Directional differentiation and identification of bone marrow-derived dendritic cells in BALB/C mice / 西安交通大学学报(医学版)

ABSTRACT

【Objective】 To establish an effective method for acquiring bone marrow-derived dendritic cells (DCs) from BALB/C mice in vitro and to establish a reservoir of DC precursor cells. 【Methods】 CD117+ hematopoietic stem cells (HSCs) were isolated and purified from bone marrow of BALB/C mice by immunomagnetic beads separation system (MACS), and then amplified in vitro with mouse stem cell factor (SCF) and interleukin-3 (IL-3). HSC was induced to differentiate into DCs by adding granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and IL-4. Different cytokines (tumor necrosis factor-alpha or IL-10) were added to control the maturity of dendritic cells. Then the morphology (electron microscopy), surface molecular markers (FACS method) and cytokine secretion level (ELISA method) were identified. 【Results】 ① The purity of CD117 + HSC isolated and purified by MACS system was over 95%. ② SCF plus IL-3 could effectively stimulate HSC amplification. ③ The morphology of mature DC (mDC) and immature DC (imDC) was significantly different under light and scanning electron microscopy. ④ In the expressions of surface markers CD40, CD80, CD86, I-A/I-E, there were significant differences between imDC group and mDC group (P<0.01). ⑤ After LPS stimulation, the secretion of IL-12 in imDC group did not change significantly (P=0.064), while the secretion of IL-12 in mDC group increased significantly (P=0.009). LPS and TNF-α had a synergistic effect in stimulating DC maturation. 【Conclusion】 Specific combinations of cytokines can effectively induce the differentiation of bone marrow HSCs into DCs in BALB/C mice, and can control the maturity of DCs. This study makes it possible to use gene modified dendritic cells in GD immunotherapy.