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

作　　者：桂连[1,2] 张倩倩[1,2] 蔡燕[1,2] 郭琪[1,2] 黄俊琪[1,2] 

机构地区：[1]中山大学附属第一医院广东省器官捐献与移植免疫重点实验室,广州510080 [2]中山大学中山医学院免疫学研究所,广州510080

出　　处：《中华微生物学和免疫学杂志》2015年第11期799-805,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30872350,31370870）;广东省自然科学基金资助项目（S2012010009050,S2013020013000,S81510008901000017）;广东省科技计划项目（2010B050700008,20118040300022,2013A020229003）;广州市科技计划项目（11C32060749,2011J4100084,2008Z1-E221）;2010年中山大学高校基本科研业务费青年培育项目（10ykvy31）;广东省器官捐献与移植免疫重点实验室建设项目（2013A061401007）

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24h、36h和48h，免疫荧光法检测其转染效率；let-7e、miR-106b及miR-20amimic分别瞬转THP-1细胞24h、36h和48h，qRT—PCR检测各miRNA表达量并筛选各miRNA的最佳转染时间；1mg／L LPS刺激各miRNA转染的THP-1细胞1h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-1d、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48h，miR-106b及miR-20amimic的最佳转染时间是24h；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。Objective To investigate the effects of a miRNA family member, let-7e, and a combination of miR-106b and miR-20a on the expression of cytokines by THP-1 cells with Luminex xMAP technology. 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 mimics （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 with 1 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）, monoeyte chemotaetic protein 1 （MCP-1） , IL-1α, IL-6, IL-10, TNF-α, IFN-α and IFN-β in 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 transfeetion times for let-7e mimic and miR-106b/miR-20a mimics were 48 h and 24 h, respectively. Compared with the corresponding negative eontrol （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 inlet-7e transfected THP-1 cells, but were inhibited in miR-106b and miR-20a co-transfected THP-1 cells.

关 键 词：液相芯片 miRNA 细胞因子 

分 类 号：R392[医药卫生—免疫学]