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目的 探索利用先扩增后诱导的“两步法”从脐血 (CB)CD34+ 细胞高效大量地获得树突细胞 (DC)。方法 免疫磁珠法从CB分选获得CD34+ 细胞 ,以干细胞因子 (SCF)、IL 3、Flt 3配体 (FL)、Tpo组合刺激 ,扩增 7d、10d和 14d(依次为Ⅰ、Ⅱ和Ⅲ组 )后以GM CSF +IL 4 +TNF α诱导 8d或 5d获得DC ,通过相差显微镜、电镜观察形态 ,流式细胞仪检测表型 ,混合淋巴细胞培养、ELISA法检测培养液上清IL 12含量评价其功能。结果 CBCD34+ 细胞经SCF +IL 3+FL +Tpo刺激扩增 7d、10d和 14d后细胞总数分别扩增了 (5 3.39± 2 0 .5 9)倍、(30 7.17± 119.5 9)倍和 (1117.2 5± 335 .4 9)倍。经GM CSF +IL 4 +TNF α诱导 8d后所得CD1a+ 细胞是扩增前细胞数的 (2 1.4 0± 16 .70 )倍、(14 3.2 0± 6 0 .35 )倍和(15 0 .80± 4 2 .16 )倍 ,Ⅱ、Ⅲ组明显多于Ⅰ组 (P <0 .0 5 ) ,但Ⅱ、Ⅲ组间无显著性差异 (P >0 .0 5 )。所得DC的形态、表型及刺激异基因T细胞增殖能力、IL 12分泌量 ,三组无显著性差异 (P >0 .0 5 )。当诱导时间缩短至 5d时 ,各组DC功能均显著下降 (P <0 .0 5 )。结论 CBCD34+ 细胞扩增 7～ 10d再诱导 8d可以高效大量获得具有正常功能的DC ,而扩增时间超过 10d并不能显著增加DC产量 ,诱导时间少于8d将降低所得DC
Objective To explore the efficient and abundant dendritic cells (DCs) obtained from cord blood (CB) CD34 + cells using the two-step induction method. Methods CD34 + cells were obtained from CB by immunomagnetic beads method. The cells were stimulated with combination of stem cell factor (SCF), IL 3, Flt 3 ligand (FL) and Tpo for 7 days, 10 days and 14 days Group), DCs were induced with GM CSF + IL 4 + TNFα for 8 days or 5 days. Morphology was observed by phase contrast microscope and electron microscope. Phenotypes were detected by flow cytometry. Mixed lymphocytes were cultured. The content of IL 12 Evaluate its function. Results The total number of CBCD34 + cells proliferated (5 3.39 ± 2.05. 5), (30 7.17 ± 119.5 9) and (1117.2) times after 7, 10 and 14 days of stimulation with SCF + IL 3 + FL + 5 ± 335. 49) times. The CD1a + cells after 8 days of induction by GM CSF + IL4 + TNFα were (2.14 0 ± 16.70) times (14 3.2 0 ± 6 0.35) times and (15 0 .80 ± 4 2 .16) times, while those in group Ⅱ and Ⅲ were significantly higher than those in group Ⅰ (P <0.05), but there was no significant difference between group Ⅱ and Ⅲ (P> 0.05). The morphology, phenotype and stimulation of proliferation of allogeneic T cells and secretion of IL 12 had no significant difference among the three groups (P> 0.05). When the induction time was shortened to 5d, the DC function of each group was significantly decreased (P <0.05). CONCLUSION: CBCD34 + cells can be regenerated in vitro for 7-10 days and then for 8 days. DCs with normal function can be efficiently and efficiently obtained. However, DCs can not be increased significantly when the expansion time exceeds 10 days. When the induction time is less than 8 days,