小鼠CD8^+记忆性T淋巴细胞的分离、纯化和体外功能检测  

Isolating,purification and function of mouse CD8+ memory T lymphocytes in vitro

作　　者：王百林[1] 刘增军[1] 翟淑萍[1] 陈义发[2] 秦欢[1] 刘秋玲[1] 

机构地区：[1]广州中医药大学第一附属医院肝胆外科,510405 [2]华中科技大学同济医学院附属同济医院肝胆外科中心,武汉430030

出　　处：《贵州医药》2010年第5期387-390,共4页Guizhou Medical Journal

基　　金：广东省自然基金资助课题;课题号:7004824;课题名称:扶正固本法对大鼠肝移植术后免疫紊乱的干预作用;广东省中医药管理局课题;课题号:2007088;课题名称:扶正固本法对大鼠肝移植术后免疫紊乱的干预作用

摘　　要：目的分离CD8+记忆性T淋巴细胞,检测其体外部分功能。方法建立小鼠皮肤移植模型,制备受体脾细胞悬液,采用免疫磁珠方法分离Tm细胞、台盼蓝细胞染色检测其细胞活性、流式细胞仪检测分选所得细胞纯度,ELISA方法检测不同抗原刺激Tm细胞后的IL-2和IL-10的浓度。结果小鼠皮肤移植存活率为93.0%(28/30),皮肤移植皮片平均存活时间(12.18±1.03)d;CD8+Tm的活细胞百分率为98.5%;流式细胞术检测表型CD8+CD4+细胞比例占(97.75±0.9)%,CD8+CD4-细胞比例占(96.0±1.02)%;在供体脾细胞、刀豆素A、Wistar大鼠脾细胞和无刺激物作用下,2×107mL-1的CD8+Tm液中细胞因子IL-2和IL-10的浓度分别为:(57.32±6.52),(38.34±5.21),(13.51±3.35),(11.32±2.51);(9.21±1.02),(11.52±2.15),(35.42±4.59),(39.21±7.3);差异有统计学意义(P<0.05,P<0.01)。结论通过免疫磁性分离技术和流式细胞技术可分离出高纯度的CD8+Tm细胞,具有良好的细胞特性,为Tm在移植免疫方面的研究提供了技术方法。Objective To establish a method for isolating CD8＋ memory T lymphocytes （Tm）,and to identify partial functions of these cells in vitro.Methods The mouse skin transplantation model was established and the splenocytes of recipients was separated.The lymphocytes were isolated by magnetic activated cell sorting （MACS）.The activity and the purity of them were detected by trypan blue staining and fluorescence activated cell sorting （FACS）,respectively.ELISA was used to measure the IL-2 and IL-10 levels in Tm by different antigens stimulated.Results The survival rate of mouse was 93.0% （28/30）,and mean survival time of skin allografts was 12.54～0.8 d.The activity of CD8＋ Tm was 98.5% approximately.The proportion of CD＋CD4＋,CD8＋CD4-lymphocytes were （97.75±0.9）% and （96.0%±1.02）%,respectively.When these CD8＋Tm cells were not stimulated or stimulated by dill%rent antigens such as donor splenocytes,Con A and Wistar rice splenocytes,the levels of IL-2 creting lymphocytes were（57.32±6.52）,（38.3±5.21）,（13.51±3.35）,（11.32±2.51）;（9.21±1.02）,（11.52±2.15）,（35.42±4.59）,（39.21±7.31）.In 2×107/mL cells,there is significance in them （P0.05,P0.01）.Conclusion Higher purification and better function of CD8＋ Tm can be isolated by MACS and FACS technology,which facilitates us technologic methods to investigate the role of Tm cells in transplantation immunity.

关 键 词：记忆性T细胞 免疫磁性分离 流式细胞术 移植 小鼠 

分 类 号：R392.12[医药卫生—免疫学]