馬卓婭 張建平 劉萍 王國兵 羅穎 鄭躍杰
【摘要】 目的:探討調(diào)節(jié)性B細(xì)胞(Regulatory B cells,Breg)在兒童哮喘發(fā)病中可能的作用。方法:選取急性期哮喘患兒23例(試驗(yàn)組),同齡健康兒童21例(正常對(duì)照組),取血備檢。采用流式細(xì)胞術(shù)分別檢測(cè)外周血調(diào)節(jié)性T細(xì)胞(Regulatory T cells,Treg)、Breg比例及Breg細(xì)胞表面CD80、CD86的表達(dá)水平;PCR檢測(cè)CD4+ T細(xì)胞中IL-17A、IL-17F IFN-γ、T-bet、GATA3、RORγt、IL-10、TGF-β mRNA表達(dá);ELISA檢測(cè)B細(xì)胞培養(yǎng)上清中IL-10濃度。結(jié)果:(1)試驗(yàn)組患兒急性發(fā)作期外周血中CD24hiCD27+Breg細(xì)胞比例及B細(xì)胞培養(yǎng)上清的IL-10濃度均比正常對(duì)照組低(P<0.05);CD80、CD86表達(dá)水平均低于正常對(duì)照組(P<0.05)。(2)試驗(yàn)組患兒CD4+CD25+Foxp3+Treg比例低于正常對(duì)照組(P<0.05);CD4+T細(xì)胞中T-bet、IFNG、RORγt、IL-17A、IL-17F表達(dá)上調(diào),差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);GATA3表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。(3)CD19+B細(xì)胞中IL-10 mRNA在試驗(yàn)組和對(duì)照組差異無統(tǒng)計(jì)學(xué)意義(P>0.05),而TGF-βmRNA在試驗(yàn)組患兒中升高,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論:Breg細(xì)胞數(shù)量及功能異常可能是哮喘發(fā)病機(jī)制之一。
【關(guān)鍵詞】 哮喘; 兒童; 調(diào)節(jié)性B細(xì)胞; 調(diào)節(jié)性T細(xì)胞; 白介素-10
Changes of Regulatory B Cells in Patients with Acute Asthma and Its Significance/MA Zhuo-ya,ZHANG Jian-ping,LIU Ping,et al.//Medical Innovation of China,2017,14(14):012-016
【Abstract】 Objective:To investigate the role of regulatory B cells in the pathogenesis in patients with asthma in children.Method:Twenty-three children with acute asthma(the experiment group) and twenty-one age-matched healthy subjects (the healthy control group)were enrolled in this study.Blood samples were collected and the proportions of Breg and CD4+CD25+Foxp3+regulatory T cells(Treg),and the expressions of co-stimulatory molecules(CD80,CD86) were analyzed by flow cytometry.The concentration of IL-10 protein was measured by enzyme-linkedimmunosorbent assay.Real-time PCR was performed to evalevaluate the expressions of T-bet,IFNG,RORγt,IL-17A,IL-17F in CD4+ T cells and IL-10 and TGF-β in CD19+B cells at mRNA level.Result:(1)The proportions of CD24hiCD27+Breg in the experiment group were significantly found to be lower than those of the healthy control group(P<0.05),and its expression levels of CD80 and CD86 were down-regulated(P<0.05),the concentrations of IL-10 protein in culture supernatant of B cells from patients were still lower than those of the healthy control group(P<0.05).(2)The proportions of CD4+CD25+Foxp3+Treg was significantly decreased in the experiment group compared with those of the healthy control group(P<0.05),and the expressions of T-bet,IFNG,RORγt,IL-17A,IL-17F at mRNA level were significantly increased inpatients compared with those of the healthy control group (P<0.05).(3)There no significant difference in the expressions of IL-10 at mRNA level in CD19+B cells between two groups and the expressions of TGF-β at mRNA level in CD19+B cells from patients were higher than those of the healthy control group(P<0.05).Conclusion:Breg cell deficiency and its impaired functiom might be one of the important factors causing immune dysfunction in patients with acute asthma.
【Key words】 Asthma; Child; Regulatory B cells; Regulatory T cells; Interleukin-10
First-authors address:Shenzhen Childrens Hospital,Shenzhen 518026,China
doi:10.3969/j.issn.1674-4985.2017.14.004
支氣管哮喘是一種反復(fù)發(fā)作的咳嗽、喘息、胸悶及呼吸困難為表現(xiàn)的慢性氣道炎癥性疾病,全國城市14歲以下哮喘兒童的累及患病率由2000年的1.97%增至2010年的3.02%[1-3]。兒童哮喘在我國占兒科門診量的3%~5%,是危害兒童健康的一種重要疾病[4]。支氣管哮喘,簡稱哮喘,是由氣道內(nèi)的炎癥細(xì)胞、結(jié)構(gòu)細(xì)胞和細(xì)胞組分共同參與的慢性炎癥性疾病,但其病因及發(fā)病機(jī)制迄今為止仍未完全闡明。以往的觀點(diǎn)認(rèn)為Th1/Th2細(xì)胞免疫失衡是哮喘發(fā)病的重要環(huán)節(jié),但是隨著研究的不斷深入,Th1/Th2細(xì)胞平衡理論未能解釋一些實(shí)驗(yàn)及臨床現(xiàn)象[5]。體內(nèi)存在具有免疫調(diào)節(jié)(或免疫抑制)功能的調(diào)節(jié)性B細(xì)胞(Breg),可通過細(xì)胞共刺激分子(CD80、CD86)或細(xì)胞因子(IL-10或TGF-β)抑制自身免疫T細(xì)胞活化,其數(shù)量或功能變化可引起自身免疫功能紊亂,導(dǎo)致免疫性疾病的發(fā)生[6-9]。本文探討哮喘患兒急性發(fā)作期外周血Breg改變,為進(jìn)一步揭示哮喘免疫發(fā)病機(jī)制提供理論基礎(chǔ)及實(shí)驗(yàn)依據(jù),現(xiàn)報(bào)道如下。
1 資料與方法
1.1 一般資料 哮喘試驗(yàn)組:選取2015年
10月-2016年5月深圳市兒童醫(yī)院呼吸??圃\斷支氣管哮喘急性期患兒23例,其中男17例,女6例,男女比例為2.83∶1;年齡3~12歲,平均(6.58±2.38)歲。診斷均符合2008年由中華醫(yī)學(xué)會(huì)兒科分會(huì)呼吸學(xué)組制定的哮喘診斷標(biāo)準(zhǔn),均為首次確診哮喘或規(guī)范吸入激素且停用>3個(gè)月后復(fù)發(fā),沒有其他傳染病和自身免疫病。正常對(duì)照組:選取在深圳市兒童醫(yī)院2015年10月-2016年5月體檢的健康兒童21例,其中男7例,女14例,男女比例為2.00∶1;年齡2~10歲,平均(5.43±1.91)歲。經(jīng)統(tǒng)計(jì)分析,兩組年齡、性別組成比較,差異均無統(tǒng)計(jì)學(xué)意義(P>0.05),具有可比性。本研究方案及步驟經(jīng)深圳市兒童醫(yī)院學(xué)術(shù)倫理委員會(huì)討論通過,參與研究的受試者或其監(jiān)護(hù)人均簽署知情同意書。
1.2 分離CD4+、CD19+B 淋巴細(xì)胞:無菌采取靜脈血3 mL,肝素抗凝,迅速送檢。聚蔗糖(Ficoll)-泛影葡胺分離外周血單個(gè)核細(xì)胞(PBMC)。參照試劑盒(美國LifeTec公司,113.31 D和111.43 D)方法,磁珠分離法分離CD4+、CD19+B淋巴細(xì)胞,檢測(cè)細(xì)胞活力>95%,純度>97%,備用。
1.3 流式細(xì)胞術(shù)檢測(cè)(flow cytometry,F(xiàn)C) 采用全血直接計(jì)數(shù)法,以CD19-eFluro450(流式抗體均購自美國eBioscience公司)設(shè)門,經(jīng)CD24-PerCP-Cy5.5、CD27-PE-Cyanine7、CD80-APC、CD86-FITC、CD1d-PE染色30 min后檢測(cè),分析CD19+CD24+CD27+Bregs比例及CD80、CD86、CD1d平均熒光強(qiáng)度(mean fluorescence intensity,MFI)。另取部分細(xì)胞,以CD4-FITC設(shè)門,固定、破膜,經(jīng)CD25-PE和Foxp3-APC抗體染色30 min,檢測(cè)CD4+ CD25+Foxp3+細(xì)胞比例。全部數(shù)據(jù)經(jīng)流式細(xì)胞儀(美國Bection Dickinson公司,CantoII)配套軟件DivaVer6.1.3獲取分析。
1.4 實(shí)時(shí)熒光定量PCR(real-time PCR) (1) 提取、定量總RNA:參照試劑盒方法(美國Ambion公司,AM1912)分離CD4+ T、CD19+ B淋巴細(xì)胞總RNA,分光光度計(jì)測(cè)定RNA OD260及OD280,評(píng)估總RNA質(zhì)量。(2)逆轉(zhuǎn)錄-PCR(RT-PCR):參照試劑盒(美國Thermo公司,K1632# )說明,RT合成cDNA。根據(jù)GenBank中IL-10、TGF-β等mRNA核酸序列合成引物(表1,引物均由廣州Invitrogen公司合成),參照試劑盒(大連寶生物,DRR036A)進(jìn)行PCR。(3)PCR產(chǎn)物鑒定:取PCR產(chǎn)物10 μL在2%瓊脂糖可回收凝膠中,90 V電泳30 min,切膠回收純化,并測(cè)序。PCR產(chǎn)物測(cè)序結(jié)果與GenBank中被檢mRNA序列一致。(4)real-time PCR:采用SYBR Green試劑盒(大連寶生物,RR091Q),使用LightCycler 480II real-time PCR儀檢測(cè),并應(yīng)用LCS 1.5軟件分析,mRNA表達(dá)水平以目的基因/β-actin的比值表示。
1.5 酶聯(lián)免疫吸附試驗(yàn)(ELISA) 取已分離的試驗(yàn)組及對(duì)照者外周血B細(xì)胞,經(jīng)DETA CHaBEAD ?CD19(美國LifeTec公司,125.06 D)去除磁珠后,調(diào)整細(xì)胞濃度至1×105/mL,接種于含10%胎牛血清的RPMI1640培養(yǎng)基中,加入LPS(美國Sigma公司,L4397)至終濃度為10 ?g/mL的,于37 ℃孵育5 min,獲取上清;ELISA(美國eBioscience公司,BMS215INST)檢測(cè)IL-10蛋白水平,具體操作參照試劑說明書。
1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS 19.0軟件對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,計(jì)量資料用(x±s)表示,F(xiàn)檢驗(yàn)后采用獨(dú)立樣本t檢驗(yàn)分析,以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2 結(jié)果
2.1 Breg比例及CD80、CD86、CD1d表達(dá)檢測(cè)結(jié)果 經(jīng)FC檢測(cè),觀察到試驗(yàn)組外周血中CD19+CD24hiCD27+Breg細(xì)胞比例明顯低于正常對(duì)照組(P<0.05)。CD19+CD24hiCD27+Breg細(xì)胞共刺激分子CD80、CD86表達(dá)水平均明顯低于正常對(duì)照組(P<0.05),而CD1d的表達(dá)水平有下降趨勢(shì),但差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表2。
2.2 B細(xì)胞培養(yǎng)上清中IL-10檢測(cè)結(jié)果 經(jīng)ELISA法檢測(cè),觀察到試驗(yàn)組患兒B細(xì)胞培養(yǎng)上清中的IL-10濃度低于正常對(duì)照組[(2.60±1.77)pg/mL vs (5.20±4.51)pg/mL,P<0.05]。
2.3 CD19+B細(xì)胞相關(guān)因子mRNA檢測(cè)結(jié)果 real-time PCR檢測(cè)觀察到試驗(yàn)組患兒外周血CD19+B細(xì)胞中IL-10 mRNA與正常對(duì)照組比較,差異無統(tǒng)計(jì)學(xué)意義(P>0.05),而TGF-β mRNA較對(duì)照組升高,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05),見表3。
2.4 調(diào)節(jié)性T細(xì)胞及相關(guān)分子檢測(cè)結(jié)果 試驗(yàn)組患兒外周血CD4+CD25+Foxp3+Treg比例明顯低于正常對(duì)照組[(4.75±2.26)% vs(7.08±4.47)%,P<0.05]。經(jīng)real-time PCR檢測(cè)發(fā)現(xiàn),試驗(yàn)組患兒CD4+T細(xì)胞中T-bet mRNA下調(diào),IFNG、RORγt、IL-17A、IL-17F mRNA上調(diào),差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);兩組GATA3 mRNA比較,差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表4。分析CD19+CD24hiCD27+Breg與CD4+CD25+Foxp3+Treg相關(guān)性,發(fā)現(xiàn)兩者成正向相關(guān)關(guān)系(r=0.491,P<0.05),見圖1。
3 討論
哮喘是由氣道內(nèi)的炎癥細(xì)胞、結(jié)構(gòu)細(xì)胞和細(xì)胞組分共同參與的慢性炎癥性疾病,但其病因及發(fā)病機(jī)制仍未完全清楚。Breg細(xì)胞是一類產(chǎn)生IL-10的B細(xì)胞亞群,參與維持體內(nèi)免疫功能動(dòng)態(tài)平衡[6-9]。
隨著Breg在哮喘中的研究不斷深入,發(fā)現(xiàn)Breg在支氣管哮喘中的發(fā)病中起重要的負(fù)向調(diào)節(jié)作用[10-13]。有研究報(bào)道,通過將局限性吸入耐受(local inhaltional tolerance,LIT)小鼠的肺門淋巴結(jié)B細(xì)胞過繼轉(zhuǎn)移到卵白蛋白(ovalbumin,OVA)致敏小鼠,發(fā)現(xiàn)OVA致敏小鼠的組織炎癥明顯減輕,支氣管肺泡灌洗液中白細(xì)胞及嗜酸性粒細(xì)胞數(shù)量減少和氣道高反應(yīng)減輕[12-13]。van der Vlugt等[10]通過對(duì)13例過敏性哮喘患者的外周血檢測(cè),發(fā)現(xiàn)產(chǎn)生IL-10的CD24hiCD27+Breg不僅數(shù)量減少而且在受LPS刺激后分泌的IL-10也減少。
Kamekura等[11]在過敏性鼻炎和支氣管哮喘的研究中發(fā)現(xiàn)過敏性鼻炎患者和過敏性鼻炎合并哮喘的患者外周血中發(fā)現(xiàn)Breg的數(shù)量比健康對(duì)照人群減少,且過敏性鼻炎合并哮喘比過敏性鼻炎Breg的數(shù)量減少程度更大。這些研究提示Breg細(xì)胞可能在哮喘發(fā)病的免疫機(jī)制中起著重要作用。近幾年在人類Breg細(xì)胞的研究主要集中于CD19+CD24hiCD27+Breg細(xì)胞[14-15]。本組研究在未加任何絲裂原體外刺激培養(yǎng),真實(shí)反映體內(nèi)免疫活性細(xì)胞活化狀態(tài)的情況下,觀察到試驗(yàn)組急性發(fā)作時(shí)外周血CD19+CD24hiCD27+Breg細(xì)胞比例比正常對(duì)照組顯著降低(P<0.05)。提示CD19+CD24hiCD27+Breg細(xì)胞可能與哮喘的免疫發(fā)病機(jī)制有關(guān)。
Breg細(xì)胞主要通過分泌IL-10和表達(dá)CD80、CD86發(fā)揮直接免疫調(diào)節(jié)功能。IL-10可抑制樹突狀細(xì)胞(dendritic cells,DC)、MC等細(xì)胞的抗原提呈功能、降低MHCⅡ類分子表達(dá)及炎性細(xì)胞因子分泌,從而抑制T細(xì)胞活化[16-17]。應(yīng)用抗CD80、CD86單克隆抗體可逆轉(zhuǎn)Breg的抑制作用,而預(yù)先刪除Treg細(xì)胞并不影響B(tài)reg抑制效果,表明細(xì)胞間接觸機(jī)制可能是Breg介導(dǎo)抑制功能的主要方式之一,但不完全依賴于Treg間接作用[6,8,18]。本組研究發(fā)現(xiàn),哮喘患兒急性發(fā)作期外周血Breg細(xì)胞上清IL-10及細(xì)胞表面CD80、CD86表達(dá)顯著下調(diào)(P<0.05),提示Breg細(xì)胞免疫調(diào)節(jié)功能失調(diào)可能是導(dǎo)致哮喘患兒急性發(fā)作機(jī)制之一。除直接發(fā)揮免疫抑制作用外,Breg細(xì)胞還可以通過IL-10,和CD80、CD86促進(jìn)CD4+CD25+Foxp3+ Treg細(xì)胞增殖、活化,間接發(fā)揮抑制作用[7,18-19]。本研究觀察到,試驗(yàn)組外周血CD4+CD25+Foxp3+Treg細(xì)胞比例明顯降低,與CD19+CD24hiCD27+Breg細(xì)胞數(shù)量呈正相關(guān)關(guān)系,提示CD4+CD25+Foxp3+Treg細(xì)胞數(shù)量及功能異??赡苁荁reg細(xì)胞間接介導(dǎo)急性期哮喘患兒免疫功能紊亂的重要途徑。
IL-10可以抑制信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3(signal transducers and activatorsof transcription 3,STAT-3)的磷酸化,導(dǎo)致維甲酸相關(guān)孤兒核受體(retinnoid-relatedorphan nuclear receptor,ROR)-γt的表達(dá)水平降低,從而抑制了Th-17的分化;IL-10也可以抑制DC分泌IL-6和IL-12,抑制Th-17的分化,阻止抗原提呈,抑制單核細(xì)胞及巨噬胞產(chǎn)生促炎因子[20]。本研究發(fā)現(xiàn)CD4+T細(xì)胞中Th17特異性轉(zhuǎn)錄因子RORγt及IL-17A、IL-17F表達(dá)均上調(diào)的。提示急性期哮喘患兒的Breg比例下降,導(dǎo)致IL-10分泌減少,減少抑制信號(hào)轉(zhuǎn)導(dǎo)和STAT-3的磷酸化,導(dǎo)致RORγt的表達(dá)水平增高,從而促進(jìn)了Th-17的分化。間接發(fā)揮免疫調(diào)節(jié)機(jī)制。
新近的研究認(rèn)為Breg也可通過表達(dá)TGF-β直接或間接介導(dǎo)部分免疫抑制功能[21],但對(duì)于B細(xì)胞中產(chǎn)生的TGF-β到底是來源于Breg還是其他亞群目前仍有爭議。本研究發(fā)現(xiàn)CD19+B細(xì)胞中TGF-βmRNA表達(dá)上調(diào),與Breg細(xì)胞比例下降的結(jié)果相反。推測(cè)可能是分泌TGF-β的其他亞群細(xì)胞在急性期哮喘增加。文獻(xiàn)[22]報(bào)道人類CD5+Breg亞群可以大量分泌TGF-β,遺憾的是本研究未涉及到該群細(xì)胞的分析。因此TGF-β在哮喘患兒Breg功能異常中發(fā)揮的作用還有待于進(jìn)一步研究。
綜上所述,Breg細(xì)胞可通過分泌IL-10和表達(dá)共刺激分子直接或間接的抑制效應(yīng)性T細(xì)胞活化和炎性細(xì)胞因子產(chǎn)生,維持機(jī)體免疫平衡。Breg細(xì)胞比例降低和功能異??赡苁菍?dǎo)致急性期哮喘患兒免疫功能異?;罨脑蛑?。
參考文獻(xiàn)
[1]全國兒科哮喘協(xié)作組.第三次中國城市兒童哮喘流行病學(xué)調(diào)查[J].中華兒科雜志,2013,51(10):729-735.
[2] Bai J,Zhao J,Shen K L,et al.Current trends of the prevalence of childhood asthma in three Chinese cities:a multicenter epidemiological survey[J].Biomed Environ Sci,2010,23(6):453-457.
[3]蘇楠,楊萌,徐靜,等.門診支氣管哮喘患者控制現(xiàn)狀的調(diào)查分析[J].中國呼吸與危重監(jiān)護(hù)雜志,2007,6(2):97-100.
[4] Braman S S.The global burden of asthma[J].Chest,2006,130(1 Suppl):4S-12S.
[5] Douwes J,Gibson P,Pekkanen J,et al.Non-eosinophilic asthma:importance and possible mechanisms[J].Thorax,2002,57(7):643-648.
[6] Evans J G,Chavez-Rueda K A,Eddaoudi A,et al.Novel suppressive function of transitional 2 B cells in experimental arthritis[J].J Immunol,2007,178(12):7868-7878.
[7] Mauri C.Regulation of immunity and autoimmunity by B cells[J].Curr Opin Immunol,2010,22(6):761-767.
[8] Matsushita T,Horikawa M,Iwata Y,et al.Regulatory B cells(B10 cells) and regulatory T cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis[J].J Immunol,2010,185(4):2240-2252.
[9] Bouaziz J D,Yanaba K,Tedder T F.Regulatory B cells as inhibitors of immune responses and inflammation[J].Immunol Rev,2008,224:201-214.
[10] van der Vlugt L E,Mlejnek E,Ozir-Fazalalikhan A,et al.
CD24hi CD27+ B cells from patients with allergic asthma have impaired regulatory activity in response to lipopolysaccharide[J].ClinExp Allergy,2014,44(4):517-528.
[11] Kamekura R,Shigehara K,Miyajima S,et al.Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma[J].Clin Immunol,2015,158(2):204-211.
[12] Natarajan P,Singh A,McNamara J T,et al.Regulatory B cells from hilar lymph nodes of tolerant mice in a murine model of allergic airway disease are CD5+,express TGF-β,and co-localize with CD4+Foxp3+ T cells[J].Mucosal Immunol,2012,5(6):691-701.
[13] Singh A,Carson W F,Secor E R,et al.Regulatory role of B cells in a murine model of allergic airway disease[J].J Immunol,2008,180(11):7318-7326.
[14] Blair P A,Nore?a L Y,F(xiàn)lores-Borja F,et al.CD19+CD24hiCD38hi B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients[J].Immunity,2010,32(1):129-140.
[15] Iwata Y,Matsushita T,Horikawa M,et al.Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells[J].Blood,2011,117(2):530-541.
[16] Fiorentino D F,Zlotnik A,Mosmann T R,et al.IL-10 inhibits cytokine production by activated macrophages[J].J Immunol,1991,147(11):3815-3822.
[17] Sun C M,Deriaud E,Leclerc C,et al.Upon TLR9 signaling,CD5+B cells control the IL-12-dependent Th1-priming capacity of neonatal DCs[J].Immunity,2005,22(4):467-477.
[18] Blair P A,Nore?a L Y,F(xiàn)lores-Borja F,et al.CD19+CD24hi CD38hi B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients[J].Immunity,2010,32(1):129-140.
[19] Francisco L M,Salinas V H,Brown K E,et al.PD-L1 regulates the development, maintenance, and function of induced regulatory T cells[J].J Exp Med,2009,206(13):3015-3029.
[20] Bolland S.A newily discovered Fc receptor that explains IgG-isotype disparities in effector responses[J].Immunity,2005,23(1):2-4.
[21] Kessel A,Haj T,Peri R,et al.Human CD19+CD25hi B regulatory cells suppress proliferation of CD4+ T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells[J].Autoimmun Rev,2012,11(9):670-677.
[22] Lee J H,Noh J,Noh G,et al.Allergen-specific transforming growth factor-β-producing CD19+CD5+ regulatory B-cell(Br3) responses in human late eczematous allergic reactions to cows milk[J].J Interferon Cytokine Res,2011,31(5):441-449.
(收稿日期:2017-03-07) (本文編輯:程旭然)