CD34免疫亲合柱在脐血造血干祖细胞分离与扩增中的应用

The application of CD34-immunoaffinity column in the isolation and expansion of hematopoietic stem and progenitor cells of cord blood

目的 :观察CD34免疫亲合柱对脐血造血干 祖细胞分离纯化的效果及纯化后CD34+ 细胞的增殖分化特性。方法 :采用CD34免疫亲合柱分离脐血单个核细胞 (MNC)中的CD34+ 细胞 ,流式细胞技术 (FACS)进行细胞表面标志测定。将分离前后的细胞加入造血生长因子进行液态扩增培养和多向祖细胞集落 (CFU GEMM)培养。结果 :经CD34免疫亲合柱分离后脐血中CD34+ 细胞为 49 6 2 %± 17 6 9% ,明显高于脐血MNC(1 17%± 0 6 8% ) ,细胞回收率为 5 4 38%± 11 91%。分离后CD34+ 细胞和脐血MNC经造血生长因子刺激培养 2 0d分别扩增 5 6 1 0 0倍和 44 44倍。培养至 12d时 ,免疫亲合柱分离组CD34+ 细胞阳性率为 5 3 38% ,对照组为 7 91%。分离组CFU GEMM产率明显高于对照组 (P <0 0 0 1)。结论 :CD34免疫亲合柱应用于脐血造血干 祖细胞的分离可充分富集CD34+ 细胞 ,且分离后的CD34+ 细胞具有明显的增殖效应和CFU GEMM形成能力。

Objective:To observe the effect of CD34 immunoaffinity column on the isolation of hematopoietic stem and progenitor cells of cord blood and the proliferatory and expansion characteristics of CD34 + cells in vitro.Methods:CD34 +cells were isolated from cord blood using CD34 immunoaffinity column.Cell surface antigens were analysed by FACS.The separated and un separated cells were cultured with human hematopoietic growth factors(HGFS)in liquid culture system and CFU GEMM culture system.Results:CD34 + cells were enriched with a purity of 49.62%±17.69% and a recovery of 54.38%±11.91% using the immunoaffinity column.The separated CD34 +cells and cord blood MNC expanded to 561.00 folds and 44.44 folds respectively after cultured with HGFS for 20 days.The percentages of CD34 + cells in separated group and control were 53.38% and 7.91% respectively after cultured for 12 days.The number of CFU GEMM in separated group was significantly higher than that in control(P<0.001). Conclusion:Using CD34 immunoaffinity column the hematopoietic stem and progenitor cells can be enriched efficiently and the separated CD34 + cells have obvious expansion efficiency and ability of CFU GEMM forming.