小鼠脾脏来源树突状细胞的体外扩增培养_临床医学论文.docVIP

小鼠脾脏来源树突状细胞的体外扩增培养_临床医学论文 小鼠脾脏来源树突状细胞的体外扩增培养_临床医学论文 作者：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，并与骨髓来源的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，并与骨髓来源的DC进行比较。结果：小鼠脾脏来源的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，其特征与骨髓来源的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来源。 【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 become an important therapeutic strategy against tumors[12]. However, the basic and clinical applications of DCs have been limited by the low yield and purity of DCs generated by traditional bone marrow derivation. In this study, we induced mouse spleen mononuclear cells into dendritic cells in vitro, and then compared the biological characteristics of them with those of bone marrow mononuclear cells derived DCs. 　　1 Materials and methods 　　1.1 Materials 　　Main reagents: recombinant mouse granulocytemacrophage colonystimulating factor(GMCSF), recombinant mouse interleukin4(IL4), recombinant 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 weeks old female BALB/c and 129 mice were purchased from Animal Center of Guiyang Medical College. 　　1.2 Methods 　　1.2.1 Induction of DC from mouse spleen Cells were flushed out of spleen of 129 mice.Mononuclear cells at the interface were collected by centrifugation over a lympholyte gradient. They were washed and then cultured in 6well plate for 2 h with the density of 5×104 ml-1.The suspended cells were removed and the adherent cells were cultured with DC culture medium. LPS was added on day 7 and the suspended