小鼠骨髓来源树突状细胞模型的建立及功能鉴定  

Establishment and functional identification of mouse bone marrow-derived dendritic cell model

作　　者：纪俊莉 梁笑星 李小萌 杨姿萱 周宇泽 凌一绮 郝艳梅[1] JI Junli;LIANG Xiaoxing;LI Xiaomeng;YANG Zixuan;ZHOU Yuze;LING Yiqi;HAO Yanmei(Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]蚌埠医学院肿瘤基础研究与临床检验诊断重点实验室,蚌埠233030

出　　处：《山西医科大学学报》2023年第9期1247-1253,共7页Journal of Shanxi Medical University

基　　金：国家大学生创新训练项目(202210367056);安徽省教育厅重点科学项目(KJ2021A0691)。

摘　　要：目的通过快速高效获取小鼠骨髓有核细胞,建立小鼠骨髓来源树突状细胞(bone marrow dendritic cell,BMDC)模型,并对BMDC进行表面特异性标志物及功能鉴定。方法利用小鼠股骨和胫骨肌肉群分布的位置特点,快速游离出C57BL/6雄性小鼠的股骨和胫骨,获取骨髓细胞,使用重组小鼠粒细胞-巨噬细胞集落刺激因子(recombinant murine granulocyte-macrophage colony stimulating factor,rmGM-CSF)和重组小鼠白细胞介素4(recombinant murine interleukin-4,rmIL-4)诱导培养成BMDC。光镜下观察细胞形态,流式细胞术检测BMDC的特异标志物CD11c,进行纯度鉴定。将BMDC分为2组:对照组和LPS组(1μg/ml),采用流式细胞术检测特异性标志物CD11c、CD86、CD80、CD40和MHCⅡ分子,CCK-8实验检测BMDC对脾脏淋巴细胞增殖能力的影响,qRT-PCR和Western blot检测BMDC中NF-κB的表达情况,进行功能鉴定。结果诱导培养7 d,镜下观察BMDC具有树突状突起的典型形态,CD11c表达可达97.4%,表明BMDC纯度能够满足后续实验需要。流式细胞术结果显示,与对照组比较,LPS组BMDC中特异性标志物CD86、CD80、CD40和MHCⅡ分子的荧光强度和阳性率显著增强(P<0.01)。CCK-8实验结果显示,与对照组比较,LPS组BMDC刺激淋巴细胞增殖的能力明显增强(P<0.01)。qRT-PCR结果显示,与对照组比较,LPS组BMDC中NF-κB的表达上调(P<0.001)。Western blot结果表明,与对照组比较,LPS组BMDC内p-NF-κB的表达上调(P<0.01)。结论成功建立了简单高效的体外诱导培养的BMDC模型,为DC免疫功能的基础研究和转化应用奠定了实验基础。Objective To establish a mouse bone marrow-derived dendritic cell(BMDC)model by rapidly and efficiently obtaining mouse bone marrow nucleated cells,and identify the BMDC surface markers and function.Methods According to the positional characteristics of the distribution of muscle groups in the femur and the tibia of C57BL/6 male mice,bone marrow cells were quickly isolated and induced into BMDC using recombinant murine granulocyte-macrophage colony stimulating factor(rmGM-CSF)and recombinant murine interleukin-4(rmIL-4).The cell morphology was observed under light microscope,and the specific marker CD11c in BMDC was detected by flow cytometry to verify the purity.BMDCs were divided into two groups:control group and LPS group(1μg/ml).The specific markers CD11c,CD86,CD80,CD40 and MHCⅡmolecules were detected by flow cytometry.The effect of BMDC on the proliferation of splenic lymphocytes was detected by CCK-8 assay.The expression of NF-κB in BMDC was detected by qRT-PCR and Western blot for functional identification.Results After seven days of culture,a typical dendritic morphology was obviously found on the surface of BMDC under microscope,and the expression of CD11c was 97.4%,indicating that the purity of BMDC could meet the needs of subsequent experiments.Flow cytometry results showed that compared with control group,the fluorescence intensity and the positive rates of specific markers CD86,CD80,CD40 and MHCⅡmolecules in LPS group were significantly increased(P<0.01).The results of CCK-8 experiment showed that compared with control group,BMDC significantly enhanced the ability to stimulate lymphocyte proliferation in LPS group(P<0.01).The qRT-PCR results showed that compared with control group,the expression of NF-κB in BMDC was upregulated in LPS group(P<0.001).Western blot results showed that compared with control group,the expression of p-NF-κB in BMDC was upregulated in LPS group(P<0.01).Conclusion A simple and efficient method for BMDC culture in vitro is successfully established,which provides an ex

关 键 词：股骨 胫骨 骨髓细胞 诱导培养 树突状细胞 脂多糖 表面标志物 

分 类 号：R593[医药卫生—内科学]