体外培养人树突状细胞分化成熟特性分析

The Character of Differentiation and Maturity of Cultured Human Dendritic Cells

目的利用人脐血CD34+干细胞诱导分化树突状细胞(DC),并对DC分化发育过程的生物学特性进行鉴定和分析。方法通过SCF、GM-CSF、TGF-β1、F lt-3L及TNF-α体外培养体系,从脐血CD34+造血干细胞中诱导扩增获得DC。采用倒置显微镜和电镜观察DC形态学特征,流式细胞仪检测DC细胞表型及胞内活性氧(ROS)水平,MTT比色法检测DC刺激同种异体T细胞增殖能力;ELISA法检测DC培养上清液中IL-12含量。结果CD34+细胞培养5至7 d,细胞呈现DC典型树突状形态,处未成熟状态,再经TNF-α诱导及继续培养至14 d,DC发育成熟。未成熟DC表达模式识别受体CD209(DC-SIGN),且胞内蓄积适量ROS,具备了细胞吞噬能力。成熟DC除仍高表达DC-SIGN,其表面黏附共刺激分子CD llc、CD54、CD83、CD80、CD86表达上调,细胞因子IL-12分泌增加,且具明显的体外刺激T细胞增殖能力,符合于抗原递呈细胞特征。此外,未成熟和成熟DC基本不表达黏附分子P-、E-选择素,但未成熟和成熟DC分别高表达和低表达L-选择素。结论建立人DC体外模型及其分化发育生物学特性分析,为进一步研究DC功能,以及利用DC调控免疫应答用于疾病防治提供了基础。

Objective Dendritic cells（DCs） are professional antigen-presenting cells with the ability to initiate primary T cell responses. In this study we observed the morphologic features, phenotype and function of cultured human DCs. Methods Cord blood CD34 ^＋ stem cells were isolated and cultured in 20% LMDM medium with SCF, GM-CSF, TGF-β1 ,Flt-3L for 7 days, or followed by stimulating with TNF-α for additional 7 days. The cell phenotype and reactive oxygen species（ROS） were assayed by flow cytometry. IL-12p70 secreted in culture medium was detected by ELISA. Results The expression of CDllc,CD54,CD83,CD80 and CD86 was lower in immaturer DCs group than in mature DCs group. There is no expression of P-or E-selectin in immature and mature gurops. The expression of L-selectin was higher in immature group than in mature group. CD209/DC-SIGN was expressed significantly in both groups. The secretion of ROS was lower in mature group than in immature group. The secretion of IL-12p70 was higher in mature group than in immature group. MLR suggested that mature group had more potency to activate allogenic T cell proliferation. Conclusion The study of differentiation and maturity of cultured human dendritic cells has great value to exploring how to regulate DCs in immunological diseases.