水牛外周血来源树突状细胞体外诱导培养与鉴定  

Induction culture and identification of dendritic cells from buffalo peripheral blood in vitro

作　　者：杨曦 梅雪芳[1] 曾子轩 王冬英[1] 黄维义[1] YANG Xi;MEI Xuefang;ZENG Zixuan;WANG Dongying;HUANG Weiyi(College of Animal Science and Technology,Guangxi University,Nanning 530000,China)

机构地区：[1]广西大学动物科学技术学院,南宁530000

出　　处：《黑龙江畜牧兽医》2020年第19期16-20,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31560679);广西创新驱动发展专项(桂科AA17204057)。

摘　　要：为了建立一种体外诱导培养水牛外周血来源树突状细胞(dendritic cell,DC)的方法,试验分离了水牛外周血来源的单个核细胞(PBMC),在粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL-4)联合作用下进行诱导分化,并以LPS诱导的未成熟树突状细胞发育为成熟树突状细胞作对照组,通过显微镜(光学显微镜、扫描电子显微镜、透射电子显微镜)和流式细胞术分别对获得的细胞进行形态学观察和表型鉴定。结果表明:PBMC在50 ng/mL GM-CSF和50 ng/mL IL-4体外持续作用6 d后,流式细胞术检测表现出CD11chighMHCⅡmedCD40medCD86low的表型特征,与未成熟树突状细胞的一般表型特征相符。培养至第4天,圆形的PBMC开始呈现出不规则状态;培养至第6天,可在细胞膜上观察到小的微绒毛、突起和细胞聚集等未成熟树突状细胞的形态特征,与表型鉴定结果一致。在LPS的进一步作用下,PBMC呈现出成熟树突状细胞的典型形态。说明GM-CSF和IL-4联合作用可以获得未成熟树突状细胞,并且该细胞可以在LPS的进一步作用下发育成熟,通过该方法可以获得足量的水牛树突状细胞以满足试验需求。In order to establish an in vitro culture method of dendritic cells(DC)derived from buffalo peripheral blood,buffalo-derived peripheral blood mononuclear cells(PBMC)were isolated in this experiment,and were induced to differentiate in a combination of granulocyte-macrophage colony(GM-CSF)and interleukin(IL-4).Immature dendritic cells were induced to develop into mature dendritic cells by LPS as a control group.Morphological observation and phenotype identification of the obtained cells were performed by microscopy(optical microscope,scanning electron microscope,transmission electron microscope)and flow cytometry,respectively.The results showed that after continuous action of PBMC at 50 ng/mL GM-CSF and 50 ng/mL IL-4 for 6 days in vitro,the phenotypic characteristics of CD11 chighMHCⅡmedCD40medCD86low were found by flow cytometry,and comply with the general phenotypic characteristics of immature dendritic cells.On the 4 th day of culture,the round PBMC began to show irregularity.On the 6 th day of culture,the morphological characteristics of immature dendritic cells,such as small microvilli,projections and cell aggregation,were observed on the cell membrane,which were consistent with the results of phenotypic identification.Under the further action of LPS,PBMC exhibited the typical morphology of mature dendritic cells.It indicated that the combined action of GM-CSF and IL-4 could obtain immature dendritic cells,and the cells could develop and mature under the further action of LPS,enough buffalo dendritic cells could be obtained through this method to meet the test requirements.

关 键 词：外周血单个核细胞 树突状细胞 水牛 流式细胞术 光学显微镜 扫描电子显微镜 透射电子显微镜 

分 类 号：S855.9[农业科学—临床兽医学]