小鼠脾脏来源树突状细胞的体外扩增培养.doc

　　小鼠脾脏来源树突状细胞的体外扩增培养 ：黎辉，曹宇皎 ，黄军华 ，刘俊峰 【摘要】 目的：对体外诱导小鼠脾脏单个核细胞分化成的树突状细胞(DC)的生物学表型及功能进行检测，并与骨髓的DC进行比较。方法：分离小鼠脾脏单个核细胞，在体外培养的条件下通过添加25 ng·ml-1的粒单核细胞集落刺激因子和25 ng·ml-1的白细胞介素4(IL4)将其诱导为DC，分别从细胞形态、表型以及功能3个方面对其进行检测，并与骨髓的DC进行比较。结果：小鼠脾脏的DC经磷酸脂多糖(LPS)刺激成熟后其表面显示出明显的树枝状突起，高表达CD11c、CD86及MHCⅡ类分子，并具有极强的刺激同种异基因淋巴细胞增殖的能力，其特征与骨髓的DC相差无几。结论：小鼠脾脏单个核细胞能够被诱导为具有正常形态、表型、功能的DC，为DC的功能研究以及进一步制备相应的肿瘤疫苗提供又一可靠。 【关键词】 脾脏; 骨髓; 树突状细胞; 诱导; 小鼠 　Dendritic cells(DC) are the most potent antigen presenting cells that are responsible for priming native T cells in the immune response. Immunotherapy of DC/tumor vaccine has bee an important therapeutic strategy against tumors[12]. Hoited by the loarroouse spleen mononuclear cells into dendritic cells in vitro, and then pared the biological characteristics of them arroononuclear cells derived DCs. 　　1 Materials and methods 　　1.1 Materials 　　Main reagents: rebinant mouse granulocytemacrophage colonystimulating factor(GMCSF), rebinant mouse interleukin4(IL4), rebinant mouse leukemia inhibitory factor(LIF,Peprotech), lipopolysaccharide(LPS,Sigma); mitomycin C(MMC) and methabenzthiazuron(MTT,Duchfo). DCs culture medium: high glucose DMEM plus 15% fetal calf serum plus 25 mg·L-1 rmGMCSF and rmIL4. Mice: six to eight ale BALB/c and 129 mice Animal Center of Guiyang Medical College. 　　1.2 Methods 　　1.2.1 Induction of DC from mouse spleen Cells ice.Mononuclear cells at the interface pholyte gradient. They l-1.The suspended cells oved and the adherent cells edium. LPS e time mouse bone mononuclear cells icroscope daily and suspended cells icroscopy. 　　1.2.3 Cell surface marker analysis Cells in. After being etry. 　　1.2.4 Mixed lymphocyte reactions(MLR) Collected spleen derived DC l-1 MMC as a stimulator for 45 min and plates at various ratios for 4 d.At the same time, ulators. 4 h before harvesting, cells g·ml-1), then added easured. The stimulation index ula: SI= absorbency of stimulation groupabsorbency of contrast/the absorbency of contrast. 　　1