改良法分离小鼠移植心内浸润淋巴细胞  

A modified method for isolation of infiltrating lymphocytes in cardiac grafts

作　　者：方泽民[1] 何文涛[1] 王升[1] 周鸿敏[1] 陈忠华 明长生[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所教育部/卫生部重点实验室,武汉430030

出　　处：《中华实验外科杂志》2010年第9期1222-1223,共2页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30772052,30972794）

摘　　要：目的 建立简便而高效的分离小鼠心脏移植物内浸润淋巴细胞的方法.方法 移植物剪碎后用Ⅱ型胶原酶（250 U/ml,30～40 min）消化,Ficoll密度梯度离心（800 g/min,20 min）分离单个核细胞,荧光标记抗体行表面抗原或胞内细胞因子染色后用流式细胞仪分析其亚群.结果 移植物分离的单个核细胞数量稳定在1 × 10^6个以上,淋巴细胞的比例为（31.9±2.3）%,活性（95.1±2.1）%,流式细胞仪能同时检测到荧光标记的淋巴细胞表面和胞内的抗原.结论 本法单用胶原酶消化移植心,减少了对心脏移植物内浸润淋巴细胞的损伤,获得的细胞可进一步用于流式表型分析.Objective To introduce a simple and efficient method for isolation of intragraft infiltrating lymphocytes. Methods Cardiac allografts were cut into small pieces that were then digested with collagenase Ⅱ （250 U/ml, 30-40 min）. Mononuclear cells were isolated by Ficoll density gradient centrifugation （800 g/min, 20 min）. Subsets of lymphocytes were identified by flow cytometry after surface antigen and intracellular cytokine staining. Results The harvested mononuclear cells were more than 1 × 10^6, which contained （31.9 ±2. 3）% lymphocytes with viability of （95.1 ±2. 1 ）%. Flow cytometry could analyze surface and intracellular antigens of the lymphocytes at the same time. Conclusion The modified method of isolating intragraft infiltrating lymphocytes with high viability is simple, reliable and efficient.

关 键 词：心脏移植 淋巴细胞 移植物 胶原酶Ⅱ 流式细胞仪 

分 类 号：R654.2[医药卫生—外科学]