两步法从慢性髓系白血病患者骨髓CD34^+细胞体外扩增诱导树突状细胞

Expansion of dendritic cells from chronic myelogenous leukemic CD34^+ progenitor cells by a two-step process

目的探讨从慢性髓系白血病(CML)患者骨髓CD34^+细胞体外扩增诱导树突状细胞(DC)的可行性,并比较CML-DC和正常DC的生物学特性。方法免疫磁珠法从CML患者和正常供者骨髓纯化CD34^+细胞,在有血清条件下应用两步法:干细胞生长因子(SCF)+FLT3配体(FL)+促血小板生成素(TPO)+白细胞介素-3(IL-3)扩增2周,然后以粒细胞巨噬细胞集落刺激因子(GM-CSF)+白细胞介素-4(IL-4)+肿瘤坏死因子-α(TNF-α)(GI方案)诱导DC。通过相差显微镜、电子显微镜、流式细胞仪分析DC的生物学特性,荧光原位杂交(FISH)检测培养前后CML细胞的bcr/abl融合基因表达。结果诱导后细胞较0d或诱导前细胞高表达DC相关抗原(CD1a,CD80,CD86,CD40,CD54,HLA-DR)。CML患者和正常供者CD34+细胞经GI方案诱导10d,CD1a阳性率分别为36.90%±26.94%和54.35%±16.34%,CD1a^+DC数是0d接种细胞的(54±54)倍和(122±129)倍。两组相比,总细胞扩增倍数、DC扩增倍数、细胞表型均无明显差异,诱导后的DC具有相似的超微结构。CML患者CD34^+细胞诱导10d后bcr/abl融合基因阳性率为43.67%±21.55%,具有典型DC形态的细胞bcr/abl融合基因阳性率为53.2%。结论两步法GI方案能诱导CMLCD34^+细胞产生大量DC,诱导生成的DC不但具有正常DC的典型形态、表型,而且起源于CML细胞。

Objective To study expansion efficiency of dendritic cells （DC） from chronic myelogenous leukemic （CML） CD34^＋ progenitor cells by a two-step process and to investigate biological properties of CML-derived DC and normal DC. Methods CD34^＋ cells were purified with CD34 microbeads from CML and normal bone marrow and cultured in serum-dependent medium by a two-step culture method： primary in the presence of stem cell factor （SCF） ,Flt-3 ligand （FL） ,thrombopoietin （TPO）, interleukin-3 （IL-3） for 14 days ,and then further cultured with granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-4 （IL-4） for 7 days in addition to tumor necrosis factor-α （TNF-α） for another 3 - 4 days （group GI）. The biological properties of CML-derived DC and normal DC were examined by phase contrast microscopy ,electron microscope, and flow cytometry. The expression of bcr/abl fusion gene of uncultured and cultured CML cells was detected with fluorescence in situ hybridization （FISH）. Results These cells showed a typical dendritic phenotype （highly positive for DC marker CDla, costimulatory molecule CD80,CD86 and CD40,adhesion molecule CD54,and HLA-DR and negative for CD3,CD19）. The proportion of CDla^＋ cells was 36.90 % ± 26.94 % and 54.35 % ± 16.34 % ,respectively. The number of CDla~ DC generated represented （54 ± 54） fold and （122 ± 129） fold that of the input,respectively. There were no significant differences between CML group and normal group in the number of total cell, CDla^＋ DC generated and cell surface markers. Electron-microscopic analysis showed no major morphological differences between normal and CML-derived DC. （43.67 %± 21.55 %） of unpurified CML-derived DC were bcr/abl gene positive and 53.2 % of cells having dendritic profiles were positive. Conclusion A plenty of DC could be obtained from CML CD34^＋ cells with the GI scheme. CML-derived DC not only had characteristic morphology, phenotype of normal DC, but also were originated from native CML cells.