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目的探讨miR-148b/DUSP1信号通路调节巨噬细胞分泌细胞因子CD206的表达对肝癌发生的影响。方法利用全自动磁珠提取纯化系统提取外周血单核细胞并培养,用粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)和巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)分别诱导生成M1型和M2型巨噬细胞,CD68、CD206进行表型鉴定,ELISA检测M1和M2型巨噬细胞分泌的细胞因子CD206的表达,CCK-8和Transwell实验检测巨噬细胞分泌细胞因子CD206对肝癌细胞(Hep G2和Huh7细胞)增殖、侵袭、转移的影响,双荧光素酶报告基因系统验证miR-148b与DUSP1的靶向结合。结果初步分离并鉴定M1和M2型巨噬细胞,M2型巨噬细胞分泌的细胞因子CD206促进肝癌细胞的生长和侵袭、转移,双荧光素酶报告基因证实DUSP1为miR-148b的靶基因,miR-148b/DUSP1信号通路促进肝癌的发生、发展,巨噬细胞标志物与肝癌患者的临床病理特征相关。结论 miR-148b/DUSP1信号通路影响巨噬细胞分泌的细胞因子促进肝癌发生、发展。
Objective To investigate the effect of miR-148b / DUSP1 signaling pathway on the occurrence of hepatocellular carcinoma (HCC) by regulating the expression of macrophage cytokine CD206. Methods Peripheral blood mononuclear cells (PBMCs) were isolated and purified by a magnetic beads extraction and purification system. The granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor stimulated factor (M-CSF) induced the formation of macrophages M1 and M2, CD68 and CD206 respectively. The expression of CD206 secreted by M1 and M2 macrophages was detected by ELISA. The expressions of CD206, The effect of macrophage secreting cytokine CD206 on the proliferation, invasion and metastasis of hepatoma cells (Hep G2 and Huh7 cells) was examined. Dual luciferase reporter gene system was used to verify the targeted binding between miR-148b and DUSP1. Results The M1 and M2 macrophages were initially isolated and identified. The growth factor M2c secreted by M2 macrophages promoted the invasion and metastasis of hepatocarcinoma cells. Dual luciferase reporter gene confirmed that DUSP1 was the target gene of miR-148b and miR -148b / DUSP1 signaling pathway to promote the occurrence and development of hepatocellular carcinoma, macrophage markers and liver cancer patients with clinical and pathological features. Conclusion The miR-148b / DUSP1 signaling pathway affects the secretion of cytokines by macrophages and promotes the development and progression of HCC.