丁酸钠诱导人单核细胞源性未成熟树突状细胞的形态及免疫功能变化

背景：未成熟树突状细胞（dendriti ccells，DC）可以诱导免疫耐受形成，在器官移植领域具有广阔应用前景。目前采用的诱导未成熟树突状细胞的方法仍存在一些不足，寻求新的诱导方法十分必要。目的：观察丁酸钠对人外周血来源的树突状细胞的成熟状态和免疫学功能的影响。设计：观察对比，体外细胞学实验。单位：中山大学附属第二医院肝胆外科。材料：人外周血样品取自中山大学身体健康的大学生志愿捐献者（年龄20—23岁），共5份，每份100mL，均在献血后2h内进行外周血单个核细胞和淋巴细胞分离。方法：实验于2006—09／2007—03在中山大学附属第二医院医学研究中心完成。①人外周血单个核细胞采用粒细胞-巨噬细胞集落刺激因子和白细胞介素4诱导成为未成熟树突状细胞。②诱导6d后，加入组蛋白去乙酰化酶抑制剂丁酸钠1mmol／L诱导，以促成熟因子脂多糖1mg／L为对照；设不加促成熟因子为空白对照组。③在诱导0d时加入丁酸钠，6d时加入脂多糖再次刺激。主要观察指标：动态观察细胞形态学变化，流式细胞仪检测DC表型，FITC标记的Dextran检测DC内吞能力，采用酶联免疫吸附法检测白细胞介素12分泌，混合淋巴细胞反应检测DC刺激淋巴细胞增殖能力。结果：①DC形态：在丁酸钠作用下，DC聚集现象明显减少。②细胞表型检测结果：丁酸钠促成熟组常规方法诱导的未成熟DC的CD80，CD83，HLA-DR表达均低于脂多糖组，差异有显著性意义（P〈0．01）。采用丁酸钠法诱导的未成熟DC，在第6天时加入促成熟因子脂多糖后CD80，CD83，HKA-DR表达仍处于低水平。③DC内吞能力：常规方法诱导的未成熟DC，用LPS诱导成熟后，其内吞能力均低于空白对照组和丁酸钠促成熟组，差异有非常显著性意义（P〈0．01）；而采用丁酸钠法诱导的DC，其内吞能力最强，高于其他各组，差异有非常显著性意义（P〈0．01）。④DC分泌白细胞介素12：常规诱导法用LPS诱导DC成熟后的白细胞介素12分泌水平高于对照组、丁酸钠促成熟组及丁酸钠诱导法，差异有非常显著性意义（P〈0．01）。⑤DC诱导的MLR：常规诱导法用LPS诱导的成熟DC刺激淋巴细胞的增殖能力显著高于对照组、丁酸钠促成熟组及丁酸钠诱导法，差异有非常显著性意义（P〈0．01）。结论：丁酸钠可以抑制DC成熟，稳定诱导未成熟DC生成，该DC不能被促成熟因子激活，在诱导移植免疫耐受方面具有潜在应用前景。

Morphology and immunological function of immature dendritic cells induced by sodium butyrate in human monocytes

BACKGROUND: The immature dendritic cell (imDC) can induce immunological tolerance and has widely application in the field of organ transplant. At present, the methods of inducing imDC are insufficient, so the new induction method is demanding.OBJECTIVE: To investigate the effect of sodium butyrate (SB) on the maturation and immunological function of human peripheral blood-derived imDC.DESIGN: Controlled observation and in vitro cytological trial.SETTING: Department of Hepatobiliary Surgery in the Second Affiliated Hospital of Sun Yat-sen University.MATERIALS: Five samples of human peripheral blood were obtained from the healthy volunteers (aged 20-23 years) of Sun Yat-sen University, totally 500 mL. Then peripheral blood mononuclear cells (PBMCs) and lymphocytes were isolated within 2 hours.METHODS: The experiment was carried out in the Medical Research Center of the Second Hospital Affiliated to Sun (1 mmol/L) was added for induction, while those supplemented with maturation promoting factor lipopolysaccharide (LPS)the beginning of induction, while LPS was added on the sixth day for second stimulation.MAIN OUTCOME MEASURES: Cell morphological change, flow cytometry was used to detect DC phenotype,FITC-labeled Dextran was used to detect the endocytosis of DC, the production of IL-12 was determined by means of enzyme-linked immunosorbent assay, and the proliferation of lymphocyte induced by DC was assayed with mixed lymphocyte reaction.expressions of CD80, CD83 and HLA-DR were significantly lower in the imDC of routine induction group following SB maturity promoting, compared with LPS group (P＜0.01). On the sixth day, LPS was added into the SB-induced imDC,Endocytosis of DC: The imDC of routine induction group possessed a significantly lower endocytic activity after induced by LPS, and there were extremely significant differences compared with blank control group and SB maturation Production of IL-12: The production of IL-12 in the mDC induced by LPS was significantly higher than that in control group, SB maturation promoting group and SB induction group, the mDC induced by LPS in routine induction group stimulated significantly stronger proliferation of lymphocyte (P＜0.01).