中文摘要：

小鼠miR-499基因包含在心肌重链肌球蛋白Myh7b基因的第19内含子中,并且在心肌细胞中特异表达,然而其在心肌细胞中表达的生物学功能和意义尚不清楚.利用可体外分化为心肌细胞的P19CL6细胞建立稳定表达miR-499的细胞株对研究miR-499的生物学功能具有重要意义.根据小鼠miR-499基因序列,设计PCR引物并将扩增产物定向克隆到pcDNA3.1中构建为真核表达载体并转染小鼠P19CL6细胞,经G418筛选建立了稳定转染的P19CL6-miR-499细胞株.用TaqMan探针实时定量PCR检测表明,P19CL6-miR-499细胞可成功表达成熟的miR-499.经1%DMSO诱导分化,RT-PCR、real-time PCR和激光共聚焦显微镜免疫荧光分析,结果表明,稳定转染miR-499对细胞分化早期的分子标志Isl1和Tbx5的表达没有明显影响,但是对分化晚期的特异性分子心肌α-肌动蛋白（α-cardiac actin）和心肌肌钙蛋白（troponin I）的表达有明显抑制作用,这提示miR-499主要对心肌细胞分化晚期有影响.

英文摘要：

MiR-499 is encoded by the intron 19 of the mouse Myh7b gene and highly expressed in cardiomyocytes.However,the key roles of miR-499 in cardiomyocytes remained unclear.It is important to establish a stably transfected P19CL6 cell line（an in vitro cardiomyocyte differentiation system） with miR-499 for investigating the biological functions of miR-499 in cardiomyocytes.The miR-499 gene fragment was amplified by PCR using specific primers and then inserted into pcDNA3.1 plasmid to generate a eukaryotic expressing vector of miR-499（pcDNA3.1-miR-499）.Stable transfectants were obtained by G418 selection and real-time PCR using TaqMan probes demonstrated that the P19CL6 celllines expressing miR-499 have been established successfully.P19CL6-miR-499 cells were induced forcardiac differentiation by 1%DMSO.RT-PCR,real-time PCR and confocal immunofluorescent analysiswere performed.The results showed that the levels of cardiac specific transcription factors Isl1 and Tbx5expressed in P19CL6-miR-499 cells at the early stage of differentiation were not significantly differentfrom wild-type P19CL6 cells;while the levels of cardiac specific contractile proteins,α-cardiac actin andtroponin I,expressed by P19CL6-miR-499 cells in the late stage of differentiation were lower than thosein wild-type P19CL6 cells.These results suggest that miR-499 might play important roles in cardiacdifferentiation at the late stage.