基于CFSE染色的小鼠淋巴细胞体内示踪方法的建立  

A tracking method for CFSE-labeled lymphocytes in vivo

作　　者：汤永永[1] 唐颖[2] 张玉华[1] 段文元[1] 赵敬湘[1] 魏广智[1] 张秀媛[1] 李鹏飞[1] 穆思媛[1] 王字玲[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850 [2]军事医学科学院科技部

出　　处：《中国输血杂志》2011年第10期850-853,共4页Chinese Journal of Blood Transfusion

基　　金：全军医药卫生科研基金(09MA023)

摘　　要：目的确定CFSE标记小鼠淋巴细胞的条件,建立简单快速、稳定可行的淋巴细胞体内示踪方法。方法利用流式细胞术分别检测不同浓度CFSE、不同悬浮溶液(1640培养基、PBS溶液)对小鼠淋巴细胞标记效果的影响,确定合适的标记条件;输注同品系CFSE标记的小鼠淋巴细胞,于不同时间点检测受鼠主要脏器内CFSE+细胞在淋巴细胞中的比例,并确定供鼠细胞的注射剂量;按照合适的注射剂量分别在注射后不同时间点测定供鼠细胞在受鼠体内的分布,考察本方法的稳定性及阳性信号的持续时间。结果 PBS组CFSE+细胞平均荧光强度分别为596、793、1123、1596、1737,明显高于1640培养基组(P<0.05);1640培养基组CFSE+细胞死亡率分别为8.7%、8.4%、8.6%、9.0%(P>0.05),而PBS组CFSE+细胞死亡率随着CFSE终浓度的上升而增加(P<0.05);按照不同剂量输注同品系淋巴细胞24h后,在受鼠各组织中检测到CFSE+细胞;每只小鼠尾静脉注射5×106个CFSE+淋巴细胞后,到d63仍可在组织中检测到阳性信号细胞。结论以PBS作为孵育液、CFSE终浓度选择10μmol/L对小鼠淋巴细胞标记、注射剂量为细胞5×106个/只,可使CFSE+细胞信号在同系小鼠体内持续2个月。Objective To establish a sensitive method for tracking mouse lymphocytes labeled with carboxyfluorescein diacetate succinimidyl ester（CFSE） in vivo.Methods Mouse lymphocytes were labeled with various concentrations of CFSE in different buffers.Based on fluorescence intensity,the optimal labeling condition was selected.Labeled lymphocytes were transfused into mouse and the distribution and duration of the cells in various organs were measured by flow cytometry.Results The mean fluorescence intensity of CFSE＋ cell in PBS groups were 596,793,1 123,1 596,and 1 737,which were significantly higher than those in RPMI-1640 groups（P0.05） .The death rates of CFSE＋ cells were 8.7%,8.4%,8.6%,and 9.0%（P0.05） at different concentrations of CFSE in RPMI-1640 groups,increasing along with the increasing concentrations of CFSE in PBS groups（P0.05） .The CFSE-labeled lymphocytes can be detected for as long as two months in many organs after transfusion.Conclusion Under optimal conditions,CFSE-labeling is reliable,stable and convenient for lymphocyte tracing in vivo.

关 键 词：CFSE 淋巴细胞 流式细胞术 细胞标记 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R446[医药卫生—基础医学]