【摘要】：目的研究microRNA-27a(miR-27a)對脂多糖(lipopolysaccharide,LPS)刺激的小鼠骨髓來源樹突狀細(xì)胞(dendritic cell,DC)的成熟及其細(xì)胞因子分泌的影響。方法小鼠骨髓來源的未成熟樹突狀細(xì)胞(immature dendritic cell,imDC)經(jīng)免疫磁珠提純后,用LPS刺激24小時,流式細(xì)胞術(shù)檢測其表面分子的表達(dá),驗證LPS誘導(dǎo)樹突狀細(xì)胞成熟的模型的建立。RT-PCR檢測LPS刺激骨髓來源的樹突狀細(xì)胞(bone marrow derived dendritic cell,BMDC)成熟前后miR-27a的表達(dá)情況。小鼠骨髓來源的imDC轉(zhuǎn)染miR-27a的模擬物(miR-27a mimics)、抑制物(miR-27a inhibitors)后,用LPS刺激24小時,采用流式細(xì)胞術(shù)檢測其表面共刺激分子CD80、CD86及MHCII表達(dá),ELISA法檢測其上清中的IL-12及IL-10蛋白水平,RT-PCR方法檢測其細(xì)胞內(nèi)IL-12及IL-10 mRNA水平,混合淋巴細(xì)胞反應(yīng)檢測其刺激T細(xì)胞增殖能力。結(jié)果純化后的BMDC純度CD11c大于90%(94.8%±4.6%),與未處理組比較,LPS刺激24小時后的DC表面的共刺激分子CD80、CD86及MHCII表達(dá)均顯著增高(P0.001);LPS刺激24小時后,與對照組及轉(zhuǎn)染miR-27a inhibitors組比較,轉(zhuǎn)染mi R-27a mimics細(xì)胞的共刺激分子CD80、CD86及MHCII表達(dá)均顯著降低(P0.001),且顯著抑制IL-12分泌(P0.01)、促進(jìn)IL-10分泌(P0.05),并顯著減弱LPS刺激的DC促CD4+T細(xì)胞增殖的能力(P0.01)。結(jié)論miR-27a抑制小鼠樹突狀細(xì)胞的表型成熟,促進(jìn)IL-10分泌、抑制IL-12分泌,且減弱LPS刺激的DC促CD4+T細(xì)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% 鹵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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R617

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