人外周血NK细胞纯化、扩增及克隆化的初步探索

Preliminary Study in Purification,Proliferation and Cloning of Human NK Cells

分别采用Panning法、补体裂解法、绵羊红细胞花环法、Percoll不连续密度梯度离心法对NK细胞进行纯化并以流式细胞仪检测其纯度和计数探索NK细胞的体外扩增条件 ,将纯化NK细胞经有限稀释 ,在饲养细胞、IL 2、PHA及LCM等培养条件下进行克隆化培养并进行鉴定。研究表明NK细胞的纯度由纯化前 10 %左右提高到 30 %～ 70 %不等 ,以Percoll不连续密度梯度离心法所获纯度最高 ,约为 70 %。NK细胞的体外扩增在含有IL 2、PHA及条件培养基最显著。每 96孔板可获 4～ 16个CD3 CD5 6 +NK细胞克隆 ,每 96个克隆的细胞数最多可达 2 35× 10 5。

To investigate the purification,proliferation and cloning of NK cells,these cells were purified by Percoll discontinuous density gradient centrifugation,panning,CDC and SRBC rosette method By limited dilution,NK cells were cultivated with feeder cells,IL2,PHA in leukocyte condition medium(LCM) It was found that the purity of NK cells increased from 10% up to 30%～70%,and the optimal condition for NK cell proliferation was that the medium contained IL2 and PHA in LCM medium By using these methods,several clones of NK cells were obtained by limited dilution method The maximal number of the NK cell clones approached up to 235×105