猪血源树突状细胞诱导培养与鉴定

Induction and Identification of Swine Blood-derived Dendritic Cells

为规范DCs诱导培养和鉴定的方法,研究相关病毒致病机制、制定相应的治疗与预防措施奠定技术平台。分离猪外周血单个核细胞(PBMCs),贴壁细胞经一定浓度GM-CSF和IL-4诱导,不同时间观察形态变化,7天后收集所诱导的细胞,经光镜、流式细胞术及激光共聚焦显微镜鉴定其形态、表面标志物CD1a/SWC3a;利用双色流式细胞术检测DCMHC-II分子和CD80/86表达情况。试验结果显示,所诱导的细胞具有典型树突状形态;其表面CD1a/SWC3a双阳性率达到90.1%,MHC-II/CD80/86分子双阳性率达98.3%;激光共聚焦显微镜观察细胞呈集落状,细胞表面CD1a/SWC3a双阳性,表明已经成功诱导出猪血源DC,并获得诱导的标准程序,为研究以DC为靶细胞的猪病毒性疾病免疫致病机理奠定基础。

To standardize the method to induce and cultivate swine blood-derived dendritic cells（ DC） in vitro, which could provide a technology platform for the study of related-viral diseases ’ mechanism and making the measures of their prevention and treatment. Porcine peripheral blood mononuclear cells （PBMCs） were separated. The adherent cells of PBMCs on the flask were induced by GM-CSF and IL-4 and their shapes were observed by the optical microscope at different times. After 7 days, these induced cells were collected and their form and surface markers CD1aSWC3a were identified by light microscope, flow cytometry and laser confocal microscope. The MHC-II and CD80/86 molecules on the surface of these cells were identified by bicolor flow cytometry. The results showed that the induced cells were dendritic, double-positive rates of CD1a/ SWC3a and MHC-II and CD80/86 were 90.1% and 98.3% respectively. Colony-like cells were observed and the surface marker CD1a/SWC3a were double-positive under laser confocal microscope. The swine blood-derived DCs were induced successfully according to the analysis of phenotype and morphology. The standard procedure of the induction is acquired, which has laid a foundation for the research of immuno-suppression mechanism of porcine viral diseases that the target cells are DCs.