应用 Luminex xMAP 液相芯片技术研究miRNAs 对 THP-1细胞产生细胞因子的影响
Analysis of the effects of miRNAs on the expression of cytokines by THP-1 cells using Luminex xMAP technology
摘要目的:研究let-7e单独作用或miR-106b和miR-20a共同作用对THP-1细胞产生细胞因子的影响。方法用Cy3标记的miRNA阴性对照瞬转THP-1细胞24 h、36 h和48 h,免疫荧光法检测其转染效率;let-7e、miR-106b及miR-20a mimic分别瞬转THP-1细胞24 h、36 h和48 h,qRT-PCR检测各miRNA表达量并筛选各miRNA的最佳转染时间;1 mg/L LPS刺激各miRNA转染的THP-1细胞1 h后,Luminex xMAP液相芯片技术检测THP-1细胞产生的IL-8、干扰素诱导蛋白-10( IP-10)、单核细胞趋化蛋白-1( MCP-1)、IL-1α、IL-6、IL-10、TNF-α、IFN-α和 IFN-β,并进行差异性分析。结果转染后90%以上的THP-1细胞带有红色荧光;let-7e mimic的最佳转染时间是48 h,miR-106b及miR-20a mimic的最佳转染时间是24 h;相对于各自的对照组,let-7e mimic组IL-8、IP-10和MCP-1表达量增加,而在miR-106b和miR-20a mimic共转染组各趋化因子表达量减少。结论 let-7e促进THP-1细胞中IL-8、IP-10和MCP-1的表达,miR-106b和miR-20a共同抑制它们的表达。
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abstractsObjective To investigate the effects of a miRNA family member, let-7e, and a combi-nation of miR-106b and miR-20a on the expression of cytokines by THP-1 cells with Luminex xMAP technol-ogy.Methods The efficiency of transfection was evaluated by immunofluorescence assay after transfecting THP-1 cells with micrONTM mimic negative control (Cy3) for 24 h, 36 h and 48 h.The three miRNA mim-ics (let-7e, miR-106b and miR-20a) were respectively used to transfect the THP-1 cells for 24 h, 36 h and 48 h and the expression of each miRNA was analyzed by qRT-PCR analysis for screening out the optimal transfection time.The transfected THP-1 cells were stimulated with1 mg/L of LPS for 1 h.The Luminex xMAP technology was used to detect the expression of IL-8, interferon-inducible protein-10 (IP-10), mono-cyte chemotactic protein 1 (MCP-1), IL-1α, IL-6, IL-10, TNF-α, IFN-αand IFN-βin the supernatants of cell culture.A statistical analysis was performed to analyze the data obtained by using SPSS16.0 software. Results More than 90% of the transfected THP-1 cells were labeled with red fluorescence.The optimal transfection times for let-7e mimic and miR-106b/miR-20a mimics were 48 h and 24 h, respectively.Com-pared with the corresponding negative control (NC), the expression of IL-8, IP-10 and MCP-1 by THP-1 cells were enhanced after the transfection with let-7e mimic, but were inhibited after the co-transfection with miR-106b and miR-20a mimics.Conclusion The expression of IL-8, IP-10 and MCP-1 were enhanced in let-7e transfected THP-1 cells, but were inhibited in miR-106b and miR-20a co-transfected THP-1 cells.
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