耐受性CD8^+ NKT细胞体外增殖能力的鉴定  

Identification of cell division of CD8^+ NKT cells in vitro

作　　者：郭业磊[1] 陈钰[1] 钟江[2] 张世仑[2] 

机构地区：[1]解放军总医院免疫学教研室,北京100853 [2]复旦大学生命科学院微生物与微生物工程系,上海200433

出　　处：《细胞与分子免疫学杂志》2011年第2期170-172,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30671981;30571650);国家高技术研究发展计划(863)资助项目(2006AA02Z462)

摘　　要：目的:利用CFSE标记细胞,流式细胞术(FCM)检测法,解析超抗原SEB活化的耐受性CD8+ NKT细胞在体外增殖的情况。方法:利用CFSE标记新鲜分离的C57BL/J鼠脾细胞,分别与ConA和LPS共同培养3d,收集细胞进行荧光染色并用FCM解析细胞表面CD69分子的表达率和增殖能力。CFSE标记的鼠脾细胞与SEB共培养5d和10d后,荧光染色并用FCM解析细胞表面CD69的百分数和增殖能力。SEB活化的第10天细胞经CFSE标记后在IL-2的协同作用下继续培养10d,荧光染色,FCM解析这群细胞的增殖能力、活性分子CD69的表达率和NKT细胞亚群的变化情况。结果:ConA、LPS和SEB三者均可以刺激小鼠脾细胞增殖。ConA和LPS在3d内可以使细胞增殖3代,且CD69的表达率为74.19%和41.56%;SEB在5d和10d内分别可以使细胞增殖5代和7代,细胞表面CD69的表达率为32.09%和48.66%。SEB活化的10d细胞可以在IL-2的协同下继续传代培养10d,可以增殖7代;这群细胞中CD8+ NKT细胞亚群,由原始的0.36%增加到38.58%;细胞表面CD69分子由正常值的0.11%提高到83.74%。结论:超抗原SEB活化的CD8+ NKT细胞可以在体外进行增殖培养,且这些细胞是活性化的细胞。利用CFSE标记细胞,FCM可以检测耐受性CD8+ NKT细胞在体外的增殖水平。AIM： To measure the in vitro cell division ability of CD8^＋ NKT cells by CFSE staining and flow cytome. try （FCM）. METHODS： Fresh spleen lymphocytes of C57BL/J mice were separated and stained with CFSE, and then stimulated by ConA and LPS for 3 d, and by SEB for 5 d and 10 d respectively. The stimulated cells were harvested and analyzed for CD69 expression on the cell surface and the ability of cell division using FCM. The SEB-activated effector cells for 10 d further stimulated with IL-2 for the consecutive 10 days, and were analyzed for their cell division ability, CD69 expression and NKT cell subsets by FCM. RESULTS： ConA, LPS and SEB stimulated the proliferation of spleen cells. ConA and LPS made the cells divide 3 times within 3 d, and increased CD69 expression up to 74. 19% and 41.56% respectively. SEB made the cells divide 5 times within 5 d and 7 times within l0 d respectively, with increased CD69 expression of 32.09% and 48.66% respectively. Ten-day IL-2 stimulation of SEB-activated cells caused population expansion for 7 times with the CD8^＋ NKT cell subsets significantly increased from 0.36% to 38.58% and CD69 expression significantly increased from 0.11% to 83. 74%. CONCLUSION： The SEB-activated CD8^＋ NKT cells proliferated in vitro and their cell division capability could be determined by CFSE staining and FCM.

关 键 词：CD8+ NKT细胞 超抗原 SEB 流式细胞术 CFSE 

分 类 号：R392.11[医药卫生—免疫学]