脐血CD34^+细胞体外定向诱导分化为T淋巴细胞的实验研究  

The studies on the committed differentiation of human umbilical cord blood CD34^+ cells into lymphocytes ex vivo

作　　者：姚志娟[1] 乌仁娜[1] 毕东杰[1] 楼晓[1] 黄云中[1] 冯凯[1] 陈虎[1] 

机构地区：[1]军事医学科学院附属医院移植科中国人民解放军造血干细胞移植中心,北京100039

出　　处：《中国免疫学杂志》2003年第12期841-844,849,共5页Chinese Journal of Immunology

基　　金：国家高技术发展规划 863项目 ( 2 0 0 1AA2 1713 1)资助

摘　　要：目的 :建立利用人造血干 祖细胞体外定向诱导分化为T淋巴细胞的方法 ,为研究T细胞生物学特性及细胞免疫提供技术平台。方法 :MACS方法分离人脐带CD34+ 细胞接种到人胎儿胸腺基质单层细胞上 ,IMDM液体培养基含 2 0 %人AB血清并加入FL、IL 12、IL 7和IL 2细胞因子组合 ,于培养 7、14、2 1、2 8、35、4 2天取非贴壁细胞利用流式细胞仪对细胞表型进行检测 ,并进行细胞形态学分析。结果 :2周后 ,CD4 + CD8+ 非成熟T淋巴细胞占细胞总数的 0 3%～ 13 3% ,4～ 5周CD4 +CD8+ T淋巴细胞达到高峰占 16 6 %～ 2 6 5 % ,且CD3+ CD4 + CD8+ 和CD3+ CD4 - CD8+ T淋巴细胞逐渐增多 ,6周后达 2 6 5 %～6 4 9%和 11 6 %～ 38 9%。培养成熟的T淋巴细胞经PHA +IL 2刺激后瑞氏染色鉴定可见大原始淋巴细胞存在。结论 :利用人脐血CD34+ 在体外人胎儿胸腺基质单层细胞上加FL、IL 12、IL 7和IL 2细胞因子组合条件下 ,可诱导分化出T淋巴细胞 ,并且培养的T细胞对有丝分裂素刺激有增殖反应。Objective:To establish the technique for T lymphocytes inducted and differentiated by using human hematopoietic stem/progenitor cells ex vivo and to provide a technological platform for studying T cells biological characteristics and cellular immune.Methods:Human CD34-positive cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system(MACS) and CD34+ cells were seeded on human fetal thymic stromal monolayer system.Liquid culture system contains hematopoietic cytokines(FL+IL-12+IL-7+IL-2) and 20% human AB serum.Non-adhension cells were obtained after 7?14?28?35?42 days of culture respectively to analysis T cells phenotype by FACS using T cells specific monoclonal antibody.T cells morphology were also studied.Results:After 2 weeks of culture CD4+CD8+ immature T lymphocytes wre about 0 3%～13 3% and the peak value is 16 6%～26 5% at 4～5 weeks of culture.CD4-CD8+ and CD4+CD8- T cells that coexpressed CD3 cells increased at 26 5%～64 9% and 11 6%～38 9% after 6 weeks of culture.Wright staining shows that the mature T cells harvested after mitogenic stimulation have the presence of large cells with lymphoblast morphology.Conclusion:Human umbilical cord blood CD34+ cells could be differentiated into T lymphocytes by the culture conditions of human fetal thymic stromal monolayer system and the cytokine combination of FL+IL-12+IL-7+IL-2,and the T cells can be stimulated by IL-2+PHA.

关 键 词：CD34^+细胞 胸腺基质细胞 诱导分化 T淋巴细胞 

分 类 号：R392.11[医药卫生—免疫学]