黃自坤 李 雪 鄧 楨 羅 清
(南昌大學(xué)第一附屬醫(yī)院檢驗科,南昌 330006)
類風(fēng)濕關(guān)節(jié)炎(Rheumatoid arthritis,RA)是一種病因尚未闡明的慢性全身性炎癥性的自身免疫性疾病,其主要特點(diǎn)是不可逆的關(guān)節(jié)炎癥和損傷。RA病因復(fù)雜,可能與遺傳、環(huán)境、感染和體內(nèi)激素水平等因素有關(guān)[1]。雖然RA致病機(jī)制還不明確,但已有大量的研究表明單核細(xì)胞/巨噬細(xì)胞、中性粒細(xì)胞、淋巴細(xì)胞等參與RA的發(fā)生和發(fā)展[2]。單核細(xì)胞/巨噬細(xì)胞可通過分泌大量的炎性細(xì)胞因子來維持RA患者的炎性環(huán)境以及招募大量的免疫細(xì)胞遷移至關(guān)節(jié)炎,導(dǎo)致關(guān)節(jié)不可逆的損傷。近年來,人類單核細(xì)胞分為三個亞群:經(jīng)典型(CD14highCD16-)、中間型(CD14highCD16+)和非經(jīng)典型(CD14lowCD16++),不同的亞群具有不同的表型和功能[3]。簇分化抗原64(Cluster of differentiation antigen,CD64)是IgG Fc 段受體Ⅰ(FcγRⅠ),為高親和性受體。CD64 是連接機(jī)體體液免疫和細(xì)胞免疫的橋梁,在細(xì)胞吞噬、清除免疫復(fù)合物、抗原遞呈和刺激炎性介質(zhì)釋放中發(fā)揮至關(guān)重要的作用[4]。該研究通過檢測RA患者和正常對照者外周血單核細(xì)胞亞群的比例及其表面CD64的表達(dá)(包括平均熒光強(qiáng)度和百分率),并分析其與疾病活動度、炎癥程度和自身抗體的關(guān)系,探討不同單核細(xì)胞亞群中CD64在RA發(fā)病中的作用。
1.1材料
1.1.1臨床資料 選取2016年6月至2017年6月于南昌大學(xué)第一附屬醫(yī)院風(fēng)濕免疫科就診的44例RA患者,其中男8例,女36例,平均年齡(57.5±11.9)歲,臨床診斷符合美國風(fēng)濕病學(xué)會(ACR)1987年的RA診斷標(biāo)準(zhǔn)[5],并排除嚴(yán)重糖尿病、高血壓、高血脂、心腦血管疾病、肝腎疾病、血栓性疾病、血小板疾病等其他疾病。正常對照組22例,均為健康志愿者,排除炎癥或其他自身免疫性疾病,男4例,女18例,平均年齡(51.2±11.6)歲。兩組性別、年齡無顯著性差異。詳細(xì)收集RA患者臨床資料及相關(guān)實(shí)驗室檢查數(shù)據(jù),并計算其RA疾病活動性評分(Disease activity score 28,DAS28)[6]。根據(jù)病程的長短將46例RA患者分為新發(fā)病例組(病程小于6個月)5例和復(fù)發(fā)病例組(病程大于6個月)39例[7]。所有的RA患者在采集標(biāo)本前都使用了抗風(fēng)濕病藥物的治療。本實(shí)驗經(jīng)過醫(yī)院倫理委員會批準(zhǔn),患者和健康人均簽署知情同意書。
1.1.2試劑和儀器 流式細(xì)胞抗體藻紅蛋白標(biāo)記的CD64抗體(Phycoerythrin- CD64,PE- CD64)、異硫氰酸熒光素標(biāo)記的CD40抗體(Fluorescein isothiocyanate- CD40,F(xiàn)ITC- CD40)購自美國eBioscience公司,藻紅蛋白- 得克薩斯紅標(biāo)記的CD14抗體(Phycoerythrin- texas red,ECD- CD14)、藻紅蛋白- Cy5標(biāo)記的CD16抗體(PhycoerythrinCy5- CD16,PE- Cy5- CD16)及相應(yīng)的同型對照PE- IgGl和FITC- IgG1均購自美國Beckman Coulter公司。Cytomics FC 500流式細(xì)胞儀購自美國Beckman Coulter公司,分析軟件為儀器自帶的CXP分析系統(tǒng)。
1.2方法
1.2.1外周血單個核細(xì)胞(Peripheral blood mononuclear cell,PBMC)提取 采集受試者清晨空腹EDTA抗凝外周血5 ml,6 h內(nèi)送檢。采用 Ficoll- Paque 分離液 (Sigma,USA)提取RA患者和對照者的PBMC。
1.2.2流式細(xì)胞檢測 各取受試者100 μl 生理鹽水重懸的PBMC加入試管中,按以下組合加入單克隆抗體各10 μl:ECD- CD14、PC5- CD16、PE- IgG1、FITC- IgG1;ECD- CD14、PC5- CD16、FITC- CD40、PE- CD64,4℃避光孵育30 min,室溫1 500 r/min離心5 min,棄上清液,用生理鹽水洗滌2次,用500 μl生理鹽水重懸上機(jī)檢測。Cytomics FC 500流式細(xì)胞儀進(jìn)行分析,使用CXP分析軟件分析經(jīng)典型、中間型、非經(jīng)典型單核細(xì)胞的比例及各單核細(xì)胞亞群CD40和CD64的表達(dá)水平。
1.2.3細(xì)胞因子檢測 血清細(xì)胞因子IL- 6、IL- 8和IL- 10采用酶聯(lián)免疫吸附法(美國 Signalway Antibody公司)。
1.2.4RA的其他指標(biāo)檢測方法 CRP和RF采用速率散色比濁法(美國Beckman Coulter公司),ACPA抗體采用酶聯(lián)免疫吸附法(上海科新生物技術(shù)股份有限公司),ESR采用動態(tài)血沉儀法(北京普利生公司)。
1.3統(tǒng)計學(xué)處理 運(yùn)用統(tǒng)計軟件SPSS17.0軟件對數(shù)據(jù)進(jìn)行描述和分析。首先進(jìn)行正態(tài)性檢驗,符合正態(tài)性分布數(shù)據(jù),兩組間比較采用t檢驗,否則采用非參數(shù)檢驗。兩變量為正態(tài)分布數(shù)據(jù),相關(guān)性采用Pearson相關(guān)分析,否則采用Spearman 相關(guān)分析。以雙側(cè)P<0.05為差異有統(tǒng)計學(xué)意義。
2.1RA患者和對照組外周血單核細(xì)胞亞群比例的比較 外周血單個核細(xì)胞經(jīng)流式抗體標(biāo)記后,流式細(xì)胞儀采用前向散射光(FS)、側(cè)向散射光(SS)和CD14區(qū)別單核細(xì)胞(圖1A),根據(jù)CD14和CD16的表達(dá)情況測定單核細(xì)胞亞群比例(圖 1A:P1、P2、P3分別為經(jīng)典型、中間型和非經(jīng)典型單核細(xì)胞)。結(jié)果如圖1所示,RA組經(jīng)典型單核細(xì)胞亞群比例低于對照組,差異有統(tǒng)計學(xué)意義(P=0.024);中間型單核細(xì)胞亞群比例高于對照組,差異有統(tǒng)計學(xué)意義(P<0.001);非經(jīng)典型單核細(xì)胞亞群比例低于對照組,差異有統(tǒng)計學(xué)意義(P=0.004)。
2.2RA患者和對照組外周血單核細(xì)胞各亞群CD64和CD40表達(dá)水平的比較 結(jié)果如表1所示,RA組經(jīng)典型、中間型、非經(jīng)典型單核細(xì)胞CD64表達(dá)水平明顯高于對照組,差異有統(tǒng)計學(xué)意義(P=0.036,P= 0.004,P<0.001);RA組經(jīng)典型、中間型、非經(jīng)典型單核細(xì)胞CD64表達(dá)百分率與對照組比較差異無統(tǒng)計學(xué)意義(P>0.05);RA組非經(jīng)典型單核細(xì)胞CD40表達(dá)水平明顯高于對照組,差異有統(tǒng)計學(xué)意義(P<0.001);而其他各組之間比較差異無統(tǒng)計學(xué)意義(P>0.05)。
2.3RA患者外周血單核細(xì)胞亞群CD64表達(dá)水
平與DAS28評分相關(guān)性分析 結(jié)果如圖2所示,RA患者外周血經(jīng)典型單核細(xì)胞CD64表達(dá)水平、中間型單核細(xì)胞CD64表達(dá)水平與DAS28呈正相關(guān)(rs=0.308,P=0.044;rs=0.302,P=0.049);而RA患者外周血非經(jīng)典型單核細(xì)胞CD64表達(dá)水平與DAS28無明顯相關(guān)性(rs=0.262,P=0.090)。
圖1 RA患者和對照組血單核細(xì)胞亞群比例的比較Fig.1 Proportions of each monocytes subsets between RA patients and CONNote: A.Analysis of monocytes subsets by flow cytometry;B.The proportions of classical monocytes was significantly increased in RA patients compared to healthy controls;C.The proportions of classical monocytes was significantly increased in healthy controls compared to RA patients;D.The proportions of classical monocytes was significantly increased in RA patients compared to healthy controls.
表1RA患者和對照組單核細(xì)胞各亞群CD64和CD40表達(dá)的比較
Tab.1ExpressionofCD64andCD40onmonocytessubsetsinRApatientsandhealthycontrols
GroupsHealthycontrolsRAt/zvaluePvalueClassicalmonocytesCD64(%)99.83±0.4699.80±0.650.180.860CD64(MFI)29.68±8.2139.32±17.74329.000.036CD40(%)24.93±14.6339.88±28.95226.000.142CD40(MFI)2.78±2.203.07±1.700.530.598IntermediatemonocytesCD64(%)99.34±1.3699.16±2.07422.500.397CD64(MFI)29.82±8.3543.19±19.14269.000.004CD40(%)43.34±17.7443.15±27.870.030.980CD40(MFI)5.14±2.975.45±4.270.270.792NonclassicalmonocytesCD64(%)59.02±19.3666.69±22.731.230.222CD64(MFI)11.61±4.8525.87±12.33136.00<0.001CD40(%)48.14±19.5342.87±28.270.680.500CD40(MFI)4.40±1.096.66±2.4695.00<0.001
2.4RA患者外周血單核細(xì)胞亞群CD64表達(dá)水平與炎癥指標(biāo)相關(guān)性分析 結(jié)果如圖3所示,RA患者外周血各單核細(xì)胞亞群CD64表達(dá)水平與ESR,CRP呈正相關(guān)(rs=0.410,P=0.008;rs=0.475,P=0.003;rs=0.448,P=0.003;rs=0.473,P=0.004;rs=0.348,P=0.026;rs=0.340,P=0.042)。
2.5RA患者外周血單核細(xì)胞亞群CD64表達(dá)水平與自身抗體的關(guān)系 結(jié)果如圖4所示,RA患者RF陽性組、ACPA陽性組外周血經(jīng)典型單核細(xì)胞上CD64表達(dá)水平明顯高于對應(yīng)的陰性組,差異有統(tǒng)計學(xué)意義(P=0.004;P=0.046);RA患者RF陽性組、ACPA陽性組外周血中間型單核細(xì)胞上CD64表達(dá)水平明顯高于對應(yīng)的陰性組,差異有統(tǒng)計學(xué)意義(P=0.004;P=0.042);RA患者RF陽性組、ACPA陽性組外周血非經(jīng)典型單核細(xì)胞上CD64表達(dá)水平與對應(yīng)的陰性組差異無統(tǒng)計學(xué)意義(P=0.813;P=0.672)。
圖2 RA患者單核細(xì)胞亞群CD64表達(dá)水平與DAS28相關(guān)性分析Fig.2 Correlation of expression of CD64 on monocy- tes subsets in RA patients with DAS28Note: A.The expression of CD64 on classical monocytes in RA patients correlated significantly with DAS28;B.The expression of CD64 on intermediate monocytes in RA patients correlated significantly with DAS28;C.The expression of CD64 on nonclassical monocytes in RA patients did not correlate with DAS28.
2.6新發(fā)和復(fù)發(fā)RA患者外周血單核細(xì)胞亞群的CD64表達(dá)水平的比較 結(jié)果如圖5所示,外周血單核細(xì)胞亞群上CD64表達(dá)水平在新發(fā)RA患者與復(fù)發(fā)RA患者之間的差異無統(tǒng)計學(xué)意義(P>0.05)。
2.7RA患者外周血單核細(xì)胞亞群CD64表達(dá)水平與細(xì)胞因子的關(guān)系 結(jié)果如表2所示,RA患者血清細(xì)胞因子IL- 6和IL- 8水平明顯高于對照組,差異
表2RA患者和對照組血清細(xì)胞因子水平的比較
Tab.2Serumlevelsofinflammatorycytokinesatbaselineinpatientswithrheumatoidarthritis(RA)andhealthycontrols
GroupsRA(n=31)Healthycontrols(n=22)t/zvaluePvalueIL-640.94±8.9519.77±6.24159.000.001IL-8103.30±23.7645.70±9.41226.000.039IL-1022.71±2.3521.24±1.77333.500.899
圖3 RA患者單核細(xì)胞亞群CD64表達(dá)水平與炎性指標(biāo)相關(guān)性分析Fig.3 Correlation of expression of CD64 on monocytes subsets in RA patients with inflammatory markersNote: A.The expression of CD64 on classical monocytes in RA patients correlated significantly with ESR;B.The expression of CD64 on intermediate monocytes in RA patients correlated significantly with ESR;C.The expression of CD64 on nonclassical monocytes in RA patients correlated significantly with ESR;D.The expression of CD64 on classical monocytes in RA patients correlated significantly with CRP;E.The expression of CD64 on intermediate monocytes in RA patients correlated significantly with CRP;F.The expression of CD64 on nonclassical monocytes in RA patients correlated significantly with CRP.
圖4 RA患者單核細(xì)胞亞群CD64表達(dá)水平與自身抗體的關(guān)系Fig.4 Correlation of expression of CD64 on monocytes subsets in RA patients with autoantibodyNote: A.The expression of CD64 on classical monocytes was significantly increased in RA patients with positive RF compared to RA patients with negative RF;B.The expression of CD64 on intermediate monocytes was significantly increased in RA patients with positive RF compared to RA patients with negative RF;C.No significant difference was observed in the expression of CD64 on nonclassical monocytes between RA patients with positive RF and RA patients with negative RF;D.The expression of CD64 on classical monocytes was significantly increased in RA patients with positive ACPA compared to RA patients with negative ACPA;E.The expression of CD64 on intermediate monocytes was significantly increased in RA patients with positive ACPA compared to RA patients with negative ACPA;F.No significant difference was observed in the expression of CD64 on nonclassical monocytes between RA patients with positive ACPA and RA patients with negative ACPA.
表3RA患者外周血單核細(xì)胞亞群CD64表達(dá)水平與細(xì)胞因子的關(guān)系
Tab.3AssociationbetweenexpressionofCD64onmonocytesubsetsandserumcytokineconcentration
GroupsRAhighRAlowt/zvaluePvalueClassicalmonocytesIL-660.58±17.9624.77±4.6776.000.092IL-862.54±20.06136.8±38.7978.000.108IntermediatemonocytesIL-663.72±17.5022.19±4.5256.000.013IL-863.13±19.97136.30±38.8889.000.242NonclassicalmonocytesIL-627.31±4.9755.49±17.2495.000.333IL-8118.30±41.5687.25±22.13114.500.843
圖5 新發(fā)RA患者和復(fù)發(fā)RA患者單核細(xì)胞亞群CD64表達(dá)水平Fig.5 Expression of CD64 on monocytes subsets in new- onset and revisiting RA patientsNote: A.No significant difference was observed in the expression of CD64 on classical monocytes between new- onset and re- visiting RA patients;B.No significant difference was observed in the expression of CD64 on intermediate monocytes between new- onset and re- visiting RA patients;C.No significant difference was observed in the expression of CD64 on nonclassical monocytes between new- onset and re- visiting RA patients
有統(tǒng)計學(xué)意義(P<0.05);RA患者血清細(xì)胞因子IL- 10水平與對照組比較無統(tǒng)計學(xué)意義(P>0.05)。以表1中各單核細(xì)胞亞群上CD64表達(dá)水平的均數(shù)作為界限分別將RA分成兩組RAhigh(經(jīng)典型CD64>39.32;中間型CD64>43.19;非經(jīng)典型CD64>25.87)和RAlow(經(jīng)典型CD64<39.32;中間型CD64<43.19;非經(jīng)典型CD64<25.87),比較各單核細(xì)胞亞群中這兩組血清細(xì)胞因子IL- 6和IL- 8的差異,結(jié)果如表3所示,中間型單核細(xì)胞RAhigh組血清細(xì)胞因子IL- 6水平明顯高于RAlow組,差異有統(tǒng)計學(xué)意義(P=0.013),其他組間無差異。
單核細(xì)胞是一群異質(zhì)性的細(xì)胞,根據(jù)CD14和CD16表達(dá)情況其可分為三種類型:經(jīng)典型(CD14++CD16-)、中間型(CD14++CD16+)和非經(jīng)典型(CD14+CD16++)。三個亞群在表型、功能及炎癥活化潛能方面存在著明顯的差異。經(jīng)典型單核細(xì)胞具有很強(qiáng)的吞噬功能和炎癥調(diào)節(jié)能力[8,9];中間型單核細(xì)胞具有很強(qiáng)的促炎作用,在急性炎癥中可明顯上調(diào)其比例[10,11];非經(jīng)典型單核細(xì)胞能識別炎癥信號并迅速遷移到炎癥部位[12]。之前的研究已證實(shí)單核細(xì)胞在RA發(fā)生和發(fā)展中發(fā)揮著重要的作用[2],而RA的發(fā)病和嚴(yán)重程度可能與單核細(xì)胞亞群的異常有關(guān)。雖然有研究證實(shí)RA患者經(jīng)典型單核細(xì)胞比例降低、中間型單核細(xì)胞比例升高,但對于非經(jīng)典型單核細(xì)胞的比例升高還存在爭議[13- 15]。與此研究一致的是,Lacerte等[14]發(fā)現(xiàn)RA患者經(jīng)典型單核細(xì)胞比例降低、中間型單核細(xì)胞比例和非經(jīng)典型單核細(xì)胞比例升高。不同研究結(jié)果不一致的原因可能是納入研究對象的病程以及用藥情況不一致。
簇分化抗原64(cluster of differentiation antigen,CD64)是IgG Fc 段受體Ⅰ(FcγRⅠ),為高親和性受體,是連接機(jī)體體液免疫和細(xì)胞免疫的橋梁,在細(xì)胞吞噬、清除免疫復(fù)合物、抗原遞呈和刺激炎性介質(zhì)釋放中發(fā)揮至關(guān)重要的作用[4]。RA患者外周血中單核細(xì)胞CD64的表達(dá)升高,并與疾病嚴(yán)重程度相關(guān)[16,17]。RA滑膜組織CD64表達(dá)升高,并可作為RA診斷標(biāo)志物[18]。本次實(shí)驗證實(shí),RA患者外周血單核細(xì)胞亞群高表達(dá)CD64,與此研究一致的是,Rossol等[19]研究表明RA患者外周血單核細(xì)胞亞群CD64表達(dá)明顯上升。此外,通過分析RA患者外周血單核細(xì)胞亞群CD64表達(dá)的臨床意義發(fā)現(xiàn),RA患者經(jīng)典型單核細(xì)胞和中間型單核細(xì)胞上CD64的表達(dá)情況與DAS28呈正相關(guān),說明經(jīng)典型單核細(xì)胞和中間型單核細(xì)胞上CD64分子的表達(dá)水平與疾病嚴(yán)重程度呈正比,患者疾病越嚴(yán)重,CD64分子的表達(dá)水平越高;且RA患者經(jīng)典型單核細(xì)胞和中間型單核細(xì)胞上表達(dá)的 CD64 與 ESR、CRP呈正相關(guān);另外,RA 患者經(jīng)典型單核細(xì)胞和中間型單核細(xì)胞上CD64的表達(dá)水平與自身抗體 RF 及 CCP 含量相關(guān)。而并沒有發(fā)現(xiàn)非經(jīng)典型單核細(xì)胞上CD64的表達(dá)水平與DAS28、自身抗體 RF 及 CCP 含量相關(guān)。這可能的原因是:一方面各單核細(xì)胞亞群所執(zhí)行的功能不同;另一方面CD64作為一種高親和力的活性受體,能結(jié)合免疫球蛋白和CRP等分子參與炎癥的調(diào)節(jié)[16,20,21]。
本研究通過檢測RA患者和對照者血清細(xì)胞因子IL- 6、IL- 8和IL- 10水平發(fā)現(xiàn),RA患者血清IL- 6和IL- 8水平升高,與此研究一致的是,Tsukamoto等[13]研究表明RA患者血清IL- 6和IL- 8水平明顯上升。通過分析RA患者外周血單核細(xì)胞亞群上CD64的表達(dá)與血清中IL- 6和IL- 8的水平關(guān)系發(fā)現(xiàn),高表達(dá)CD64的中間型單核細(xì)胞組的IL- 6水平明顯升高,說明中間型單核細(xì)胞表達(dá)的CD64與其分泌的細(xì)胞因子IL- 6有關(guān)。IL- 6在可溶性IL- 6受體(sIL- 6R)存在下能促進(jìn)血管內(nèi)皮生長因子的產(chǎn)生,而血管內(nèi)皮生長因子可促進(jìn)內(nèi)皮細(xì)胞的增殖與遷移,從而改變血管通透性,以此介導(dǎo)炎癥反應(yīng),這與 RA 血管翳的形成密切相關(guān)[22]。RA患者血清中IL- 6在疾病活動期升高顯著,且與RA臨床表現(xiàn)相關(guān)[23],針對 IL- 6的生物制劑對部分患者有肯定的療效[24]。
此外,需要指出的是,本文尚存在一些不足:(1)新發(fā)RA患者的研究樣本不夠多;(2)沒有檢測未用藥的新發(fā) RA 患者,因此我們無法了解在沒有藥物干擾情況下,外周血單核細(xì)胞亞群上CD64在RA中的作用;(3)由于單核細(xì)胞經(jīng)過LPS等刺激劑作用后CD16會消失[25],因此未直接檢測單核細(xì)胞亞群分泌細(xì)胞因子的能力。本研究在后續(xù)的研究中將進(jìn)一步擴(kuò)大樣本量檢測未用藥新發(fā)RA 患者CD64的表達(dá)情況,使研究結(jié)果更具有可靠性。
綜上所述,RA 患者外周血經(jīng)典型單核細(xì)胞和中間型單核細(xì)胞表達(dá)CD64升高,且其表達(dá)與疾病活動度及自身抗體產(chǎn)生有關(guān),高表達(dá)CD64的中間型單核細(xì)胞分泌較多的促炎細(xì)胞因子IL- 6,參與RA的疾病過程,為臨床觀察疾病活動度、療效及預(yù)后提供新的實(shí)驗室依據(jù),為RA的治療提供新的方向。
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