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| In Vitro Culture and Comparative Study of Plasmacytoid and Myeloid Dendritic Cells from Mouse Bone Marrow |
| TAN Jianfeng, LI Dongfang, GUO Quanwei, et al |
| Shenzhen Hospital of Southern Medical University, Guangdong Shenzhen 518000, China |
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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.
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2021, Vol. 27 
