小鼠骨髓浆细胞样与髓样树突状细胞的体外培养及对比研究  

In Vitro Culture and Comparative Study of Plasmacytoid and Myeloid Dendritic Cells from Mouse Bone Marrow

作　　者：谭剑峰 李东方 郭权威 况军 张建华 TAN Jianfeng;LI Dongfang;GUO Quanwei(Shenzhen Hospital of Southern Medical University, Guangdong Shenzhen 518000, China)

机构地区：[1]南方医科大学深圳医院胸外科,广东深圳518000

出　　处：《河北医学》2021年第10期1626-1631,共6页Hebei Medicine

基　　金：南方医科大学深圳医院胸外科重点学科建设基金,(编号:ZDXK2020003)。

摘　　要：目的:建立小鼠骨髓浆细胞样与髓样树突状细胞亚群的体外培养、分离及鉴定的方法,探讨小鼠骨髓树突状细胞亚群间生物学功能的差异。方法:获取C57BL/6小鼠骨髓细胞,利用重组小鼠fms样酪氨酸激酶受体3配体(rmFlt3-L)诱导其向pDC及mDC亚群分化,并利用流式细胞仪及磁珠抗体对小鼠骨髓DC亚群进行分离与鉴定,计算不同时间点细胞数量及mDC与pDC的占比,确定最佳分选时间,然后将Lewis肺癌细胞裂解物分别刺激mDC及pDC,观察DC细胞形态、利用流式细胞仪检测各组免疫表型分子的变化、ELISA法检测细胞因子的分泌情况及CCK-8法检测淋巴细胞的增殖情况。结果:小鼠骨髓细胞经rmFlt3-L体外诱导培养8~9d,可获得表型为CD11c+CD11b-B220+pDC与CD11c+CD11b+B220-mDC。功能方面,在负载Lewis肺癌细胞裂解物条件下,mDC组分泌的IL-12及TNF-α均高于pDC组(P<0.05);mDC组刺激淋巴细胞增殖的能力均高于pDC组(P<0.05)。形态上,mDC体积较大,细胞核不规则,且细胞周边见较多的“突起”;而pDC体积较小,呈类圆形,大部分细胞表面无“突起”。免疫表型方面,负载Lewis肺癌细胞裂解物的mDC组细胞表面CD80、CD86、CD40及MHC-Ⅱ类分子平均荧光强度值高于pDC组。结论:小鼠骨髓细胞在rmFlt3-L的刺激诱导下分化成pDC与mDC亚群,负载肿瘤细胞裂解物的mDC的生物学功能强于pDC,可能与mDC的形态成熟与共刺激分子表达上调相关。Objective:To establish a method for culture,isolation and identification of mouse bone marrow plasmacytoid and myeloid dendritic cell subsets in vitro,and to explore the differences of biological functions between mouse bone marrow dendritic cell subsets.Methods:Bone marrow cells of C57BL/6 mice were obtained,and the mice fms-like tyrosine kinase receptor 3 ligand(rmFlt3-L)was used to induce them to differentiate into pDC and mDC subpopulations.The DC subpopulations of mice bone marrow were separated and identified by flow cytometry and magnetic bead antibody.The cell number and the ratio of mDC to pDC at different time points were calculated to determine the best sorting time.Then,the lysates of Lewis lung cancer cells stimulated mDC and pDC,respectively,and the morphology of DC cells was observed.The changes of immunophenotype molecules in each group were detected by flow cytometry,and the secretion of cytokines was detected by ELISA,and the proliferation of lymphocytes was detected by CCK-8.Results:Mouse bone marrow cells were cultured in vitro by rmFlt3-L for 8-9 days,and the phenotypes were CD11c+CD11b-B220+pDC and CD11c+CD11b+B220-mDC.Functionally,under the condition of loading Lewis lung cancer cell lysates,the secretion of IL-12 and TNF-αin mDC group was higher than that in pDC group(P<0.05).The ability to stimulate lymphocyte proliferation in mDC group was higher than that in pDC group(P<0.05).Morphologically,mDC has large volume,irregular nucleus and many"protrusions"around the cells.However,pDC is small and round,and most cells have no"protrusion"on their surface.Immunophenotypically,the average fluorescence intensity of CD80,CD86,CD40 and MHC-Ⅱmolecules on the cell surface of mDC group loaded with Lewis lung cancer cell lysate was higher than that of pDC group.Conclusion:Mouse bone marrow cells can differentiate into pDC and mDC subpopulations under the stimulation of rmFlt3-L.The mDC loaded with tumor cell lysates has better biological function than pDC,which may be related to the morphological ma

关 键 词：髓样树突状细胞 浆细胞样树突状细胞 肿瘤细胞裂解物 

分 类 号：R73[医药卫生—肿瘤]