猪骨髓源单核巨噬细胞诱导培养方法建立及应用  

Development and Application of a Culture Method for Bone Marrow-Derived Mononuclear Phagocytes of Piglets

作　　者：张伟 李俊辉 潘晓梅 曹剑 唐慧芬 毛丽萍 贺笋 ZHANG Wei;LI Junhui;PAN Xiaomei;CAO Jian;TANG Huifen;MAO Liping;HE Sun(Tecon bio-pharmaceutical Co.,Ltd.,Urumqi 830023,China)

机构地区：[1]天康生物制药有限公司,乌鲁木齐830023

出　　处：《中国动物传染病学报》2025年第1期50-59,共10页Chinese Journal of Animal Infectious Diseases

基　　金：兵团科技攻关项目(2023AB009-03);自治区“天山英才”培养计划青年拔尖人才项目(2023TSYCJC0029)。

摘　　要：选择不同细胞因子和培养基,比较4种培养基和细胞因子对猪骨髓细胞分化为单核巨噬细胞数量和增殖能力的影响,筛选出猪骨髓细胞诱导单核巨噬细胞的最佳培养条件。结果显示,在含10%胎牛血清的DME/F12培养基中添加粒细胞-巨噬细胞集落刺激因子(GM-CSF)(40 ng/mL)和巨噬细胞集落刺激因子(M-CSF)(50 ng/mL)能够诱导骨髓细胞诱分化成大量单核巨噬细胞,细胞数量与对照组差异显著(P<0.05),并且诱导细胞中有针对单核巨噬细胞的特异性抗原(CD64)表达,单核巨噬细胞纯度为77.57%左右。诱导后的细胞中检测到针对单核巨噬细胞的特异性Nramp1基因。猪繁殖与呼吸综合征病毒(PRRSV)能够在细胞中大量增殖,与未诱导的骨髓细胞差异显著(P<0.05)。以上结果表明,DME-F12培养基添加GM-CSF和L929细胞上清液能够显著增强猪骨髓细胞诱导分化成单核巨噬细胞的能力,并且能够获得高纯度、高密度、高活率的单核巨噬细胞。In order to explore the optimal in vitro growth condition for bone marrow-derived mononuclear phagocytes of the piglets,culture media and cytokines were optimized in the present study.To understand the infl uences of media and cytokines on the generation quantity and proliferation capacity,four media with cytokine suspensions were compared to select the optimal isolation and culture condition.The results showed that the monocyte-macrophages proliferated significantly better(P<0.05)in DME-F12 with GM-CSF(40 ng/mL),MCSF(50 ng/mL)as compared with the control media.The cell purity was about 77.57%as determined in FCM.The specific molecule(CD64)was expressed in induced monocyte-macrophages and the Nramp1 gene was also transcribed as demonstrated in PCR.The Porcine reproductive respiratory syndrome virus(PRRSV)was proliferated more actively(P<0.05)in induced monocytemacrophages as compared with the control group.These results indicated that DME-F12 media with GM-CSF(40 ng/mL)and L929 was favorable to proliferation and differentiation of bone marrow cells and beneficial to obtain monocyte-macrophages with high purity,viability and density.

关 键 词：猪 骨髓 单核巨噬细胞 体外培养 

分 类 号：S852[农业科学—基础兽医学]