RNA干扰对成肌细胞系L6中FOXO3a基因表达的影响  

RNA interference affects the feak-headbox 3a gene expression in myoblast cell line L6

作　　者：丁洁[1] 梁炳生[2] 达志峰[2] 朱志祥[2] 韦建[2] 贾英伟[2] 冯勇[3] 

机构地区：[1]山西医科大学,山西省太原市030001 [2]山西医科大学第二医院骨科,山西省太原市030001 [3]上海市第六人民医院骨科,上海市200030

出　　处：《中国组织工程研究》2013年第33期5974-5980,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学青年基金(81000805);课题名称:联合MuRF1与FOXO3a基因治疗延缓失神经肌萎缩的实验研究~~

摘　　要：背景:最近的研究发现,一些因子在失神经骨骼肌萎缩的过程中发挥关键性作用,其中"feak-headbox"(Foxo)转录因子是调控骨骼肌萎缩最关键的分子。目的:探讨 RNA 干扰技术体外抑制 FOXO3a 基因表达的效果。方法:6 孔细胞培养板中培养大鼠成肌细胞系 L6,使用 pEGFP-N1 与 siRNA 重组质粒等比例在Lipofectamine2000 介导下转染,优化与检测系统的转染效率;将 2 μg FOXO3a 基因 siRNA 重组质粒转染L6,转染 48 h 与 72 h。结果与结论:① p EGFP-N1 与 siRNA 重组质粒转染后 48 h,荧光显微镜下可见细胞中有大量明亮的绿色荧光表达,显示系统有较高的转染效率。②实时定量 PCR 分析结果显示,转染后 48 h 和 72 h,干扰序列 FOXO3a-Ⅰ、FOXO3a-Ⅱ、FOXO3a-Ⅲ、FOXO3a-Ⅳ对 FOXO3a mRNA 的抑制率与未转染对照组相比差异均有显著性意义(P < 0.05),转染 72 h 与 48 h 相比,抑制效应更为明显,并以 FOXO3a-Ⅰ的抑制效果最为明显。③Western 印迹灰度分析结果显示,转染后 48 h 和 72 h,干扰序列 FOXO3a-Ⅰ、FOXO3a-Ⅱ、FOXO3a-Ⅲ、FOXO3a-Ⅳ对 FOXO3a 蛋白表达的抑制率与对照组相比差异有显著性意义(P < 0.05),转染 72 h 与 48 h 相比,抑制效应更为明显,与 mRNA 水平的影响一致。结果可见 RNA 干扰技术在体外能够明显抑制叉头蛋白转录因子 FOXO3a 基因的表达,FOXO3a 基因 siRNA 重组质粒转染其干扰序列对 FOXO3a 的 mRNA 和蛋白抑制效果尚不明确,这可为 RNA 干扰介导的失神经骨骼肌萎缩基因治疗提供一种新的思路。BACKGROUND： Recent studies found that some factors play important role in the process of denervated muscle atrophy, especially the feak-headbox transcription factor, is the key element to regulate the denervated muscle atrophy. OBJECTIVE： To investigate the effect of RNA interference on inhibiting feak-headbox 3a gene expression in vitro. METHODS： The myoblast cell line L6 were cultured in the 6-well cell culture plates, then pEGFP-N1 and small interfering RNA recombinant plasmid with the same ratio was transfected under the Lipofectamine2000 mediation to optimize the transfection efficiency of the detection system; 2 pg small interfering RNA recombinant plasmid of feak-heedbox 3a gene were transfected with myoblast cell line L6 for 48 and 72 hours. RESULTS AND CONCLUSION： At 48 hours after pEGFP-N1 and siRNA recombinant plasmid transfection, alarge number of bright green fluorescent displayed under fluorescence microscope with higher transfection efficiency. Real-time quantitative PCR analysis showed that there were significant differences in the sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-IV on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection （P 〈 0.05）, and the inhibition effect was more significant at 72 hours after transfection when compared with that at 48 hours after transfection. Western Blot gray analysis showed that there were significant differences in sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection （P 〈 0.05）, and the inhibition effect was more significant at 72 hours after transfection when compared with that at 48 hours after transfection, which was same with the effect on mRNA level. RNA interference in vitro can significantly inhibit the fork-head transcription factor feak-headbox 3a gene expression, and the inhibition effect of feak-headbox

关 键 词：组织构建 组织构建细胞学实验 肌萎缩 FOXO3A L6 失神经 RNA 干扰 基因表达 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]