溶解10 min,2 500 r/min离心10 min后吸取淋巴细胞;行细胞表面荧光染色,固定/破膜后再行细胞内染色,设立对照管后上机检测,圈定淋巴细胞为门内细胞,要求每次获取10 000个以上门内细胞,并保存相关数据,设门检测Th17细胞分数、Treg细胞分数,并计算Th17/Treg细胞比值。采用酶联免疫吸附试验(ELISA)检测血清IL-17、IL-10水平,采用酶标仪于450 nm处读取吸光度值,依据标准曲线计算血清IL-17、IL-10水平;检测过程严格按试剂盒说明书操作。
1.3肺功能检查方法采用成都日升电气RFSJ1000肺功能仪检查所有受试者第1秒用力呼气容积占预计值百分比(FEV1%)、用力肺活量占预计值百分比(FVC%)及第1秒用力呼气容积与用力肺活量比值(FEV1/FVC)。
1.4观察指标比较4组受试者外周血Th17细胞分数、Treg细胞分数、Th17细胞/Treg细胞比值,血清IL-17、IL-10水平及肺功能指标,并分析外周血Th17细胞分数、Treg细胞分数、Th17细胞/Treg细胞比值及Th17细胞/Treg细胞比值与肺功能指标的相关性。
2结果
2.1外周血Th17细胞分数、Treg细胞分数、Th17细胞/Treg细胞比值及血清IL-17、IL-10水平4组受试者外周血Th17细胞分数、Treg细胞分数、Th17/Treg细胞比值及血清IL-17、IL-10水平比较,差异有统计学意义(P<0.05);其中急性加重期组和稳定期组患者外周血Th17细胞分数、Th17细胞/Treg细胞比值及血清IL-17水平高于吸烟对照组和正常对照组,外周血Treg细胞分数及血清IL-10水平低于吸烟对照组和正常对照组,差异有统计学意义(P<0.05);急性加重期组患者外周血Th17细胞分数、Th17细胞/Treg细胞比值及血清IL-17水平高于稳定期组,外周血Treg细胞分数及血清IL-10水平低于稳定期组,差异有统计学意义(P<0.05);吸烟对照组受试者外周血Th17细胞分数、Th17细胞/Treg细胞比值高于正常对照组,差异有统计学意义(P<0.05),而吸烟对照组与正常对照组受试者外周血Treg细胞分数及血清IL-17、IL-10水平比较,差异均无统计学意义(P>0.05,见表1)。
2.2肺功能指标4组受试者FVC%比较,差异无统计学意义(P>0.05)。4组受试者FEV1%、FEV1/FVC比较,差异有统计学意义(P<0.05);其中急性加重期组和稳定期组患者FEV1%、FEV1/FVC低于吸烟对照组和正常对照组,急性加重期组患者FEV1%、FEV1/FVC低于稳定期组,差异有统计学意义(P<0.05,见表2)。
2.3相关性分析Pearson相关性分析结果显示,外周血Th17细胞分数与FEV1%、FEV1/FVC呈负相关(P<0.01),Th17/Treg细胞比值与FEV1%、FVC%、FEV1/FVC呈负相关(P<0.01),而外周血Treg细胞分数与FEV1%、FVC%、FEV1/FVC呈正相关(P<0.01,见表3)。
Table2Comparisionofindexofpillmonaryfunctionamongthefourgroups
组别例数FEV1%FVC%FEV1/FVC正常对照组2892.47±7.99102.47±11.6789.43±8.33吸烟对照组2291.88±9.45101.90±13.4988.99±9.66稳定期组3669.33±6.51ab98.74±7.9951.77±7.64ab急性加重期组2942.10±6.08abc96.17±12.4942.94±5.39abcF值281.3661.875274.592P值0.0000.1380.000
注:与正常对照组比较,aP<0.05;与吸烟对照组比较,bP<0.05;与稳定期组比较,cP<0.05;FEV1%=第1秒用力呼气容积占预计值百分比,FVC%=用力肺活量占预计值百分比,FEV1/FVC=第1秒用力呼气容积与用力肺活量比值
表3外周血Th17细胞分数、Treg细胞分数、Th17/Treg细胞比值与肺功能指标的相关性
Table 3Correlations between Th17 cell percentage,Treg cell percentage,Th17/Treg cell ratio and index of pulmonary function
指标FEV1%r值 P值FVC%r值 P值FEV1/FVCr值 P值Th17细胞分数-0.7360.0000.0480.608-0.6940.000Treg细胞分数0.8370.0000.3680.0000.8220.000Th17/Treg细胞比值-0.8920.000-0.2220.017-0.7480.000
3讨论
近年研究表明,免疫学机制尤其是自身抗原和自身免疫应答介导的炎性反应在COPD的发生和发展中发挥着重要作用,其中Th17细胞主要通过分泌白介素6(IL-6)、IL-17及IL-22等而募集巨噬细胞,进而增强炎性反应;Treg细胞则通过分泌IL-10、转化生长因子β(TGF-β)等抑制炎性反应,进而维持自身免疫稳定。正常生理条件下,Th17/Treg细胞维持着动态平衡,而Th17/Treg细胞失衡在多种炎性疾病中具有重要作用。COPD为进展性慢性炎性疾病,因此Th17/Treg细胞可能在其发生和发展中发挥着重要的调节作用[2]。
Table1ComparisonofperipheralbloodTh17cellpercentage,Tregcellpercentage,Th17/Tregcellratio,serumlevelsofIL-17andIL-10amongthefourgroups
组别例数Th17细胞分数(%)Treg细胞分数(%)Th17细胞/Treg细胞比值IL-17(ng/L)IL-10(ng/L)正常对照组281.32±0.487.56±2.260.18±0.0519.58±6.2181.68±15.08吸烟对照组222.72±0.70a8.04±2.580.34±0.09a20.29±7.9583.06±17.84稳定期组364.05±0.82ab4.35±1.15ab0.93±0.13ab29.62±9.74ab42.93±12.64ab急性加重期组296.23±1.33abc2.07±0.75abc3.01±0.37abc38.94±11.41ab30.94±9.60abF值152.51669.562134.09227.136103.621P值0.0000.0000.0000.0000.000
注:与正常对照组比较,aP<0.05;与吸烟对照组比较,bP<0.05;与稳定期组比较,cP<0.05;Th17细胞=辅助性T细胞17,Treg细胞=调节性T细胞,IL-17=白介素17,IL-10=白介素10
本研究相关性分析结果显示,外周血Th17细胞分数与FEV1%、FEV1/FVC呈负相关,Th17/Treg细胞比值与FEV1%、FVC%、FEV1/FVC呈负相关,而外周血Treg细胞分数与FEV1%、FVC%、FEV1/FVC呈正相关,提示Th17/Treg细胞失衡与COPD患者肺功能损伤相关。自身免疫反应是导致COPD患者持续存在气道慢性炎症的主要原因,而持续存在气道慢性炎症可导致上皮细胞损伤甚至气道重塑,最终导致肺功能损伤。
综上所述,COPD患者存在Th17/Treg细胞失衡,而Th17/Treg细胞失衡与吸烟、COPD病情变化及肺功能损伤等密切相关,但本研究样本量较小、观察指标较少,而Th17/Treg细胞在COPD中的确切作用机制仍有待于进一步研究。
本文无利益冲突。
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(本文编辑:谢武英)
【中图分类号】R 563.9
【文献标识码】A
DOI:10.3969/j.issn.1008-5971.2016.06.010
(收稿日期:2016-02-20;修回日期:2016-06-18)
Change of Th17/Treg Cells and Its Correlation With Pulmonary Function of Patients With Chronic Obstructive Pulmonary Diseas
LILing-chun.
DepartmentofRespiratoryMedicine,ChengduWesternHospital,Chengdu610000,China
【Abstract】ObjectiveTo observe the change of Th17/Treg cells of patients with chronic obstructive pulmonary disease(COPD),to investigate its correlation with pulmonary function.MethodsFrom September 2013 to September 2015 in Chengdu Western Hospital,a total of 36 patients with stable COPD were selected as A group,29 patients with acute exacerbation of COPD were selected as B group,22 smoking healthy people admitted to this hospital for physical examination were selected as C group,and 28 non-smoking healthy people admitted to this hospital for physical examination were selected as D group.Peripheral blood Th17 cell percentage,Treg cell percentage,Th17/Treg cell ratio,serum levels of IL-17 and IL-10,and index of pulmonary function(FEV1%,FVC% and FEV1/FVC)were compared among the four groups,and the correlations between peripheral blood Th17 cell percentage,Treg cell percentage,Th17/Treg cell ratio and index of pulmonary function were analyzed.ResultsPeripheral blood Th17 cell percentage,Th17/Treg cell ratio and serum IL-17 level of A group and B group were statistically significantly higher than those of C group and D group,while peripheral blood Treg cell percentage and serum IL-10 level of A group and B group were statistically significantly lower than those of C group and D group(P<0.05);peripheral blood Th17 cell percentage,Th17/Treg cell ratio and serum IL-17 level of B group were statistically significantly higher than those of A group,while peripheral blood Treg cell percentage and serum IL-10 level of B group were statistically significantly lower than those of A group(P<0.05);peripheral blood Th17 cell percentage and Th17/Treg cell ratio of C group were statistically significantly higher than those of D group(P<0.05).No statistically significant differences of FVC% was found among the four groups(P>0.05);FEV1% and FEV1/FVC of A group and B group were statistically significantly lower than those of C group and D group,FEV1% and FEV1/FVC of B group were statistically significantly lower than those of A group(P<0.05).Pearson correlation analysis showed that,peripheral blood Th17 cell percentage was respectively negatively correlated with FEV1% and FEV1/FVC(r=-0.736,-0.694,P<0.01),Th17/Treg cell ratio was respectively negatively correlated with FEV1%,FVC% and FEV1/FVC(r=-0.892,-0.222,-0.748,P<0.01),while peripheral blood Treg cell percentage was respectively positively correlated with FEV1%,FVC% and FEV1/FVC(r=0.837,0.368,0.822,P<0.01).ConclusionTh17/Treg cells imbalance widely exists in patients with COPD,and it is correlated with smoking,disease evolution of COPD and lung function injury.
【Key words】Pulmonary disease,chronic obstructive;T-lymphocyte subsets;Immunity,cellular;Pulmonary function