張瑩　王靜

北京市房山區(qū)中醫(yī)醫(yī)院呼吸科,北京102400

支氣管哮喘患者外周血CD4+、CD8+T細(xì)胞、CD4+CD25+調(diào)節(jié)性T細(xì)胞表達(dá)水平與肺功能的相關(guān)性研究

張瑩王靜

北京市房山區(qū)中醫(yī)醫(yī)院呼吸科,北京102400

目的探討支氣管哮喘患者外周血CD4+、CD8+T細(xì)胞、CD4+CD25+調(diào)節(jié)性T細(xì)胞表達(dá)水平與肺功能的關(guān)系。方法收集2012年3月～2015年6月北京市房山區(qū)中醫(yī)醫(yī)院呼吸科收治的支氣管哮喘患者106例,其中急性發(fā)作（急性發(fā)作組)患者59例,非急性發(fā)作（非急性發(fā)作組)患者47例,同時以72例健康者作為對照組。對所有入組研究對象進(jìn)行肺活量（VC),一秒用力呼氣容積占用力肺活量的百分比（FEV1/FVC%)及呼氣流量峰值（PEF)檢查,采用流式細(xì)胞儀檢測外周血CD4+、CD8+T細(xì)胞及CD4+CD25+調(diào)節(jié)性T細(xì)胞水平,并對急性發(fā)作期患者CD4+、CD8+T細(xì)胞、CD4+CD25+Treg水平與肺功能的相關(guān)性進(jìn)行分析。結(jié)果①非急性發(fā)作組及急性發(fā)作組的肺功能指標(biāo)與對照組比較均顯著降低（P＜0.05)；與非急性發(fā)作組比較,急性發(fā)作組患者肺功能指標(biāo)均明顯偏低（P＜0.05)。②非急性發(fā)作組及急性發(fā)作組患者CD4+T細(xì)胞水平、CD4+CD25+Treg水平及CD4+/CD8+比值明顯低于對照組,CD8+T細(xì)胞水平、CD4+CD25+Treg/CD4+比值顯著高于對照組（P＜0.05)；與非急性發(fā)作組比較,急性發(fā)作組CD4+T細(xì)胞水平、CD4+CD25+Treg水平及CD4+/CD8+比值顯著下降,CD8+T細(xì)胞水平、CD4+CD25+Treg/CD4+比值顯著上升（P＜0.05)。③急性發(fā)作期患者外周血CD4+T細(xì)胞水平與肺功能指標(biāo)均呈正相關(guān)（r=0.47、0.50、0.45,P＜0.05)；CD8+T細(xì)胞水平與肺功能指標(biāo)均呈負(fù)相關(guān)（r=-0.54、-0.49、-0.56,P＜0.05)；CD4+CD25+Treg水平與肺功能指標(biāo)均呈正相關(guān)（r=0.39、0.42、0.40,P＜0.05)。結(jié)論CD4+、CD8+T細(xì)胞及CD4+CD25+調(diào)節(jié)性T細(xì)胞在支氣管哮喘疾病的發(fā)生、發(fā)展過程中發(fā)揮著重要作用,其水平可用來評估支氣管哮喘病情的嚴(yán)重程度。

支氣管哮喘;CD4+;CD8+;CD4+CD25+;T細(xì)胞;肺功能

支氣管哮喘（bronchial asthma)是由免疫機(jī)制介導(dǎo)的慢性肺部疾病,患者發(fā)病時常伴有體液免疫及細(xì)胞免疫功能異常[1-2]。大量研究表明,支氣管哮喘患者外周血淋巴細(xì)胞亞群水平增高與哮喘發(fā)作密切相關(guān)[3-4]。本研究通過檢測支氣管哮喘患者外周血CD4+、CD8+T細(xì)胞、CD4+CD25+調(diào)節(jié)性T細(xì)胞（CD4+CD25+Treg)水平,并對患者進(jìn)行肺功能檢測,探討支氣管哮喘患者外周血T細(xì)胞與肺功能的相關(guān)性,為支氣管哮喘的免疫預(yù)防和診斷提供理論參考。

1　資料與方法

1.1一般資料

收集2012年3月～2015年6月北京市房山區(qū)中醫(yī)醫(yī)院（以下簡稱“我院”)呼吸科收治的支氣管哮喘患者106例。入組患者均符合中華醫(yī)學(xué)會呼吸病學(xué)分會哮喘學(xué)組制訂的支氣管哮喘防治指南（支氣管哮喘的定義、診斷、治療和管理方案)診斷標(biāo)準(zhǔn)[5],其中,急性發(fā)作患者（急性發(fā)作組)59例（輕中度38例,重度21例)；非急性發(fā)作患者（非急性發(fā)作組)47例。排除患有其他肺部疾病者（包括肺纖維化、肺癌、支氣管擴(kuò)張等)；心腦血管疾病、高血壓、糖尿病患者；合并肝腎功能不全者；近期使用過免疫抑制劑者。收集同期在我院進(jìn)行體檢的72例健康者作為對照組。所有入組研究對象的年齡、性別等基本資料差異無統(tǒng)計學(xué)意義（P＞0.05),具有可比性。見表1。

1.2方法

1.2.1肺功能檢查對所有研究對象采用美國麥加菲肺功能儀測定肺功能。主要檢測指標(biāo)為肺活量（VC),一秒用力呼氣容積占用力肺活量的百分比（FEV1/ FVC%)及呼氣流量峰值（PEF)。

1.2.2外周血CD4+、CD8+T及CD4+CD25+Treg水平測定采用Beckman Coulter流式細(xì)胞儀檢測,CellQuest軟件計算陽性細(xì)胞數(shù)量。所有研究對象抽取1次清晨空腹外周靜脈全血5 mL,檢測過程嚴(yán)格按照試劑盒說明書進(jìn)行操作,CD4-FITC、CD8-FITC試劑盒購于BD Bioscience公司,CD25-PE、4-FITC/8-PE/3-PC5試劑盒購于Beckman Coulter公司。

表1　入組研究對象的一般資料比較

1.3統(tǒng)計學(xué)方法

采用SPSS 19.0統(tǒng)計學(xué)軟件進(jìn)行數(shù)據(jù)分析,計量資料數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（±s)表示,多組間比較采用單因素方差分析,組間兩兩比較采用LSD-t檢驗；計數(shù)資料用率表示,組間比較采用χ2檢驗；相關(guān)性分析采用Pearson檢驗。以P＜0.05為差異有統(tǒng)計學(xué)意義。

2　結(jié)果

2.1三組肺功能指標(biāo)比較

非急性發(fā)作組及急性發(fā)作組的VC、FEV1/FVC及PEF值與對照組比較均顯著降低（P＜0.05)；與非急性發(fā)作組比較,急性發(fā)作組患者三個指標(biāo)均明顯偏低（P＜0.05)。見表2。

表2　三組肺功能指標(biāo)比較（±s)

表2　三組肺功能指標(biāo)比較（±s)

注:與對照組比較,*P＜0.05；與非急性發(fā)作組比較,#P＜0.05；VC:肺活量；FEV1/FVC%:一秒用力呼氣容積占用力肺活量的百分比；PEF:呼氣流量峰值

組別例數(shù)V C（L ) F E V1/ F V C（% ) P E F（L / s )對照組非急性發(fā)作組急性發(fā)作組輕中度重度7 2 4 7 2 . 6 0 ± 0 . 3 9 2 . 2 9 ± 0 . 3 0*7 5 . 9 5 ± 1 0 . 1 1 6 1 . 3 8 ± 6 . 4 3*5 . 0 1 ± 0 . 6 4 3 . 4 8 ± 0 . 5 9*3 8 2 1 2 . 1 1 ± 0 . 2 3*#1 . 8 2 ± 0 . 1 9*#5 0 . 4 7 ± 5 . 0 2*#4 3 . 0 9 ± 5 . 1 1*#3 . 0 7 ± 0 . 5 5*#2 . 7 6 ± 0 . 3 5*#

2.2三組外周血CD4+、CD8+T細(xì)胞、CD4+CD25+Treg水平比較

非急性發(fā)作組及急性發(fā)作組CD4+T細(xì)胞水平、CD4+CD25+Treg水平及CD4+/CD8+比值明顯低于對照組,CD8+T細(xì)胞水平、CD4+CD25+Treg/CD4+比值顯著高于對照組（P＜0.05)；與非急性發(fā)作組比較,急性發(fā)作組CD4+T細(xì)胞水平、CD4+CD25+Treg水平及CD4+/ CD8+比值顯著降低,CD8+T細(xì)胞水平、CD4+CD25+Treg/CD4+比值顯著升高（P＜0.05)。見表3。

2.3急性發(fā)作期患者CD4+、CD8+T細(xì)胞、CD4+CD25+Treg水平與肺功能的相關(guān)性分析

急性發(fā)作期患者外周血CD4+T細(xì)胞水平與VC、FEV1/FVC及PEF均呈正相關(guān)關(guān)系（P＜0.05)；CD8+T細(xì)胞水平與VC、FEV1/FVC及PEF均呈負(fù)相關(guān)關(guān)系（P＜0.05)；CD4+CD25+Treg水平與VC、FEV1/FVC及PEF均呈正相關(guān)關(guān)系（P＜0.05)。見表4。

表3　三組外周血CD4+、CD8+T細(xì)胞、CD4+CD25+Treg水平比較（±s)

表3　三組外周血CD4+、CD8+T細(xì)胞、CD4+CD25+Treg水平比較（±s)

注:與對照組比較,*P＜0.05；與非急性發(fā)作組比較,#P＜0.05

組別例數(shù)CD4+（%)CD8+（%)CD4+CD25+Treg（%)CD4+/CD8+CD4+CD25+Treg/CD4+對照組非急性發(fā)作組急性發(fā)作組輕中度重度72 47 40.05±4.49 31.82±3.76*20.33±3.97 25.18±3.58*3.56±0.63 2.79±0.49*1.84±0.31 1.37±0.27*5.06±1.22 7.17±1.45*38 21 27.35±3.89*#22.19±3.01*#29.04±3.99*#37.46±4.20*#1.98±0.53*#1.74±0.46*#1.02±0.29*#0.75±0.20*#8.09±1.38*#9.50±2.34*#

表4　急性發(fā)作期CD4+、CD8+T細(xì)胞、CD4+CD25+Treg水平與肺功能的相關(guān)性分析

3　討論

哮喘是一種由多種細(xì)胞參與的以慢性呼吸道炎性反應(yīng)為特點的免疫變態(tài)反應(yīng)性疾病[6-8]。迄今為止,哮喘的發(fā)病機(jī)制尚未完全研究清楚,病理研究發(fā)現(xiàn),免疫反應(yīng)是哮喘患者產(chǎn)生氣道慢性炎癥的主要原因[9-11],患者氣道受到刺激時生成并釋放炎癥介質(zhì),呼吸道分泌物增多,影響肺通氣與換氣功能[12]。

T淋巴細(xì)胞的主要功能是調(diào)節(jié)蛋白質(zhì)抗原引起的免疫應(yīng)答,其中最重要的是CD4+T細(xì)胞和CD8+T細(xì)胞,兩者比例失調(diào)影響機(jī)體免疫系統(tǒng)[13-14]。Lin等[15]研究顯示,當(dāng)CD4+T細(xì)胞或CD8+T細(xì)胞的水平或功能出現(xiàn)異常,T淋巴細(xì)胞調(diào)節(jié)就失去平衡而導(dǎo)致機(jī)體發(fā)病。本研究顯示,非急性發(fā)作組與急性發(fā)作組的CD8+T細(xì)胞水平顯著高于對照組,CD4+T細(xì)胞水平、CD4+/CD8+比值低于對照組,提示哮喘患者體內(nèi)T細(xì)胞水平的高低可反映機(jī)體免疫系統(tǒng)內(nèi)環(huán)境穩(wěn)定情況。

近年來研究認(rèn)為,CD4+CD25+Treg細(xì)胞在維持機(jī)體免疫穩(wěn)態(tài)及抑制炎癥方面起著重要作用,與哮喘氣道炎癥密切相關(guān)[16-18]。Mamessier等[19]研究表明,哮喘患者外周血CD4+CD25+Treg數(shù)量減少,導(dǎo)致其免疫抑制作用降低,不能平衡Th1/Th2免疫應(yīng)答和抑制Th2細(xì)胞優(yōu)勢應(yīng)答,進(jìn)一步導(dǎo)致哮喘患者體內(nèi)Treg細(xì)胞數(shù)量下降。本研究中,非急性發(fā)作組與急性發(fā)作組CD4+CD25+Treg水平顯著低于對照組,CD4+CD25+Treg/ CD4+比值顯著高于對照組（P＜0.05)；急性發(fā)作組與非急性發(fā)作組相比,CD4+CD25+Treg水平明顯下降, CD4+CD25+Treg/CD4+比值顯著上升（P＜0.05),說明支氣管哮喘患者外周血中CD4+CD25+Treg細(xì)胞缺乏,在急性發(fā)作期時下降更明顯,該結(jié)果亦與黃花榮等[20]的研究結(jié)論一致。

有研究顯示,哮喘急性發(fā)作期患者體內(nèi)CD4+CD25+Treg水平越低,其肺部的變態(tài)反應(yīng)越嚴(yán)重,與患者肺功能呈負(fù)相關(guān)[21]。本研究對急性發(fā)作期患者CD4+T、CD8+T細(xì)胞及CD4+CD25+Treg與肺功能的相關(guān)性分析顯示,患者肺功能指標(biāo)VC、FEV1/FVC及PEF與CD4+T、CD8+T細(xì)胞、CD4+CD25+Treg具有一定的相關(guān)性,患者體內(nèi)CD4+T細(xì)胞及CD4+CD25+Treg細(xì)胞水平越低,CD8+T細(xì)胞水平越高,患者的肺功能越差。

綜上所述,支氣管哮喘患者外周血CD4+、CD8+T細(xì)胞、CD4+CD25+Treg表達(dá)水平可作為評估支氣管哮喘病情變化的監(jiān)測指標(biāo),支氣管哮喘病癥具有獨特的臨床表現(xiàn)及機(jī)體內(nèi)環(huán)境變化,臨床診斷中應(yīng)對疾病的特點予以充分考慮,給出科學(xué)的治療方案。

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Changes of peripheral blood CD4+,CD8+T cells,CD4+CD25+regulatory T cells levels in patients with bronchial asthma and its correlation with lung function

ZHANG YingWANG Jing
Department of Respiratory,Fangshan Hospital of Traditional Chinese Medicine,Beijing102400,China

Objective To investigate the relationship between lung function and the levels of CD4+,CD8+T cells,CD4+CD25+regulate T cells of peripheral blood in patients with bronchial asthma.Methods 106 patients with bronchial asthma in Fangshan Hospital of Traditional Chinese Medicine from March 2012 to June 2015 were selected as the observation subjects,59 cases had acute asthma(acute asthma group)and 47 cases had non-acute asthma(non-acute asthma group),72 healthy human with physical examination at the same period were selected as the control group.The lung function indexes of vital capacity(VC),forced expiratory volume in one second/forced vital capacity(FEV1/FVC%) and peak expiratory flow(PEF)of all subjects were detected.The levels of CD4+,CD8+T cells,CD4+CD25+regulatory T cells were determined by flow cytometry.The correlation between lung function and the expression levels of CD4+,CD8+T cells,CD4+CD25+regulate T cells in patients with acute asthma were analyzed.Results①The lung function indexes of VC,FEV1/FVC%and PEF in the non-acute asthma group and the acute asthma group were significantly lower than those in the control group(P＜0.05);VC,FEV1/FVC%and PEF in the acute asthma group were obviously lower than those in the non-acute asthma group(P＜0.05).②CD4+T cells,CD4+CD25+Treg cells and CD4+/CD8+ratio inthe non-acute asthma group and the acute asthma group were significantly lower than those in the control group,while CD8+T cells and CD4+CD25+Treg/CD4+ratio were obviously higher than those in the control group(P＜0.05);compared with the non-acute asthma group,CD4+T cells,CD4+CD25+Treg cells and CD4+/CD8+ratio in the acute asthma group were significantly decreased,while CD8+T cells,CD4+CD25+Treg/CD4+ratio were significantly increased(P＜0.05).③CD4+T cells in the acute asthma group were positively correlated with lung function parameters(r=0.47,0.50,0.45,P＜0.05);CD8+T cells were negatively correlated with lung function parameters(r=-0.54,-0.49,-0.56,P＜0.05);CD4+CD25+Treg cells were positively correlated with lung function parameters(r=0.39,0.42,0.40,P＜0.05).Conclusion CD4+,CD8+T cells and CD4+CD25+regulatory T cells play important roles in the occurrence and development of asthma,which can be used to assess the severity of bronchial asthma.

Bronchial asthma;CD4+;CD8+;CD4+CD25+;T cells;Lung function

R562.25

A

1673-7210(2016)06(b)-0093-04

北京市科技計劃課題“首都臨床特色應(yīng)用研究”（Z141107002514083)。

（2016-03-14本文編輯:程銘)