【摘要】 【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。更多还原

【Abstract】 [Objective]To introduce a new method to induce large number of predominant immature CD8a+ DC (DC2) from murine bone marrow cells. [Methods] Bone marrow cells of C57BL/6 mice were obtained and cultured in vitro at 37℃, 5%CO2 in the presence of 5 ng/mL GM-CSF, 25 ng/mL Flt3L, I20 ng/mL L-4, and 100 ng/mL SCF. On day 3, 7, and16, 4-color flow cytometry analyses were carried out to analyze cell phenotype, and total cell count was used to analyze cell yield. Immunofluorescence, phase contrast observations, and electron microscope analysis were used to observe cell morphology. [Results] On day 3, murine bone marrow cells induced by GM-CSF+IL-4+Flt3L+SCF generated 97.09% CD8a+ predominant DC2 (34.6% of total cells), which 61.77% of them were CD8a+MHCⅡ- immature DC. The proportion decreased in prolonged culture but on day 16 CD8a+MHCⅡ- immature DC were still predominant. Total cell count showed that the total cell decreased with prolonged culture. Immunofluorescence, phase contrast observations, and electron microscope analysis showed that those predominant DC2 bore typical morphological feature of dendritic cell. [Conclusion] GM-CSF+IL-4+Flt3L+SCF could quickly induce large quantity of predominant immature CD8a+ DC in vitro from murine bone marrow cells, which is a valuable new method in preparing immature DC2 in vitro.更多还原