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【目的】探索从小鼠骨髓细胞体外诱导大量优势不成熟CD8a+DC(DC2)的新方法。【方法】取小鼠骨髓细胞,采用GM-CSF5ng/mL,IL-420ng/mL,Flt3L25ng/mL,SCF100ng/mL4种细胞因子组合,37℃,5%CO2体外培养诱导。第3、7、16天流式细胞术4色荧光标记法分析细胞表型,细胞计数分析总细胞产量,采用电镜、免疫荧光及相差摄影观察细胞形态。【结果】小鼠骨髓细胞经GM-CSF+IL-4+Flt3L+SCF诱导第3天在CD11c+的DC细胞群中可产生97.09%的CD8a+优势DC2细胞(占总细胞的34.6%),其中CD8a+MHCⅡ-的不成熟DC占61.77%;随培养时间延长比例下降,但培养第16天时CD8a+的不成熟DC仍占优势。总细胞计数分析显示随培养时间延长,细胞总数下降。电镜、免疫荧光及相差摄影等形态学检查显示所诱导的DC呈现典型树突状形态特征。【结论】GM-CSF+IL-4+Flt3L+SCF可以体外快速诱导小鼠骨髓细胞产生大量优势不成熟CD8a+DC,是体外制备不成熟DC2的一种较好的新方法。
【Objective】 To explore a new method for inducing a large number of dominant immature CD8a + DCs from mouse bone marrow cells in vitro. 【Method】 Mouse bone marrow cells were obtained and cultured in vitro with 5% GM-CSF 5ng / mL, IL-420ng / mL, Flt3L25ng / mL and SCF100ng / mL. Cell phenotypes were analyzed by flow cytometry on day 3, day 7, and day 16, and the total cell yield was analyzed by cell counting. Cell morphology was observed by electron microscopy, immunofluorescence and phase contrast photography. 【Results】 The results showed that 97.09% of CD8a + predominant DC2 cells (34.6% of the total cells) were produced in the DCs of CD11c + on the third day after induced by GM-CSF + IL-4 + Flt3L + SCF. Among them, CD8a + MHCⅡ-immature DC accounted for 61.77%; with the proportion of prolonged incubation time decreased, but cultured on the 16th day CD8a + immature DC is still dominant. Total cell count analysis showed that as the culture time prolonged, the total number of cells decreased. Electron microscopy, immunofluorescence and phase contrast photography and other morphological examination showed that the induced DC showed typical dendritic morphology. 【Conclusion】 GM-CSF + IL-4 + Flt3L + SCF can rapidly induce a large number of mouse bone marrow cells to produce a large number of premature CD8a + DCs in vitro, which is a good new method for preparing immature DC2 in vitro.