奶牛外周血树突状细胞体外诱导培养与鉴定

摘要

通过粒－巨噬细胞集落刺激因子（ GM⁃CSF）和白细胞介素－4（ IL⁃4）体外诱导外周血单核细胞为树突状细胞，为利用树突状细胞免疫疗法治疗奶牛乳房炎奠定基础和提供细胞模型。利用淋巴细胞分离液分离获得奶牛外周血单核细胞，在6孔板内培养2h后，弃掉含有大量的T细胞和B细胞上清液，贴壁的基本上是单核细胞，磷酸盐缓冲液清洗5次，加入含有GM⁃CSF和IL⁃4的2 mL培养基进行3 d诱导。之后，从培养基顶部小心吸弃1．4 mL的培养基，然后再补加含有GM⁃CSF和IL⁃4的1．8 mL培养基继续诱导3 d。每天通过显微镜观察细胞形态。第7天经流式检测细胞表面抗原 CD11c、CD14、主要组织相容性复合体Ⅱ（ MHCⅡ）、CD40、CD80、CD86的表达。结果表明：1）第2天，一些细胞表面可以生长出刺突并伴随着伪足的生长。第3天，细胞表面的刺突和伪足越来越多。第4、5天，一些带有刺突和伪足的细胞开始聚集和融合。第6天，单核细胞基本被诱导为树突状细胞，细胞表面含有大量清晰可见的刺突和伪足。2）经流式检测，CD14、CD11c、MHCⅡ阳性表达细胞分别占诱导细胞的6．8％、65．0％、75．9％，CD80和CD86阳性表达细胞分别占诱导细胞的2．0％和1．2％。综上所述，采用奶牛外周血单核细胞经体外诱导能够获得一定纯度的奶牛树突状细胞。%This study aimed to induce bovine peripheral blood monocyte derived dendritic cell by granulocyte⁃macrophage colony stimulating factor ( GM⁃CSF) and interleukin⁃4 ( IL⁃4) cytokines, which could lay founda⁃tion and provide cell model for dairy cow mastitis treatment using cell immunotherapy. The bovine peripheral blood monocyte was acquired by lymphocyte separation medium and seeded in 6⁃proe plate to culture for 2 h. Then, suspended cells containing an amount of B and T cells were discarded, and adherent cells were mostly monocyte. The cells were washed for 5 times using phosphate buffer, and 2 mL culture medium containing GM⁃CSF and IL⁃4 cytokines was added to culture for 3 d. After that, 1.4 mL culture medium was discard from medium top, and 1.8 mL culture medium containing GM⁃CSF and IL⁃4 was added to culture for another 3 d. The cell was observed under microscope every day. On days 7, the cells were harvested to detect CD11c, CD14, major histocompatibility complex Ⅱ ( MHCⅡ) , CD40, CD80 and CD86 molecules by flow cytome⁃try. The result showed as follows:1) on days 2, some of the cells started to extend spikes and accompanied by pseudopodia stretching. On days 3, the spikes and pseudopodia grew more and more. On days 4 and 5, cells with spikes and pseudopodia started aggregation and fusion. On days6, most of monocytes were derived to den⁃dritic cells, on which plenty of spikes and pseudopodia could be clearly seen. 2) CD14, CD11c, MHCⅡ, CD80 and CD86 positively expressed cells accounts for 6. 8%, 65. 0%, 75. 9%, 2. 0% and 1. 2% of induced cells by flow cytometry, respectively. In conclusion, bovine peripheral blood monocytes can be derived to den⁃dritic cells with certain purity.