免疫磁性分选系统大量分选CD34+细胞的实验研究

CD3 4 细胞的分离纯化在自体外周血干细胞、异基因骨髓移植 /外周血干细胞移植及干细胞研究中具有重要的应用价值。为了摸索大量分选CD3 4 细胞的方法 ,本研究应用Isolex3 0 0i磁性分选系统富集CD3 4 细胞 ,采用流式细胞术监测分选前后细胞表面标志 ,经CD3 4 细胞体外增殖实验及克隆形成实验验证分选获得的CD3 4 细胞生物学活性。结果显示 ,所完成的 5例次自体外周血富集CD3 4 细胞时 ,先收获单个核细胞约 ( 3 .5 -6.0 )× 10 10 ,CD3 4 细胞占单个核细胞百分率为 ( 0 .5 5 - 1.2 ) % ;收获的CD3 4 阳性细胞总数为 ( 2 - 3 )× 10 8,纯度为 ( 75 - 85 ) % ,回收率为 ( 4 0 - 65 ) %。体外实验表明 ,在SCF IL 3 FL TPO EPO存在下 ,经过 3 - 4天培养 ,可扩增 2 - 3倍 ;经CFU GM、BFU E集落形成实验显示具有形成集落的能力 ,证实分选后细胞具有造血祖细胞生物活性。结论 :应用Isolex3 0 0iCD3 4 细胞分选仪可以高效大量富集CD3 4 细胞 ,适于临床应用。

Study on Large-scale CD34+ Cell Enrichment Using Immunomagnetic Isolation Apparatus

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.