改良法分离小鼠移植心内浸润淋巴细胞

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 × 106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.

A modified method for isolation of infiltrating lymphocytes in cardiac grafts

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 × 106, 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.