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为了比较先扩增、后诱导的两步法从脐血(CB)CD34+细胞和动员外周血(MPB)CD34+细胞诱导所得DC的产量及功能,将免疫磁珠分选获得的CB-CD34+细胞和MPB-CD34+细胞用FL、TPO、SCF、GM-CSF等细胞因子先扩增10天,然后加入GM-CSF、IL-4及TNF-α、CD40Ab、PGE2等细胞因子组合诱导获得DC。采用流式细胞仪检测DC表型,混合淋巴细胞培养检测DC刺激异基因T细胞增殖能力,ELISA法检测DC分泌IL-12能力,Transwell板检测DC在次级淋巴组织趋化因子(SLC)介导下的趋化功能。结果表明①扩增10天时CB组、MPB组细胞中CD14+CD1a-细胞含量无显著差异[(40.48±16.85)%vs(28.07±23.19)%,P>0.05]。但由于CB组细胞扩增倍数显著高于MPB组(388.88±84.63倍vs79.67±10.32倍,P<0.01),CB组CD14+CD1a-细胞扩增倍数显著高于MPB组(189.42±25.02倍vs28.74±23.27倍,P<0.01);②TNF-α/CD40Ab/PGE2条件下与TNF-α条件下相比,CB组和MPB组所得DC均表达更高的CD83[分别为(34.52±11.22)%vs(3.70±2.27)%、(36.69±13.36)%vs(7.34±3.364)%,P均<0.01];③CB组与MPB组在TNF-α/CD40Ab/PGE2诱导条件下所得DC均高水平表达CD83、CD86、HLA-DR、CD11c、CD54、CD40,CB组所得CD83+细胞的扩增倍数显著高于MPB组(198.72±117.53倍vs33.95±6.19倍,P<0.01);④CD40Ab/PGE2/TNF-α条件下CB与MPB来源的DC在刺激异基因T细胞增殖、IL-12的分泌[(16.2±4.31)pg/mlvs(13.5±4.1)pg/ml]以及SLC介导的迁移率[(28.09±7.76)%vs(18.5±3·47)%]上均无显著差别(P均>0.05)。结论在两步法培养体系下,CB-CD34+细胞与MPB-CD34+细胞来源的DC具有相同的功能,而前者产量显著高于后者。
In order to compare the yield and function of DC obtained from CD34 + cells and mobilized peripheral blood (MPB) CD34 + cells induced by pre-amplification and post-induction in a two-step method, CB-CD34 + cells obtained by immunomagnetic bead sorting and MPB-CD34 + cells were expanded with cytokines such as FL, TPO, SCF and GM-CSF for 10 days. Then DCs were induced by adding cytokines such as GM-CSF, IL-4 and TNF-α, CD40Ab and PGE2. The DC phenotype was detected by flow cytometry. The proliferation of allogeneic T cells stimulated by DCs was detected by mixed lymphocyte culture. The ability of DCs to secrete IL-12 was detected by ELISA. The expression of DCs in secondary lymphoid tissue chemotaxis (SLC) Lead chemotaxis function. The results showed that there was no significant difference in the content of CD14 + CD1a- cells between CB group and MPB group [(40.48 ± 16.85)% vs (28.07 ± 23.19)%, P> 0.05] at 10 days. However, the expansion ratio of CD14 + CD1a cells in CB group was significantly higher than that in MPB group (38.48 +/- 84.63 times vs79.67 +/- 10.32 times, P <0.01) vs 28.74 ± 23.27 folds, P <0.01). ② Compared with TNF-α in TNF-α / CD40Ab / PGE2 group, DCs in CB group and MPB group expressed higher CD83 [(34.52 ± 11.22 ) were significantly higher than those in MPB group (P <0.05); (3) Compared with MPB group, the DCs induced by TNF-α / CD40Ab / PGE2 were all higher The amplification of CD83 + cells from CD83, CD86, HLA-DR, CD11c, CD54, CD40 and CB groups was significantly higher than MPB group (198.72 ± 117.53 vs 33.95 ± 6.19, P <0.01) / CB-MPB-derived DCs stimulated the proliferation of allogeneic T cells and the secretion of IL-12 [(16.2 ± 4.31) pg / ml vs (13.5 ± 4.1) pg / ml] [(28.09 ± 7.76)% vs (18.5 ± 3.47)%], respectively (all P> 0.05). Conclusions Under the two-step culture system, CB-CD34 + cells have the same function as DCs derived from MPB-CD34 + cells, while the yield of the former is significantly higher than the latter.