猪骨髓源树突状细胞体外诱导培养与鉴定

摘要

Dendritic cell (DC) is a very important antigen presenting cells of the body.It plays an important role in innate and adaptive immunity.In order to explore the DC function and inducing differentiation methods of DC,pigs were anesthetized by the epidural space using procaine,the bone marrow was aspirated from the hip bone cavity by bone marrow puncture.The precursor cells were collected by differential adherence method after lysing the red blood cells.The precursor cells were cultured in the medium with recombinant porcine interleukin (rpIL-4) and granulocyte-macrophage colony-stimulating factor (rpGM-CSF) in vitro.Then the DC was identified by morphologic and phenotypic characteristic using light microscope and electron microscope.The results showed that the differentiated cells displayed a typical morphology of DC.The expression of CD1 in the surface of the cells was 65.13％ (p＜0.01) and 56.5％ (p＜0.01) at the 6th and 8th days after the differentiation of the bone marrow precursor cells,the expression of SLA-Ⅱ-DR was 86.87％ (p＜0.01) and 84.60％ (p＜0.01) at the 6th and 8th days,respectively.A large quantity of porcine bone marrow-derived DC with higher purity could be prepared in vitro by this way used in this study.%树突状细胞(DC)是机体主要的抗原递呈细胞,在固有免疫及适应性免疫中发挥重要作用.为进一步探究DC功能及诱导其分化的方法,本研究采用硬膜外腔麻醉法麻醉受试猪,以骨髓采集针从髋骨处抽取骨髓,裂解红细胞后,差速贴壁法获得前体细胞后,添加重组猪粒细胞-巨噬细胞集落刺激因子和重组猪白细胞介素4诱导其分化,以形态学方法和流式细胞术鉴定其特征.结果显示,骨髓前体细胞经诱导分化后具有典型的DC形态,其标记分子分化抗原簇l在第6d和8d阳性率分别达65.13％(p＜0.01)和56.5％(p＜0.01),标记分子二型猪白细胞抗原(SLA-IL-DR)在诱导后第6d和8d阳性率分别为86.87％(p＜0.01)和84.60％(p＜0.01).本研究建立了活体猪骨髓源DC的体外分离培养和诱导分化方法,为基于DC的疾病防控研究提供依据.