慢性阻塞性肺疾病患者輔助性T細(xì)胞17/調(diào)節(jié)性T細(xì)胞變化及其與肺功能的相關(guān)性研究

李玲春

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·論著·

慢性阻塞性肺疾病患者輔助性T細(xì)胞17/調(diào)節(jié)性T細(xì)胞變化及其與肺功能的相關(guān)性研究

李玲春

610000四川省成都市西區(qū)醫(yī)院呼吸內(nèi)科

【摘要】目的觀察慢性阻塞性肺疾病(COPD)患者輔助性T細(xì)胞17(Th17細(xì)胞)/調(diào)節(jié)性T細(xì)胞(Treg細(xì)胞)變化，探討其與肺功能的相關(guān)性。方法選取2013年9月—2015年9月四川省成都市西區(qū)醫(yī)院收治的老年COPD穩(wěn)定期患者36例作為穩(wěn)定期組，COPD急性加重期患者29例作為急性加重期組；另選取同期在本院體檢健康者50例作為對(duì)照組，其中吸煙者22例作為吸煙對(duì)照組，不吸煙者28例作為正常對(duì)照組。比較4組受試者外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值，血清IL-17、IL-10水平及肺功能指標(biāo)〔第1秒用力呼氣容積占預(yù)計(jì)值百分比(FEV1%)、用力肺活量占預(yù)計(jì)值百分比(FVC%)及第1秒用力呼氣容積與用力肺活量比值(FEV1/FVC)〕，并分析外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)及Th17細(xì)胞/Treg細(xì)胞比值與肺功能指標(biāo)的相關(guān)性。結(jié)果急性加重期組和穩(wěn)定期組患者外周血Th17細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值及血清IL-17水平高于吸煙對(duì)照組和正常對(duì)照組，外周血Treg細(xì)胞分?jǐn)?shù)及血清IL-10水平低于吸煙對(duì)照組和正常對(duì)照組(P<0.05)；急性加重期組患者外周血Th17細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值及血清IL-17水平高于穩(wěn)定期組，外周血Treg細(xì)胞分?jǐn)?shù)及血清IL-10水平低于穩(wěn)定期組(P<0.05)；吸煙對(duì)照組受試者外周血Th17細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值高于正常對(duì)照組(P<0.05)。4組受試者FVC%比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)；急性加重期組和穩(wěn)定期組患者FEV1%、FEV1/FVC低于吸煙對(duì)照組和正常對(duì)照組，急性加重期組患者FEV1%、FEV1/FVC低于穩(wěn)定期組(P<0.05)。Pearson相關(guān)性分析結(jié)果顯示，外周血Th17細(xì)胞分?jǐn)?shù)與FEV1、FEV1%/FVC呈負(fù)相關(guān)(r值分別為-0.736、-0.694，P<0.05)，Th17/Treg細(xì)胞比值與FEV1、FVC%、FEV1%/FVC呈負(fù)相關(guān)(r值分別為-0.892、-0.222、-0.748，P<0.01)，而外周血Treg細(xì)胞分?jǐn)?shù)與FEV1、FVC%、FEV1%/FVC呈正相關(guān)(r值分別為0.837、0.368、0.822，P<0.01)。結(jié)論COPD患者存在Th17/Treg細(xì)胞失衡，而Th17/Treg細(xì)胞失衡與吸煙、COPD病情變化及肺功能損傷等密切相關(guān)。

【關(guān)鍵詞】肺疾病，慢性阻塞性；T淋巴細(xì)胞亞群；免疫，細(xì)胞；肺功能

李玲春.慢性阻塞性肺疾病患者輔助性T細(xì)胞17/調(diào)節(jié)性T細(xì)胞變化及其與肺功能的相關(guān)性研究[J].實(shí)用心腦肺血管病雜志，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資料與方法

1.1一般資料選取2013年9月—2015年9月四川省成都市西區(qū)醫(yī)院收治的老年COPD穩(wěn)定期患者36例作為穩(wěn)定期組，COPD急性加重期患者29例作為急性加重期組。納入標(biāo)準(zhǔn)：(1)符合2007年中華醫(yī)學(xué)會(huì)制定的“慢性阻塞性肺病診斷指南”中的COPD診斷標(biāo)準(zhǔn)；(2)年齡≥60歲；(3)患者能配合完成肺功能檢查并簽署知情同意書。排除標(biāo)準(zhǔn)：(1)合并支氣管哮喘；(2)合并其他肺部疾病，如支氣管擴(kuò)張、肺纖維化、肺結(jié)核、胸腔積液等；(3)合并心力衰竭；(4)合并自身免疫性疾病，如系統(tǒng)性紅斑狼瘡、類風(fēng)濕關(guān)節(jié)炎、系統(tǒng)性硬化癥、炎癥性腸病等。另選取同期在本院體檢健康者50例作為對(duì)照組，其中吸煙者22例作為吸煙對(duì)照組，不吸煙者28例作為正常對(duì)照組。穩(wěn)定期組中男31例，女5例；年齡60～77歲，平均年齡(66.3±3.4)歲。急性加重期組中男26例，女3例；年齡60～79歲，平均年齡(66.7±3.9)歲。吸煙對(duì)照組中男20例，女2例；年齡60～78歲，平均年齡(67.7±3.8)歲。正常對(duì)照組中男25例，女3例；年齡60～79歲，平均年齡(67.2±3.8)歲。各組受試者性別、年齡間具有均衡性。本研究經(jīng)醫(yī)院倫理委員會(huì)審查批準(zhǔn)。

1.2Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)及血清IL-17、IL-10水平檢測(cè)方法

1.2.1主要試劑及儀器抗人IL-17、IL-10 ELISA試劑盒(美國(guó)ADL公司)、離子霉素、氟波酯、布雷桿氏菌素A(Sigma公司)、FIX & PERM試劑盒(固定/破膜劑)、異硫氰基標(biāo)記的CD4、別藻藍(lán)蛋白標(biāo)記的CD25、藻紅蛋白標(biāo)記的Foxp3試劑盒、PE標(biāo)記的IL-17A、APC標(biāo)記的CD3、FITC標(biāo)記的CD8(eBioscience公司)、RPMI 1640培養(yǎng)基(Hyclone公司)、淋巴細(xì)胞分離液、胎牛血清、流式細(xì)胞分析儀、Bio-Rad 550型酶標(biāo)儀等。

1.2.2檢測(cè)方法所有受試者在測(cè)定肺功能當(dāng)天采集外周血5 ml，分別放入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離心10 min，吸取血清并置于-20 ℃冰箱保存待檢；取100 μl全血并加入2 ml溶血?jiǎng)?7 ℃溶解10 min，2 500 r/min離心10 min后吸取淋巴細(xì)胞；行細(xì)胞表面熒光染色，固定/破膜后再行細(xì)胞內(nèi)染色，設(shè)立對(duì)照管后上機(jī)檢測(cè)，圈定淋巴細(xì)胞為門內(nèi)細(xì)胞，要求每次獲取10 000個(gè)以上門內(nèi)細(xì)胞，并保存相關(guān)數(shù)據(jù)，設(shè)門檢測(cè)Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)，并計(jì)算Th17/Treg細(xì)胞比值。采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)血清IL-17、IL-10水平，采用酶標(biāo)儀于450 nm處讀取吸光度值，依據(jù)標(biāo)準(zhǔn)曲線計(jì)算血清IL-17、IL-10水平；檢測(cè)過(guò)程嚴(yán)格按試劑盒說(shuō)明書操作。

1.3肺功能檢查方法采用成都日升電氣RFSJ1000肺功能儀檢查所有受試者第1秒用力呼氣容積占預(yù)計(jì)值百分比(FEV1%)、用力肺活量占預(yù)計(jì)值百分比(FVC%)及第1秒用力呼氣容積與用力肺活量比值(FEV1/FVC)。

1.4觀察指標(biāo)比較4組受試者外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值，血清IL-17、IL-10水平及肺功能指標(biāo)，并分析外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值及Th17細(xì)胞/Treg細(xì)胞比值與肺功能指標(biāo)的相關(guān)性。

2結(jié)果

2.1外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值及血清IL-17、IL-10水平4組受試者外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)、Th17/Treg細(xì)胞比值及血清IL-17、IL-10水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中急性加重期組和穩(wěn)定期組患者外周血Th17細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值及血清IL-17水平高于吸煙對(duì)照組和正常對(duì)照組，外周血Treg細(xì)胞分?jǐn)?shù)及血清IL-10水平低于吸煙對(duì)照組和正常對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；急性加重期組患者外周血Th17細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值及血清IL-17水平高于穩(wěn)定期組，外周血Treg細(xì)胞分?jǐn)?shù)及血清IL-10水平低于穩(wěn)定期組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；吸煙對(duì)照組受試者外周血Th17細(xì)胞分?jǐn)?shù)、Th17細(xì)胞/Treg細(xì)胞比值高于正常對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，而吸煙對(duì)照組與正常對(duì)照組受試者外周血Treg細(xì)胞分?jǐn)?shù)及血清IL-17、IL-10水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05，見(jiàn)表1)。

2.2肺功能指標(biāo)4組受試者FVC%比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。4組受試者FEV1%、FEV1/FVC比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中急性加重期組和穩(wěn)定期組患者FEV1%、FEV1/FVC低于吸煙對(duì)照組和正常對(duì)照組，急性加重期組患者FEV1%、FEV1/FVC低于穩(wěn)定期組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表2)。

2.3相關(guān)性分析Pearson相關(guān)性分析結(jié)果顯示，外周血Th17細(xì)胞分?jǐn)?shù)與FEV1%、FEV1/FVC呈負(fù)相關(guān)(P<0.01)，Th17/Treg細(xì)胞比值與FEV1%、FVC%、FEV1/FVC呈負(fù)相關(guān)(P<0.01)，而外周血Treg細(xì)胞分?jǐn)?shù)與FEV1%、FVC%、FEV1/FVC呈正相關(guān)(P<0.01，見(jiàn)表3)。

Table2Comparisionofindexofpillmonaryfunctionamongthefourgroups

組別例數(shù)FEV1%FVC%FEV1/FVC正常對(duì)照組2892.47±7.99102.47±11.6789.43±8.33吸煙對(duì)照組2291.88±9.45101.90±13.4988.99±9.66穩(wěn)定期組3669.33±6.51ab98.74±7.9951.77±7.64ab急性加重期組2942.10±6.08abc96.17±12.4942.94±5.39abcF值281.3661.875274.592P值0.0000.1380.000

注：與正常對(duì)照組比較，aP<0.05；與吸煙對(duì)照組比較，bP<0.05；與穩(wěn)定期組比較，cP<0.05；FEV1%=第1秒用力呼氣容積占預(yù)計(jì)值百分比，F(xiàn)VC%=用力肺活量占預(yù)計(jì)值百分比，F(xiàn)EV1/FVC=第1秒用力呼氣容積與用力肺活量比值

表3外周血Th17細(xì)胞分?jǐn)?shù)、Treg細(xì)胞分?jǐn)?shù)、Th17/Treg細(xì)胞比值與肺功能指標(biāo)的相關(guān)性

Table 3Correlations between Th17 cell percentage，Treg cell percentage，Th17/Treg cell ratio and index of pulmonary function

指標(biāo)FEV1%r值 P值FVC%r值 P值FEV1/FVCr值 P值Th17細(xì)胞分?jǐn)?shù)-0.7360.0000.0480.608-0.6940.000Treg細(xì)胞分?jǐn)?shù)0.8370.0000.3680.0000.8220.000Th17/Treg細(xì)胞比值-0.8920.000-0.2220.017-0.7480.000

3討論

近年研究表明，免疫學(xué)機(jī)制尤其是自身抗原和自身免疫應(yīng)答介導(dǎo)的炎性反應(yīng)在COPD的發(fā)生和發(fā)展中發(fā)揮著重要作用，其中Th17細(xì)胞主要通過(guò)分泌白介素6(IL-6)、IL-17及IL-22等而募集巨噬細(xì)胞，進(jìn)而增強(qiáng)炎性反應(yīng)；Treg細(xì)胞則通過(guò)分泌IL-10、轉(zhuǎn)化生長(zhǎng)因子β(TGF-β)等抑制炎性反應(yīng)，進(jìn)而維持自身免疫穩(wěn)定。正常生理?xiàng)l件下，Th17/Treg細(xì)胞維持著動(dòng)態(tài)平衡，而Th17/Treg細(xì)胞失衡在多種炎性疾病中具有重要作用。COPD為進(jìn)展性慢性炎性疾病，因此Th17/Treg細(xì)胞可能在其發(fā)生和發(fā)展中發(fā)揮著重要的調(diào)節(jié)作用[2]。

Table1ComparisonofperipheralbloodTh17cellpercentage，Tregcellpercentage，Th17/Tregcellratio，serumlevelsofIL-17andIL-10amongthefourgroups

組別例數(shù)Th17細(xì)胞分?jǐn)?shù)(%)Treg細(xì)胞分?jǐn)?shù)(%)Th17細(xì)胞/Treg細(xì)胞比值IL-17(ng/L)IL-10(ng/L)正常對(duì)照組281.32±0.487.56±2.260.18±0.0519.58±6.2181.68±15.08吸煙對(duì)照組222.72±0.70a8.04±2.580.34±0.09a20.29±7.9583.06±17.84穩(wěn)定期組364.05±0.82ab4.35±1.15ab0.93±0.13ab29.62±9.74ab42.93±12.64ab急性加重期組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：與正常對(duì)照組比較，aP<0.05；與吸煙對(duì)照組比較，bP<0.05；與穩(wěn)定期組比較，cP<0.05；Th17細(xì)胞=輔助性T細(xì)胞17，Treg細(xì)胞=調(diào)節(jié)性T細(xì)胞，IL-17=白介素17，IL-10=白介素10

本研究相關(guān)性分析結(jié)果顯示，外周血Th17細(xì)胞分?jǐn)?shù)與FEV1%、FEV1/FVC呈負(fù)相關(guān)，Th17/Treg細(xì)胞比值與FEV1%、FVC%、FEV1/FVC呈負(fù)相關(guān)，而外周血Treg細(xì)胞分?jǐn)?shù)與FEV1%、FVC%、FEV1/FVC呈正相關(guān)，提示Th17/Treg細(xì)胞失衡與COPD患者肺功能損傷相關(guān)。自身免疫反應(yīng)是導(dǎo)致COPD患者持續(xù)存在氣道慢性炎癥的主要原因，而持續(xù)存在氣道慢性炎癥可導(dǎo)致上皮細(xì)胞損傷甚至氣道重塑，最終導(dǎo)致肺功能損傷。

綜上所述，COPD患者存在Th17/Treg細(xì)胞失衡，而Th17/Treg細(xì)胞失衡與吸煙、COPD病情變化及肺功能損傷等密切相關(guān)，但本研究樣本量較小、觀察指標(biāo)較少，而Th17/Treg細(xì)胞在COPD中的確切作用機(jī)制仍有待于進(jìn)一步研究。

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(本文編輯：謝武英)

【中圖分類號(hào)】R 563.9

【文獻(xiàn)標(biāo)識(shí)碼】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%，F(xiàn)VC% 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，F(xiàn)EV1% 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%，F(xiàn)VC% 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%，F(xiàn)VC% 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