miRNA-133a过表达对体外L6成肌细胞增殖分化作用机制的研究

Overexpression of miRNA-133a on the in vitro proliferation and differentiation of L6 myoblasts

目的 构建微小RNA-133a重组慢病毒载体,观察其转染后对L6成肌细胞增殖、分化及转录因子MEF2A的影响.方法 构建表达含微小RNA-133a基因的重组慢病毒载体,转染L6成肌细胞,以实时定量PCR（Taqman探针法）对微小RNA-133a基因表达水平进行检测;细胞计数试剂盒（CCK-8）试验评价微小RNA-133a过表达后对L6成肌细胞增殖的影响;倒置荧光显微镜观察L6成肌细胞增殖、分化的影响;Western blot法检测转录因子MEF2A表达水平的变化.结果 构建的微小RNA-133a重组慢病毒载体经质粒酶切和测序鉴定正确;与对照组比较,转染L6成肌细胞24h后,微小RNA-133a基因的相对表达量明显增加（P＜0.01）;L6细胞增殖数量明显增加（P＜0.01）,对L6成肌细胞的分化有明显抑制作用（P＜0.01）;MEF2A的表达量逐渐下降（P＜0.01）.结论 成功构建微小RNA-133a重组慢病毒载体并转染16成肌细胞后可高效表达微小RNA-133a,促进体外成肌细胞的增殖并抑制分化.

Objective To construct recombinant lentiviral vector of micro RNA-133a and observe the proliferation,differentiation and expression of transcription factor MEF2A of L6 myoblasts transfected with the vector system.Methods Recombinant lentiviral vector containing micro RNA-133a gene was constructed and transfected into L6 myoblasts.The expression of micro RNA-133a gene was detected by real-time PCR （Taqman probe）.The effect of micro RNA-133a overexpression on L6 myoblast proliferation was quantified using cell counting kit （CCK-8）.Its effect on cell differentiation was detected by inverted fluorescence microscope.Western blot assay was used to detect the expression level of transcription factor MEF2A in these cells.Results The successful construction of micro RNA-133a recombinant lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing.Compared with the control group,relative expression of micro RNA-133a gene in L6 myoblasts was significantly increased （P 〈 0.01） 24h after the vector transfection.L6 cell proliferation was increased significantly （P 〈 0.01）,while its differentiation was effectively inhibited.The expression level of MEF2A was significantly reduced （P 〈 0.01）.Conclusion Micro RNA-133a recombinant lentiviral vector can successfully transfect L6 myoblasts causing the cells to overexpress micro RNA-133a.This overexpression promotes L6 myoblast proliferation and inhibits its differentiation in an in vitro cell culture system.