两步法扩增诱导脐血及动员外周血CD34^+细胞来源树突状细胞的比较

Comparison of Expanding Dendritic Cells Derived from Cord Blood and Mobilized Peripheral Blood by Two-step Culture Method

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。

To compare the expansion efficiency and function of dendritic cells derived from CB-CD34^＋ cells and MPB-CD34^＋ cells by using two-step culture method, enriched CB-CD34^＋ cells or MPB-CD34^＋ cells with immunoadsorption were primarily cultured in the presence of FL, SCF, TPO, GM-CSF for 10 days, and then further cultured with a combination of GM-CSF,IL-4 ,TNF-α,CD40Ab and PGE2 to induce DC. The DC phenotypes were detected by flow cytometry, the expansion efficiency and cell function were evaluated by mix-lymphocyte reaction （MLR）, IL-12 level was detected by using ELISA and the chemotactic function mediated by secondary lymphoid tissue chemokine （ SLC ） was determined with Transwell plate. The results indicated that after 10 days of expansion, there were no significant difference in the percentage of CD14^＋ CD1a^- cells between CB and MPB [ （40.48 ± 16.85 ）% vs （28.07 ± 23.19 ）% , P 〉 0. 05 ], but the expansion of total cells in CB was higher than that in MPB （388.88 ± 84.63- fold vs 79.67 ± 10.32- fold, P 〈0.01 ）, so the yield of CD14^＋ CD1a^- cells from CB was significantly higher than that from MPB too （ 189.42 ±25.02- fold vs 28.74 ±23.27-fold, P 〈0.01 ）. The percentage of CD83^＋ DCs cultured with CD40Ab/PGE2 derived from CB were higher than those cultured with TNF-α derived from MPB respectively [（34.52 ± 11.22） % vs （3.70 ± 2. 27 ） % and （36.69 ± 13.36） % vs （7.34 ± 3. 364） % respectively, P 〈 0.011. In the same circumstance, the yield of CD83^＋ DCs derived from CB was much more than that from MPB （ 198.72 ± 117.53 times vs 33.95 ± 6. 19 times, P 〈 0.01 ） . There were no difference in stimulating capacity, IL-12 secretion and migration capacity between DCs derived from CB and MPB. It is concluded that DCs induced from CB-CD34^＋ cells by two-step culture possesse similar functions with that from MPB-CD34^＋ cells, but the yield of DCs from CB CD34^＋ cells is much more than that from MPB CD34^＋ cells.