MicroRNA-27a对小鼠骨髓来源树突状细胞成熟及其细胞因子分泌的影响

发布时间：2018-09-12 05:34

【摘要】：目的研究microRNA-27a(miR-27a)对脂多糖(lipopolysaccharide,LPS)刺激的小鼠骨髓来源树突状细胞(dendritic cell,DC)的成熟及其细胞因子分泌的影响。方法小鼠骨髓来源的未成熟树突状细胞(immature dendritic cell,imDC)经免疫磁珠提纯后,用LPS刺激24小时,流式细胞术检测其表面分子的表达,验证LPS诱导树突状细胞成熟的模型的建立。RT-PCR检测LPS刺激骨髓来源的树突状细胞(bone marrow derived dendritic cell,BMDC)成熟前后miR-27a的表达情况。小鼠骨髓来源的imDC转染miR-27a的模拟物(miR-27a mimics)、抑制物(miR-27a inhibitors)后,用LPS刺激24小时,采用流式细胞术检测其表面共刺激分子CD80、CD86及MHCII表达,ELISA法检测其上清中的IL-12及IL-10蛋白水平,RT-PCR方法检测其细胞内IL-12及IL-10 mRNA水平,混合淋巴细胞反应检测其刺激T细胞增殖能力。结果纯化后的BMDC纯度CD11c大于90%(94.8%±4.6%),与未处理组比较,LPS刺激24小时后的DC表面的共刺激分子CD80、CD86及MHCII表达均显著增高(P0.001);LPS刺激24小时后,与对照组及转染miR-27a inhibitors组比较,转染mi R-27a mimics细胞的共刺激分子CD80、CD86及MHCII表达均显著降低(P0.001),且显著抑制IL-12分泌(P0.01)、促进IL-10分泌(P0.05),并显著减弱LPS刺激的DC促CD4+T细胞增殖的能力(P0.01)。结论miR-27a抑制小鼠树突状细胞的表型成熟,促进IL-10分泌、抑制IL-12分泌,且减弱LPS刺激的DC促CD4+T细胞增殖的能力。
[Abstract]:Objective to study the effects of microRNA-27a (miR-27a) on maturation and cytokine secretion of murine bone marrow-derived dendritic cells (dendritic cell,DC) stimulated by lipopolysaccharide (lipopolysaccharide,LPS). Methods immature dendritic cells (immature dendritic cell,imDC) derived from mouse bone marrow were purified by immunomagnetic beads and stimulated with LPS for 24 hours. The surface molecules were detected by flow cytometry. To verify the establishment of LPS induced dendritic cell maturation model. RT-PCR was used to detect the expression of miR-27a before and after (bone marrow derived dendritic cell,BMDC maturation stimulated by LPS. Mouse bone marrow-derived imDC was transfected with miR-27a mimics), inhibitor of miR-27a (miR-27a inhibitors) and stimulated with LPS for 24 hours. The surface costimulatory molecule CD80,CD86 and MHCII expression were detected by flow cytometry and the IL-12 and IL-10 protein levels in the supernatant were detected by Elisa. The levels of IL-12 and IL-10 mRNA in the supernatant were detected by RT-PCR and the proliferation ability of T cells stimulated by mixed lymphocyte reaction was detected. Results the purified BMDC CD11c was more than 90% (94.8% 卤4.6%). Compared with the untreated group, the expression of CD80,CD86 and MHCII on the DC surface after 24 hours was significantly increased (P0.001). Compared with the control group and the transfected miR-27a inhibitors group, the expression of CD80,CD86 and MHCII on the surface of the purified DC was significantly higher than that of the control group and the transfected miR-27a inhibitors group. The expression of CD80,CD86 and MHCII in mi R-27a mimics cells was significantly decreased (P0.001), IL-12 secretion was inhibited (P0.01), IL-10 secretion was promoted (P0.05), and the ability of LPS stimulated DC to stimulate CD4 T cell proliferation was significantly decreased (P0.01). Conclusion miR-27a inhibits the phenotypic maturation of mouse dendritic cells, promotes the secretion of IL-10, inhibits the secretion of IL-12, and weakens the ability of DC stimulated by LPS to promote the proliferation of CD4 T cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R617

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