胶原诱导关节炎模型小鼠CCP抗原特异性T细胞的量子点标记检测及其与CCP特异性淋巴细胞增殖的关系  

Relationship of quantum dots-labeling CCP antigen specific T cells and the proliferation of CCP specific lymphocytes in the mice with collagen-induced arthritis

作　　者：张敏杰[1,2] 张晶[2] 夏海萍[2] 刘国瑞[2] 严孝岭[2] 虞伟[2] 李晓军[2] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013 [2]南京军区南京总医院解放军临床检验医学研究所,南京210002

出　　处：《临床检验杂志》2014年第6期450-453,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81071419);江苏省科技支撑计划-社会发展项目(BE2011786);江苏省"六大人才高峰"项目(内科卫生D203);江苏省生物材料与器件重点实验室开放研究基金(2010LBMD01)

摘　　要：目的建立基于量子点(quantum dots,QDs)标记环瓜氨酸肽(CCP)抗原特异性T细胞(CCP-AST)的流式细胞分析术(FCM),研究胶原诱导关节炎(collagen-induced arthritis,CIA)模型小鼠CCP-AST的阳性率及其与CCP抗原特异性淋巴细胞增殖的关系。方法构建CIA小鼠模型,用QDs标记链霉亲合素(QD705-SA)结合生物素化的CCP对CCP-AST(CD4+CD8-CCP-TCR+)进行体外标记与FCM检测,同时将CCP与小鼠脾单个核细胞(MSMC)进行共培养,并对其增殖刺激指数(SI)、CCP-AST阳性率及其他参数做相关分析。结果成功建立检测QDs标记CCP-AST的FCM术,发现CIA组小鼠CCP-AST阳性率[中位数(第25分位数,第75分位数)]为0.575%(0.475%,1.185%),显著高于PD2B对照肽的0.165%(0.113%,0.240%)和健康组小鼠的0.085%(0.000%,0.205%),差异均有统计学意义(P均<0.01)。CIA组小鼠经CCP刺激,MSMC出现明显的增殖反应,SI值为4.710,高于健康组小鼠CCP刺激的SI值0.860,差异有统计学意义(P<0.01)。相关性分析显示,CIA组小鼠CCP-AST阳性率与CCP刺激MSMC的SI值呈显著正相关关系(r=0.734,P<0.05),与关节炎症指数之间亦呈正相关(r=0.611,P<0.05)。结论 QD705-SA结合生物素化的CCP,可实现对CCP-AST的有效标记和FCM检测,CIA组小鼠CCP-AST阳性率与CCP抗原刺激的特异性淋巴细胞增殖结果显著相关,并与CIA小鼠关节肿胀和炎症程度关系密切。Objective To establish the quantum dots -labeling flow cytometry（FCM） to detect the cyclic citrullinated peptide （CCP） antigen specific T lymphocytes（CCP-AST） in the collagen-induced arthritis （CIA） mice, and investigate the correlation between the percentage of CCP-AST and the stimulation index （SI） of CCP antigen specific lymphocyte proliferation. Methods The CIA mouse model was established. Quantum dots-labeling streptavidin（QD705-SA） was conjuncted with biotinylated CCP, and then was used to label CCP-AST（ CD4 ＋ CD8-CCP-TCR^＋ ） in vitro for the FCM detection. Meanwhile, CCP was co-incubated with mouse spleen mono- nuclear cells（ MSMCs）, and the SI was obtained. The correlations of SI with the percentage of CCP-AST and other parameters were an- alyzed. Results The quantum dots-labeling FCM for the detection of CCP-AST was successfully established. The percentage of the CCP-AST [ median（ P25, P75 ） ] in the CIA mice [ 0. 575 % （0. 475 %, 1. 185 % ） ] was significantly higher than those in the mice treated with the control peptide PD2B [ 0. 165% （0. 113%, 0. 240% ） ] and the healthy mice [ 0. 085% （0. 000%, 0. 205% ） ] （ P 〈 0.01 ）. The SI in the CIA mice stimulated with CCP（4. 710） was significantly higher than that in healthy mice（0. 860, P 〈0.01 ）. In addition, the percentage of CCP-AST in CIA mice was positively related to the SI stimulated with CCP（r = 0. 734, P 〈 0.05 ） and the joint inflammation index（ r = 0.611, P 〈 0.05）. Conclusion QD705-SA combined with biotinylated CCP can be used to label CCP- AST effectively for the FCM detection. The positive rate of CCP-AST in CIA mice was significantly related to the proliferation response of CCP antigen specific lymphocytes and the joint swelling and inflammation degree.

关 键 词：量子点 环瓜氨酸肽 抗原特异性T细胞 胶原诱导性滑膜炎 增殖 

分 类 号：R392-33[医药卫生—免疫学]