骨髓CD34^+细胞体外扩增诱导树突状细胞实验研究

Expansion of dendritic cells from human bone marrow CD34^+ progenitor cells

目的探讨应用不同细胞因子组合方案从骨髓CD34+细胞体外扩增诱导树突状细胞(DC)的可行性及评价不同诱导方案诱导DC的效果。方法免疫磁珠法纯化骨髓CD34+细胞。在有血清条件下应用两步法:SCF+FL+TPO+IL-3扩增2周,然后以GM-CSF+IL-4+TNF-α(GI方案)或GM-CSF+TNF-α(GT方案)诱导DC;或者一步法:SCF+FL+TPO+IL-3+GM-CSF+TNF-α直接作用2周扩增诱导DC。通过相差显微镜、电子显微镜、流式细胞仪分析、异硫氰酸荧光素标记的葡聚糖(FITC-DX)内吞实验检测DC的生物学特性。结果诱导后细胞较0d或诱导前细胞高表达DC相关标记(CD1a、CD80、CD86、CD40、CD54、HLA-DR)。两步法GI方案诱导10d,总细胞扩增倍数、CD1a+DC扩增倍数分别为(198±178)倍和(122±129)倍,与GT方案比较无统计学意义,但诱导细胞CD1a、CD80、CD86的表达明显高于后者。一步法扩增诱导2周时总细胞数扩增(43±16)倍,CD1a+DC数是0d接种细胞的(4±2)倍。结论两步法能从正常CD34+细胞诱导产生大量DC,GI方案优于GT方案。两者扩增效率均优于一步法。

Objective To study the ex vivo expansion efficiency of dendritic cells (DCs) from human bone marrow CD34+ progenitor cells and this biological properties. Methods CD34+ cells were purified by using CD34 microbeads from normal bone marrow and cultured in serum-dependent medium by two ways:①Two-step culture method,expension in the presence of stem cell factor(SCF),Flt-3 ligand(FL),thrombopoietin(TPO) and interleukin-3(IL-3) for 14 days,and then 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),or with GM-CSF plus TNF-a for 10 or more days (group GT);②One-step culture method,cultured in SCF,FL,TPO,IL-3,GM-CSF and TNF-α for 14 days. The biological properties of DCs were examined by phase contrast microscope,electron microscope,flow cytometry and fluorescein isothiocyanate conjugated dextran (FITC-DX) uptake test. Results These cells showed a typical dendritic phenotype:they were highly positive for DC marker CD1a,costimulatory molecule CD80,CD86,CD40,adhesion molecule CD54, and HLA-DR and negative for CD3 and CD19. After inducing for 10 days in group GI,the number of total cell and CD1a+ DCs generated increasingly (198±178) fold and (122±129) fold,respectively. Comparing with group GT,group GI produced a similar total cell number and CD1a+DCs number, but induced a higher expression of CD1a,CD80 and CD86. The number of total cell and CD1a+ DCs generated increasingly (43±16) fold and (4±2) fold,respectively. Conclusions A large number of DCs could be induced from normal CD34+ cells in a two-step process. The GI scheme is superior to GT scheme on the quality of DCs and both are superior to one-step culture method.