摘要
目的 探索体外诱导脐血单个核细胞 (MNC)扩增分化为T/NK细胞的最佳培养体系。方法 脐血MNC在 6种培养体系中培养 4周 ,于各时间点用流式细胞仪测定细胞表面T/NK标志抗原的表达 ;测定有核细胞 (NC)数 ;并行细胞形态学鉴定及细胞毒功能实验。结果 脐血MNC在SCF +FLT 3L +IL 7+IL 15 +TNF α +IL 2体系中培养 ,第 2 2天NC数达 (2 0～ 2 6 )× 10 6/ml;淋巴细胞占NC的比例和CD3 + T细胞占淋巴细胞的比例均达到 90 %以上 ,以CD8+ T细胞亚群为主 ,CD4+ T细胞亚群比例下降 ;CD56+ CD3 + NKT细胞和TCRγδ+ 细胞的比例分别由不足 2 %升高到 30 %～ 4 0 %和 10 %～ 15 %。CD3 -CD56+ NK细胞无扩增。培养后细胞在效靶比为 5 0∶1时对K5 6 2细胞和Raji细胞的杀伤率分别达75 %以上和 32 %～ 6 5 %。结论 本实验中体外诱导脐血MNC扩增分化为T/NK细胞的最佳培养体系为SCF +FLT 3L +IL 7+IL 15 +TNF α+IL 2 ,最佳扩增时间是培养后第 2 2天。
Objective To explore the most efficient culture system which can induce cord blood(CB)-mononuclear cells(MNC) to differentiate into mature T/NK cells in vitro. Methods The CB MNCs were cultured in six culture systems respectively for 4 weeks. The T/NK cell surface phenotypes were analyzed by flow cytometry and the absolute numbers of nucleated cells(NCs) were counted at each time point. Moreover, cell morphology was identified by Giemsa-Wright staining, and cytotoxicity of the cultured cells to K562 and Raji tumor cells was also evaluated by MTT method. Results Cultured in the cytokine cocktail of SCF+FLT-3L+IL-7+IL-15+TNF-α+IL-2, the NCs were (20～26)×10 6/ml in numbers at day 22. The percentage of lymphocytes in the NCs and that of CD_3 + T cells in the lymphocytes both exceeded 90% at the same time. Most of the CD_3 +T cells were CD_3 +CD_8 + and the percentage of CD_3 +CD_4 + T cells declined gradually. The percentage of CD_3 +CD_ 56 + NKT cells and γδ +T cells in the lymphocytes arised from lower than 2% to 30%～40% and 10%～15%, respectively. CD_3 -CD_ 56 + NK cells were not expanded. The cytotoxic activity of the cultured cells to K562 and Raji cells at an effector∶target(E∶T) ratio of 50∶1 was over 75% and about 32%～65%, respectively. Conclusion The most efficient culture system which can induce CB MNC to differentiate into mature T/NK cells in vitro is the cytokines cocktail of SCF+FLT-3L+IL-7+IL-15+TNF-α+IL-2, and the optimum culture time is 22 days.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2003年第11期576-579,共4页
Chinese Journal of Hematology
