Th1/Th2/Th17/Treg對(duì)鑒別兒童支氣管哮喘與單純性肺炎的意義

袁穎志　賀宇彤　吳成　張蕊　劉江惠

071700　河北省容城縣醫(yī)院兒科(袁穎志)，外一科(吳成)，婦一科(張蕊)；河北醫(yī)科大學(xué)第四醫(yī)院(賀宇彤、劉江惠)

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·論著·

Th1/Th2/Th17/Treg對(duì)鑒別兒童支氣管哮喘與單純性肺炎的意義

袁穎志賀宇彤吳成張蕊劉江惠

071700河北省容城縣醫(yī)院兒科(袁穎志)，外一科(吳成)，婦一科(張蕊)；河北醫(yī)科大學(xué)第四醫(yī)院(賀宇彤、劉江惠)

【摘要】目的檢測Th1(IFN-γ+、調(diào)節(jié)T細(xì)胞Foxp3+)、輔助T細(xì)胞17(Th17)及細(xì)胞因子IL-2、IL-4、IL-17和IL-21在兒童支氣管哮喘以及肺炎患兒外周血中的表達(dá),探討其在兒童支氣管哮喘發(fā)病中的作用以及與肺炎鑒別的意義。方法收集哮喘患兒32 例，肺炎患兒37例作為研究對(duì)象，選取同期體檢的22例健康兒童為對(duì)照組。分離外周血單個(gè)核細(xì)胞,采用流式細(xì)胞術(shù)檢測Th17+細(xì)胞百分率。采用酶聯(lián)免疫吸咐(ELISA)法檢測3組IL-2、IL-4、IL-17、IL-21表達(dá)水平。結(jié)果哮喘組患兒外周血Foxp3+細(xì)胞占T細(xì)胞的比率明顯低于肺炎組及對(duì)照組；哮喘組患兒者外周血IFN-γ+細(xì)胞比例明顯低于肺炎組和對(duì)照組；哮喘組患兒者外周血Th17+細(xì)胞所占比例高于肺炎組及對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。哮喘組患兒外周血IL-2的濃度明顯低于對(duì)照組(P<0.05)，肺炎組患兒外周血IL-2濃度明顯低于對(duì)照組(P<0.05)，但與哮喘組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。哮喘組患兒外周血IL-4、IL-17和IL-21的濃度明顯低于肺炎組和對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論Th1/Th2/Th17/Treg功能失調(diào)參與了兒童支氣管哮喘的發(fā)病機(jī)制，臨床上可以通過檢測Th1/Th2/Th17/Treg相關(guān)細(xì)胞因子的變化評(píng)估兒童支氣管哮喘病情并輔助與肺炎的鑒別。

【關(guān)鍵詞】支氣管哮喘；流式細(xì)胞術(shù)；Th1/Th2/Th17/Treg；兒童

1資料與方法

1.1一般資料收集2013年1月至2014年12月在容城縣醫(yī)院兒科住院治療的哮喘患兒32例和肺炎患兒37例，所有患兒全部根據(jù)中華醫(yī)學(xué)會(huì)兒科學(xué)分會(huì)呼吸學(xué)組制定的支氣管哮喘與肺炎常規(guī)診斷標(biāo)準(zhǔn)確診[6]，不伴有其他心肺疾病、自身免疫性疾病、自身過敏及過敏性家族史、腫瘤等病史。前1個(gè)月內(nèi)均未使用糖皮質(zhì)激素及其他免疫抑制劑或免疫調(diào)節(jié)劑等。哮喘組32例，其中男19例，女13例；平均年齡(3.3±1.2)歲。肺炎組37例，其中男21例，女16例；平均年齡(3.2±1.2)歲。選取同期容城縣醫(yī)院兒科行體檢的健康兒童22例為對(duì)照組，其中男12例，女10例；平均年齡(3.1±1.3)歲。3組性別比、年齡比較差異均無統(tǒng)計(jì)學(xué)意義(F=0.262,P=0.770)。本研究醫(yī)院倫理委員會(huì)審核通過，并獲患兒家長的知情同意。

1.2標(biāo)本采集用5 ml肝素鈉抗凝負(fù)壓真空采血管采集患兒清晨空腹靜脈血4 ml,在4 h內(nèi)進(jìn)行標(biāo)本處理。取300 μl全血做流式細(xì)胞分析；剩余全血300 G/min離心10 min，上層血漿用于檢測血漿細(xì)胞因子水平，沉淀部分用于分離PBMC，標(biāo)本于-80℃保存。

1.4酶聯(lián)免疫吸附(ELISA)檢測細(xì)胞因子水平取已分離的血漿，4℃解凍。用雙抗夾心ELISA 法檢測血漿總IL-2、IL-4、IL-17、IL-21濃度水平，按照說明書進(jìn)行操作，ELISA試劑盒均購自美國Life Science公司,產(chǎn)品編號(hào)分別為：SEA073Hu、SEA077Hu、SEA063Hu和SEB688Hu。

2結(jié)果

組別CD+4CD+25CD+4CD+25Foxp3+哮喘組(n=32)38.63±15.662.09±1.16肺炎組(n=37)41.12±18.735.13±2.41*對(duì)照組(n=22)40.73±13.135.67±1.79*

注：與哮喘組比較，*P<0.05

組別CD+4IFN-γ+CD+4Th17+哮喘組(n=32)8.47±3.237.40±2.69肺炎組(n=37)15.48±3.00*2.86±1.62*對(duì)照組(n=22)14.84±3.74*2.56±1.18*

注：與哮喘組比較，*P<0.05

2.33組患兒外周血血漿細(xì)胞因子水平比較3組患兒外周血IL-2濃度差異有統(tǒng)計(jì)學(xué)意義(F=4.644，P=0.012)。其中哮喘組患兒外周血IL-2的濃度明顯低于對(duì)照組，肺炎組患兒外周血IL-2濃度，明顯低于對(duì)照組，但與哮喘組差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。3組患兒外周血IL-4濃度差異有統(tǒng)計(jì)學(xué)意義(F=28.412，P=0.000)。其中哮喘組患兒外周血IL-4的濃度[(24.32±6.31)%]明顯低于肺炎組[(11.13±3.31)%]和對(duì)照組[(10.57±2.59)%]，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。3組患兒外周血IL-17和IL-21濃度差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。哮喘組患兒外周血IL-17和IL-21的濃度明顯低于肺炎組和對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見表3。

表3　3組患兒外周血中血漿細(xì)胞因子比較　±s

注：與哮喘組比較，*P<0.05；與肺炎組比較，#P<0.05

3討論

本研究結(jié)果表明哮喘組患兒Th1分泌的IFN-γ及IL-2均明顯低于對(duì)照組，Th2分泌的IL-4明顯高于肺炎組和對(duì)照組，與既往研究結(jié)果[9,10]相同，提示TTh1/Th2細(xì)胞因子失衡在兒童哮喘發(fā)病過程中起重要作用。Th1/Th2免疫失衡有可能是兒童哮喘發(fā)病的始動(dòng)因素之一[11]，也有可能是發(fā)病之后相關(guān)免疫因子所引發(fā)的繼發(fā)反應(yīng)。Th1/Th2免疫平衡失調(diào)引發(fā)IL-4、IL-10等促炎性因子的過度表達(dá)及IFN-γ等炎性抑制因子活性的降低可能是兒童支氣管哮喘發(fā)病的因素之一。本研究發(fā)現(xiàn)肺炎患兒表現(xiàn)為IL-2明顯低于對(duì)照組，IFN-γ與IL-4則沒有變化。提示Th1/Th2免疫失衡在兒童哮喘與肺炎中作用不同，可以以此輔助鑒別診斷。

2006年，日本學(xué)者發(fā)現(xiàn)了一種不同于Th1、Th2細(xì)胞的新型Th細(xì)胞亞群，即Th17細(xì)胞。這類細(xì)胞可分泌IL-17A-F,其中以IL-17為主，并不分泌IL-4及IFN-γ，所以有別于Th1及Th2細(xì)胞。Th17在機(jī)體免疫反應(yīng)中主要起促炎作用，該作用通過對(duì)中性粒細(xì)胞的募集作用、對(duì)不同組織或者細(xì)胞誘導(dǎo)產(chǎn)生炎性細(xì)胞因子、金屬蛋白酶以及剌激樹突狀細(xì)胞產(chǎn)生等方面體現(xiàn)[17]。研究表明,哮喘患者在支氣管活檢、支氣管肺泡灌洗液和痰標(biāo)本中, IL-17的局部表達(dá)水平明顯高于對(duì)照組[18]。有許多研究發(fā)現(xiàn)，在中、重度支氣管哮喘患者的外周血中，Th17及其分泌的IL-17表達(dá)均顯著增加[19,20]。可見Th17在哮喘的發(fā)病機(jī)制中發(fā)揮重要作用，其發(fā)揮效應(yīng)主要是通過特征性細(xì)胞因子IL-17來完成。本研究表明,哮喘組患兒與對(duì)照組相比,其外周血IL-17表達(dá)增高, 肺炎組患兒與對(duì)照相比則沒有統(tǒng)計(jì)學(xué)差異， 提示Th17可以作為輔助鑒別兒童哮喘與肺炎的指標(biāo)之一。

本研究顯示哮喘組患兒外周血IL-21水平高于對(duì)照組，肺炎組患兒則與對(duì)照組無統(tǒng)計(jì)學(xué)差異。提示IL-21 在兒童哮喘發(fā)病過程中起到一定的保護(hù)作用。IL-21是新近發(fā)現(xiàn)一種的細(xì)胞因子，由Th17分泌產(chǎn)生。IL-21與其受體結(jié)合后可以促進(jìn)T淋巴細(xì)胞的增殖,促進(jìn)B淋巴細(xì)胞、NK細(xì)胞的增殖、分化并能提高NK細(xì)胞的殺傷活性，從而對(duì)免疫系統(tǒng)發(fā)揮重要調(diào)節(jié)作用。近年來研究顯示IL-21的水平下降,可能會(huì)導(dǎo)致有抗過敏作用的IgG4水平下降[21]。還有研究發(fā)現(xiàn)IL-21能使IL-4誘導(dǎo)的IgE產(chǎn)生減少[22]。Tang等[23]研究發(fā)現(xiàn)哮喘患兒外周血IL-21明顯高于對(duì)照組，本研究結(jié)果與其一致，同時(shí)發(fā)現(xiàn)哮喘患兒與肺炎患兒外周血IL-21存在統(tǒng)計(jì)學(xué)差異。

本研究結(jié)果表明Th1/Th2/Th17/Treg功能失調(diào)不僅參與哮喘的發(fā)病機(jī)制，而且是哮喘發(fā)展和病理改變的結(jié)果。臨床上通過檢測兒童哮喘細(xì)胞因子水平的變化，結(jié)合臨床表現(xiàn)，可以評(píng)估兒童哮喘病情，了解體內(nèi)炎癥水平，并與肺炎進(jìn)行鑒別，進(jìn)而可以探討通過使用細(xì)胞因子調(diào)控劑或細(xì)胞因子拮抗劑來佐治兒童哮喘，為兒童哮喘的早期診斷和治療提供科學(xué)依據(jù)。

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The significance of Thl/Th2 /Thl7/Treg in distinguishing asthma from pneumonia in children

YUANYingzhi*,HEYutong,WUCheng,etal.

*DepartmentofPediatrics,RongchengCountyHospital,Hebei,Rongcheng071700,China

【Abstract】ObjectiveTo investigate the levels of Th1 (IFN-γ+), regulatory T cells Foxp3+), helper T cells 17 (Th17), and cytokines-IL-2, IL-4, IL-17, IL-21 in peripheral blood of children with asthma and pneumonia,and to explore their significance in the pathogenesis of asthma and the differential diagnosis from pneumonia.MethodsThirty-two children with asthma (asthma group) and thirty-seven children with pneumonia (pneumonia group) were enrolled in the study,moreover, the other twenty-two healthy children were served as control group. The peripheral blood mononuclear cells (PBMC) were separated, then the percentages of Th17+ in PMBC were detected by flow cytometry. The expression levels of IL-2,IL-4,IL-17 and IL-21 in plasma were detected by enzyme-linked mimunosorbent assay (ELISA).ResultsThe ratio[(2.09±1.16)%] of Foxp3+ T cell in asthma group was obviously lower than that [(5.13±2.41)%] in pneumonia group and that [(5.67±2.79)%] in control group. The cell percentage of IFN-γ+ in asthma group [(8.47±3.23)%] was obviously lower than that in pneumonia group [(15.48±3.00)%] and that in control group[(14.84±3.74)%].The percentage [(7.40±2.69)%] of Th17+ in asthma group was significantly higher than that [(2.86±1.62)%] in pneumonia group and that [(2.56 ± 1.18)%] in control group (P<0.05). The levels of IL-2 in asthma group and pneumonia group were significantly lower than those in control group (P<0.05).However there were no significant differences in the levels of IL-2 between asthma group and pneumonia group (P>0.05).The concentrations of IL-4, IL-17 and IL-21 in asthma group were significantly lower than those in pneumonia group and those in control group (P<0.05).ConclusionThe functional disorder of Th1/Th2 /Th17/Treg may be involved in the pathogenesis of asthma in children,which can be used as the indexes to evaluate pathogenetic condition of bronchial asthma in children and can distinguish it from pneumonia.

【Key words】bronchial asthma;flow cytometry;Th1/Th2/Th17/Treg;children

(收稿日期：2015-12-29)

【中圖分類號(hào)】R 562.25

【文獻(xiàn)標(biāo)識(shí)碼】A

【文章編號(hào)】1002-7386(2016)09-1285-04

通訊作者：劉江惠，050011石家莊市，河北醫(yī)科大學(xué)第四醫(yī)院；

doi:10.3969/j.issn.1002-7386.2016.09.001

項(xiàng)目來源：國家自然科學(xué)基金項(xiàng)目(編號(hào)：81272682)；河北省科學(xué)技術(shù)研究與發(fā)展計(jì)劃項(xiàng)目(編號(hào)：132777259)

E-mail:ljh707@yeah.net