中文摘要：

目的探讨RNAi技术体外抑制MuRF-1或FOXO3a基因表达的效果，为RNAi介导的失神经骨骼肌萎缩基因治疗奠定基础。方法体外培养大鼠成肌细胞系L6，将MuRF-1和（或）FOXO3asiRNA重组质粒在Lipofectamine2000介导下转染，优化与检测系统的转染效率；将2ggMuRF-1或FOXO3a基因siRNA重组质粒转染L6，转染后48h与72h，采用实时定量PCR检测siRNA重组质粒对MuRF-1和F0xO3a的mRNA的抑制效果，使用Western印迹检测siRNA重组质粒对MuRF-1和FOXO3a蛋白水平的抑制效果。用单因素方差分析方法和LSD法进行组问比较。结果（1）质粒转染后24h，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：MuRF-1与FOXO3a各自的siRNA重组质粒转染后48h，二者干扰序列在mRNA水平分别明显抑制了MuRF-1和FOXO3a的表达，抑制率达67％和54％，与对照组相比差异均有统计学意义（P〈0．05），联合MuRF-1与FOXO3a两基因siRNA重组质粒转染后48h，干扰序列在mRNA水平明显抑制了MuRF-1及FOXO3a的表达，抑制率达61％及58％，与对照组相比差异有统计学意义（P〈0．05）；转染后72h，MuRF-1与FOXO3a各自的siRNA重组质粒干扰序列对MuRF-1和FOXO3a的mRNA的抑制率分别达79％和81％，联合MuRF-1与FOXO3a两基因siRNA重组质粒干扰序列对MuRF-1和FOXO3a的mRNA的抑制率达77％及72％，与对照组相比差异均有统计学意义（P〈0．05），与48h相比，抑制效应更为明显。（3）Western印迹灰度分析结果显示：转染后48h，干扰序列MuRF-1明显抑制了MuRF-1蛋白的表达，抑制率达61％，与对照组相比差异有统计学意义（P〈0．05），干扰序列FOXO3a明显抑制了FOXO3a蛋白的表达，抑制率达46％，与对照组相比差异有统计学意义（P〈0．05），联合MuRF-1与FOXO3a两基因siRNA重组质粒干扰序列对MuRF-1和FOXO3a蛋白表达的抑制率达64?

英文摘要：

Objective To investigate the effect of RNAi technology in inhibiting gene expression of MuRF-1 or FOXO3a in vitro, establishing a foundation for RNAi mediated gene therapy for denervated skeletal muscle atrophy. Methods Rat muscle cell line L6 was transfected with MuRF-1 or FOXO3a siRNA plasmid using Lipofectamine 2000. After transfection was optimized, 2 pg of MuRF-1 or FOXO3a siRNA recombinant plasmid was transfted. At 48 h and 72 h after transfection, the real-time quantitative PCR was used to detect the inhibition effect of MuRF-1 and FOXO3a. Protein level was evaluated using Western blotting. Results （1） At 24 h after transfection, strong bright green fluorescence was observed under fluorescence microscopy, indicating high transfection efficiency. （2） Real time quantitative PCR analysis showed mRNA expression was significantly inhibited for both MuRF-1 and FOXO3a after 48 h, with an inhibition rate of 67 % and 54 % respectively. When MuRF- 1 and FOXO3a siRNA recombinant plasmids were co-transfectied, MuRF-1 and FOXO3a expression was inhibite by 61% and 58 % respectively compared with the control （P 〈 0.05 for both）. At 72 h after transfection, the inhibitory rates for MuRF-1 and FOXO3a siRNA recombinant plasmids at mRNA level were 79 % and 81% respectively. When MuRF-1 and FOXO3a siRNA recombinant plasmids were co-transfected, MuRF-1 and FOXO3a mRNA was inhibited by 77 % and 72 % respectively, significantly higher than the control group （P 〈 0.05） and higher than that at 48 h. （3） Western blotting showed MuRF-1 and FOXO3a were inhibited by 61% and 46 % respectively. When MuRF-1 and FOXO3a siRNA recombinant plasmids were co-transfected, MuRF-1 and FOXO3a protein expression was inhibited by 64 % and 42 % respectively, both higher than the control （P 〈 0.05）. At 72 h, MuRF-1 and FOXO3a protein expression was inhibited by 70% and 72% respectively, significantly greater than the control. Whenunited MuRF-1 and FOXO3a siRNA recombinant plasmids were co-transfected, MuRF-1 and FOXO3a in