Abstract：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-α in 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 maturation and the up-regulation of costimulatory molecules of mDC.
谭剑峰, 李东方, 郭权威, 况军, 张建华. 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[J]. 河北医学, 2021, 27(10): 1626-1631.
TAN Jianfeng, LI Dongfang, GUO Quanwei, et al. In Vitro Culture and Comparative Study of Plasmacytoid and Myeloid Dendritic Cells from Mouse Bone Marrow. HeBei Med, 2021, 27(10): 1626-1631.
[1] Son YI,Egawa S,Tatsumi T,et al.A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells[J].Immunol Methods,2002,262(1-2):145~157. [2] Puhr S,Lee J,Zvezdova E,et al.Dendritic cell development-History advances and open questions[J].Semin Immunol,2015,27(6):388~396. [3] Zhang R,Yuan F,Shu Y,et al.Personalized neoantigen-pulsed dendritic cell vaccines show superior immunogenicity to neoantigen-adjuvant vaccines in mouse tumor models[J].Cancer Immunology Immunotherapy,2020,69(1):135~145. [4] Kohnepoushi C,Nejati V,Delirezh N,et al.Poly lactic-co-glycolic acid nanoparticles containing human gastric tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy[J].Immunol Invest,2019,48(8):794~808. [5] Galati D,Zanotta S.Empowering dendritic cell cancer vaccination:the role of combinatorial strategies[J].Cytotherapy,2018,20(11):1309~1323. [6] Piccioli D,Sammicheli C,Tavarini S,et al.Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation[J].Blood,2009,113(18):4232-4239. [7] Sathe P,Wu L.The network of cytokines,receptors and transcription factors governing the development of dendritic cell subsets[J].Protein Cell,2011,2(8):620~630. [8] 李辉,安军,谷力加.小鼠骨髓浆细胞样树突状细胞亚群的体外诱导及其鉴定[J].第三军医大学学报,2013,35(10):931~937. [9] Palma G,De Laurenzi V,De Marco M,et al.Plasmacytoids dendritic cells are a therapeutic target in anticancer immunity[J].Biochimica et Biophysica Acta (BBA) - Reviews on Cancer,2012,1826(2):407~414.
2021, Vol. 27 
冀公网安备13080202000677号