黃琳惠,黃奕江,蔡興俊,莫儒冰
(海南省人民醫(yī)院呼吸內(nèi)科,海南海口570311)
支氣管哮喘小鼠外周血CD8+CD28-T細(xì)胞群和CD8+CD56+T細(xì)胞群作用機(jī)制研究
黃琳惠,黃奕江,蔡興俊,莫儒冰
(海南省人民醫(yī)院呼吸內(nèi)科,海南???70311)
目的研究哮喘小鼠外周血細(xì)胞與細(xì)胞間的關(guān)系及兩種細(xì)胞群在哮喘中的作用。方法30只BALB/c小鼠隨機(jī)分為對(duì)照組和哮喘組,每組各15只。測(cè)定小鼠的氣道反應(yīng)性,對(duì)支氣管肺泡灌洗液行細(xì)胞學(xué)分類,觀察肺組織的病理變化,流式檢測(cè)小鼠外周血細(xì)胞及細(xì)胞的比例,分析哮喘中兩種細(xì)胞之間的相關(guān)性。結(jié)果成功建立小鼠哮喘模型。①哮喘組小鼠BALF中細(xì)胞總數(shù)和嗜酸性粒細(xì)胞(%)較對(duì)照組明顯增高;②哮喘組小鼠外周血細(xì)胞及細(xì)胞比例均較對(duì)照組升高;③細(xì)胞與細(xì)胞數(shù)量上呈正相關(guān)(r=0.737,P=0.002)。結(jié)論哮喘時(shí)外周血細(xì)胞和細(xì)胞數(shù)目異常,兩種細(xì)胞正相關(guān),可能在哮喘發(fā)病中起重要作用。
哮喘小鼠模型;氣道反應(yīng)性;支氣管肺泡灌洗液;外周血;細(xì)胞
臨床研究顯示,長(zhǎng)期使用皮質(zhì)激素可能會(huì)抑制免疫系統(tǒng)的調(diào)節(jié)功能,破壞免疫穩(wěn)定,造成哮喘難治,因此,在哮喘的發(fā)病源頭或其上游抑制其變態(tài)反應(yīng)或建立調(diào)節(jié)性T細(xì)胞(Regulatory T cell,Tr)的抑制作用可能是治療哮喘的研究方。哮喘中氣道有大量Tr細(xì)胞浸潤(rùn)。Tr細(xì)胞中主要的細(xì)胞表型為,后者能產(chǎn)生各種細(xì)胞因子發(fā)揮細(xì)胞毒效應(yīng)[3],并與各種細(xì)胞相互作用。本文通過新的方法建立哮喘小鼠模型,研究其外周血T細(xì)胞在哮喘發(fā)病中的作用及其臨床意義。
1.1 實(shí)驗(yàn)材料
1.1.1 動(dòng)物SPF級(jí)雌性BALB/c小鼠30只,3~5周齡,體重16~18 g,海南醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供。
1.1.2 主要試劑和儀器卵蛋白(OVA):美國(guó)Sigma公司;FITC anti-mouse CD8a,PE anti-mouse CD28,PE anti-mouse CD56a:美國(guó)biolegend公司;超聲波霧化器:廣東粵華牌。
1.2 實(shí)驗(yàn)方法
1.2.1 實(shí)驗(yàn)分組及模型制備30只SPF級(jí)雌性BALB/c小鼠隨機(jī)分為哮喘組(n=15)和對(duì)照組(n=15)。哮喘組第0、14天給予小鼠腹腔注射致敏,每只每次給予含Ⅱ級(jí)OVA 5 mg及氫氧化鋁5 mg的生理鹽水混懸液200μl。第21天開始對(duì)小鼠給予4%OVA生理鹽水霧化液40 ml進(jìn)行霧化激發(fā),1次/d,每次40 min,連續(xù)7 d。對(duì)照組第0、14天腹腔注射致敏,每只每次給予含氫氧化鋁5 mg的生理鹽水混懸液200μl。于第21天開始對(duì)小鼠給予生理鹽水40 ml進(jìn)行霧化激發(fā),頻次和持續(xù)時(shí)間同哮喘組。
1.2.2 肺功能檢查末次激發(fā)48 h后,Buxco小鼠肺功能儀無創(chuàng)肺阻抗法測(cè)定小鼠的氣道反應(yīng)性。
1.2.3 肺泡灌洗液(BALF)細(xì)胞計(jì)數(shù)對(duì)小鼠氣管插管,PBS灌洗。BALF細(xì)胞涂片。固定后染色,連續(xù)計(jì)數(shù)400個(gè)細(xì)胞并行細(xì)胞分類,計(jì)算各細(xì)胞百分比。
1.2.4 肺組織病理學(xué)觀察將左肺上葉固定后包埋、切片、HE染色。
1.2.5 流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面分子采集小鼠外周血,處理后行相應(yīng)熒光抗體染色,流式檢測(cè),設(shè)立同型對(duì)照。
1.3 統(tǒng)計(jì)學(xué)方法數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x-±s)表示。采用SPSS13.0統(tǒng)計(jì)軟件分析。相關(guān)性檢驗(yàn)采用Pearson直線相關(guān)性分析;兩組間樣本均數(shù)的比較行t檢驗(yàn)。以P<0.05表示差別有統(tǒng)計(jì)學(xué)意義。
2.1 肺功能檢查結(jié)果采用無創(chuàng)肺阻抗法,通過小鼠肺功能儀測(cè)定小鼠的氣道反應(yīng)性。給予300 μl生理鹽水霧化1 min,然后再測(cè)定濃度遞增的乙酰甲膽堿(Mch;3.12 mg/ml、6.25 mg/ml、12.50 mg/ml、25.00 mg/ml、50.00 mg/ml)霧化激發(fā)小鼠氣道反應(yīng)性的變化,每次霧化1 min,記錄3 min,見表1。
表1 氣道反應(yīng)性(mg/ml)
表1 氣道反應(yīng)性(mg/ml)
組別對(duì)照組哮喘組t值P值生理鹽水0.542±0.087 0.573±0.109 0.714 0.484 Mch3.12 0.628±0.073 0.684±0.076 1.678 0.111 Mch 6.25 0.675±0.176 1.260±0.086 9.463 0.000 Mch 12.5 1.085±0.370 2.585±0.423 8.437 0.000 Mch 25.00 1.887±0.491 3.980±1.051 5.705 0.000 Mch 50.00 2.606±0.793 7.895±1.415 10.314 0.000
2.2 BALF細(xì)胞總數(shù)和分類比較①BALF細(xì)胞總數(shù):哮喘組顯著高于對(duì)照組,兩組間差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。②BALF嗜酸性粒細(xì)胞百分率:哮喘組顯著高于對(duì)照組,兩組間差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。③BALF中性粒細(xì)胞百分率和淋巴細(xì)胞百分率:哮喘組顯著高于對(duì)照組,兩組間差異有統(tǒng)計(jì)學(xué)意義(P<0.01),見表2。
表2 兩組BALF中細(xì)胞總數(shù)和分類的比較(n=15,%)
表2 兩組BALF中細(xì)胞總數(shù)和分類的比較(n=15,%)
組別對(duì)照組哮喘組t值P值例數(shù)15 15 Neu 5.67±1.45 30.63±4.88 -11.479 0.000細(xì)胞總數(shù)(×105/ml) 0.52±0.43 1.72±0.15 -14.152 0.000 Eos 0.72±1.09 27.11±4.06 23.328 0.000 Lym 10.52±3.11 21.48±4.30 5.129 0.000
2.3 肺組織病理學(xué)觀察結(jié)果哮喘組支氣管、血管周圍、肺泡腔內(nèi)可見嗜酸性粒細(xì)胞、淋巴細(xì)胞浸潤(rùn),氣道上皮損傷,杯狀細(xì)胞肥大增生,氣道內(nèi)黏液栓形成。對(duì)照組肺組織氣道結(jié)構(gòu)清晰,氣道纖毛上皮排列整齊,無明顯炎癥改變,見圖1。
2.4 外周血CD8+CD28-T、CD8+CD56+T細(xì)胞流式檢測(cè)結(jié)果哮喘組和對(duì)照組CD8+CD28-T細(xì)胞分別為(13.65±3.075)%和(10.32±2.318)%,CD8+CD56+T細(xì)胞分別為(4.11±1.47)%和(2.59±1.19)%,兩組比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);CD8+CD28-T細(xì)胞與CD8+CD56+T細(xì)胞存在正相關(guān)(r=0.737,P=0.002),見圖2、圖3和圖4。
圖1 病理切片(×200)
圖2 外周血T細(xì)胞流式檢測(cè)結(jié)果
圖3 外周血CD8+CD56+T細(xì)胞流式檢測(cè)結(jié)果
圖4 CD8+CD28-T細(xì)胞與CD8+CD56+T細(xì)胞的相關(guān)分析
支氣管哮喘是T淋巴細(xì)胞參與的體液免疫過程,也是T淋巴細(xì)胞活化、增殖過強(qiáng),凋亡減弱的過程。Filaci等[2]發(fā)現(xiàn),CD8+調(diào)節(jié)T淋巴細(xì)胞有三種不同亞群,Ⅰ、Ⅱ型T細(xì)胞表型為CD8+CD28-。本研究通過建立哮喘小鼠模型,檢測(cè)小鼠外周血CD8+CD28-T細(xì)胞及CD8+CD56+T細(xì)胞的比例,分析兩種細(xì)胞亞型之間的相關(guān)性,探討CD8+CD28-T細(xì)胞和CD8+CD56+T細(xì)胞在哮喘發(fā)病中的作用。
哮喘是以大量表達(dá)CD8+CD56+細(xì)胞和CD8+CD28-T細(xì)胞為特點(diǎn)[4],我們的實(shí)驗(yàn)也證明了這點(diǎn)。上述兩種細(xì)胞亞群都存在于健康和哮喘小鼠,但在哮喘小鼠外周血中,CD8+CD28-T細(xì)胞及CD8+CD56+T細(xì)胞亞型的數(shù)量均較對(duì)照組明顯增加。Chamberlain等[5]發(fā)現(xiàn),CD8+T細(xì)胞效應(yīng)性細(xì)胞毒作用主要存在于CD28-細(xì)胞群,CD28表達(dá)缺失標(biāo)志著作用上分化成為細(xì)胞毒性記憶細(xì)胞。其他研究也證明,外周血CD8+CD28-T細(xì)胞克隆有著較短的質(zhì)粒,對(duì)凋亡有著更強(qiáng)的抵抗[6]。在哮喘和COPD患者及其他自身免疫異常的慢性炎癥,如多發(fā)性硬化[7-8]患者外周血都有CD8+CD28-T細(xì)胞和穿孔素的表達(dá)較健康人的明顯增加,CD8+CD28-T細(xì)胞即是以大量產(chǎn)生穿孔素、TNF-α、IFN-γ從而在自身免疫疾病中發(fā)揮調(diào)節(jié)作用為特點(diǎn)的細(xì)胞群[9]。哮喘加劇期間,炎癥的表現(xiàn)更傾向于抗病毒反應(yīng),而不是對(duì)抗嗜酸性粒細(xì)胞為主的免疫應(yīng)答[10],提示T細(xì)胞在介導(dǎo)哮喘及決定糖皮質(zhì)激素治療的有效性方面有著獨(dú)立作用。
CD8+CD56+細(xì)胞是哮喘中另一主要的細(xì)胞亞型。它們與CD8+CD28-細(xì)胞在哮喘中存在著密切的正相關(guān)(r=0.737)。CD56,CD158b等標(biāo)志是常見自然殺傷(NKT)細(xì)胞的受體,Umetsu等[11]證明NKT細(xì)胞和T細(xì)胞在哮喘中的啟動(dòng)作用及它們?cè)跈C(jī)體中同時(shí)對(duì)TH2細(xì)胞因子、CD8+細(xì)胞、NKT細(xì)胞進(jìn)行調(diào)控的關(guān)鍵步驟。因此,CD8+CD56+和CD8+CD28-T細(xì)胞在哮喘的發(fā)病過程中是存在密切聯(lián)系的,但是要了解這兩種細(xì)胞在哮喘中的相互作用細(xì)節(jié),還需要更進(jìn)一步的研究。
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Function and mechanism of CD8+CD28-T cells and CD8+CD28-T cells in peripheral blood of asthmatic mouse model.
HUANG Lin-hui,HUANG Yi-jiang,CAI Xing-jun,MO Ru-bing.Department of Respiratory Medicine,People's Hospital of Hainan Province,Haikou 570311,Hainan,CHINA
ObjectiveTo examine the relationship betweenT cells in peripheral blood of mice and the function of the two subsets in the development of asthma.MethodsThirty Balb/c mice were randomly divided into two groups:asthma group,control group,with 15 mice in each group.The airway responsiveness were measured,bronchoalveolar lavage cytology was performed and lung tissue sections were prepared for histopathologic examination to evaluate the airway inflammation,examine the fraction ofT cells by flow cytometry and correlation analysis.ResultsThe mouse models established successfully.①The total cell number and the percentages of eosinophils in BALF of the asthmatic group were significantly higher than that in the control group;②The percentages ofT cells in peripheral blood[(13.65±3.075)vs(4.11± 1.47)]in asthmatic group were significantly higher than that in control group[(10.32±2.318)vs(2.59±1.19)].③There was a strong positive correlation betweenT cells(r=0.737,P=0.002).ConclusionThe fractions ofT cells are associated with asthma ongoing closely,and there is a strong positive correlation between these two subsets.
Airway responsiveness;Bronchoalveolar lavage fluid;Peripheral blood;CD56+T cell
R-332
A
1003—6350(2013)18—2658—03
10.3969/j.issn.1003-6350.2013.18.1107
2013-05-15)
黃琳惠。E-mail:rochelle-11@163.com