中文摘要：

目的 探索小鼠脾脏NKT细胞的分离、纯化及体外培养扩增的方法。方法 分离小鼠脾细胞，制成单细胞悬液，α-半乳糖神经鞘胺醇（α-galactosylceramide，α-Galcer）和rhIL-2作用下刺激其活化增殖，不同时间点取样，利用双色免疫荧光抗体标记技术和流式细胞术（FACS）检测NKT细胞纯度，并用MTT法检测其对靶细胞K562的杀伤活性。结果 脾NKT细胞在第0，4，8，12天计数细胞总数为0．5×10^7、（1．3±0．1）×10^7、（1．9±0．2）×10^7和（2．2±0．2）×10^7，第0,4,8,12天纯度分别为（6．27±1．04）％、（8．54±1．58）％、（10．65±1．95）％和（21．36±4．34）％；α-Galcer和rhIL-2作用下，纯化的NKT细胞较脾新鲜分离的单个核细胞对靶细胞K562的杀伤作用强。在效靶比20：1时，培养时间为4、8，12天的NKT细胞杀伤率分别为52．7％、71．2％和85．7％，明显高于脾MNC33．8％（P〈0．05）。结论 在α-Galcer和rhIL-2作用下，小鼠NKT细胞得到扩增，细胞纯度约为22％。在效靶比20：1时，NKT细胞对K562细胞杀伤率约为86％。

英文摘要：

Objective To develop a method to isolate, purify and culture mouse spleen natural killer T cells in vitro. Methods Mouse spleen was isolated and made into singal cell suspension. Cells were activated and expanded in the presence of α-Galcer and rhIL-2. NKT cells were harvested at different time and the purity of NKT cells was determined by two-tone immunofluorescence sign and flow cytometrx（FACS）. The cytotoxicity to K562 targets were detected by MTT assay. Results At the 0,4th, 8th, 12th day, the total number of the spleen NKT cells was 0.5 × 10^7 ,（1.3 ±0.1） × 10^7 ,（1.9±0.2） × 10^7 and （2.2±0.2） × 10^7 ,and the percentage of NKT cells at the 0,4th,Sth, 12th day was （6.27 ± 1.04）%, （8.54 ± 1.58）%, （10.65 ± 1.95）% and （21.36± 4.34）% respectively. The cytotoxicity to K562 targets was significantly higher than that of newly separated splenic MNC （P 〈0.05）,and increased from 33.8% up to 52.7% ,71.2% and 85.7% respectively at 20：1 of effector： target ratio. Conclusion Mouse NKT cells were expanded in the presence of α-Galcer and rhIL-2, and the purity reaches around 20% .The cytotoxicity to K562 targets is about 86% at 20：1 of effector： target ratio.