CAJ | 학술논문

目的：探讨慢病毒转染RNAi技术介导的FoxO3a基因下调延缓大鼠失神经骨骼肌萎缩的疗效。方法将36只SD大鼠随机分为对照组和实验组，各18只。切断两组大鼠右侧坐骨神经，建立右下趾长伸肌失神经肌萎缩模型。建立模型后于实验组大鼠失神经趾长伸肌注射50μl重组慢病毒载体Lenti-GFP-FoxO3a；对照组注射Lenti-GFP溶液50μl，注射后1、3周，每个时间点两组大鼠分别取3只，完整切除右侧趾长伸肌，于荧光显微镜下观察绿色荧光信号，每个时间点两组大鼠分别取6只，完整切除右侧趾长伸肌，观察肌细胞超微结构，检测肌肉总蛋白含量，测肌肉湿重维持率，RT-PCR检测FoxO3a基因，检测肌细胞横截面积。结果转染后1和3周，两组趾长伸肌中均可观察到大量GFP荧光信号。转染后1周实验组的肌肉湿重维持率、肌细胞横截面积、趾长伸肌肌肉总蛋白含量分别为（90.87±1.56）％、（937.63.42±17.63）μm2和（93.20±1.33） mg/ml，均明显高于对照组（77.73±2.21）%、（721.50±14.40）μm2和（74.74±1.45） mg/ml，差异均有统计学意义（均P<0.01）。RT-PCR检测实验组FoxO3a基因与对照组相比明显下调（P<0.01）。转染后3周，上述各指标分别为（86.69±1.31）%、（843.10±16.44）μm2和（80.39±2.34） mg/ml，仍高于对照组，对照组各指标为（53.42±2.01）%、（633.90±12.90）μm2和（65.22±2.72） mg/ml（均P<0.01）。超微结构显示，术后1周和3周，实验组退变的细胞核均少于对照组，核质染色均匀。结论 FoxO3a基因siRNA重组慢病毒在大鼠体内有较高的转染率，可有效抑制FoxO3a基因的表达。RNAi介导的FoxO3a基因下调可在大鼠失神经早期延缓失神经骨骼肌的萎缩。
목적：탐토만병독전염RNAi기술개도적FoxO3a기인하조연완대서실신경골격기위축적료효。방법장36지SD대서수궤분위대조조화실험조，각18지。절단량조대서우측좌골신경，건립우하지장신기실신경기위축모형。건립모형후우실험조대서실신경지장신기주사50μl중조만병독재체Lenti-GFP-FoxO3a；대조조주사Lenti-GFP용액50μl，주사후1、3주，매개시간점량조대서분별취3지，완정절제우측지장신기，우형광현미경하관찰록색형광신호，매개시간점량조대서분별취6지，완정절제우측지장신기，관찰기세포초미결구，검측기육총단백함량，측기육습중유지솔，RT-PCR검측FoxO3a기인，검측기세포횡절면적。결과전염후1화3주，량조지장신기중균가관찰도대량GFP형광신호。전염후1주실험조적기육습중유지솔、기세포횡절면적、지장신기기육총단백함량분별위（90.87±1.56）％、（937.63.42±17.63）μm2화（93.20±1.33） mg/ml，균명현고우대조조（77.73±2.21）%、（721.50±14.40）μm2화（74.74±1.45） mg/ml，차이균유통계학의의（균P<0.01）。RT-PCR검측실험조FoxO3a기인여대조조상비명현하조（P<0.01）。전염후3주，상술각지표분별위（86.69±1.31）%、（843.10±16.44）μm2화（80.39±2.34） mg/ml，잉고우대조조，대조조각지표위（53.42±2.01）%、（633.90±12.90）μm2화（65.22±2.72） mg/ml（균P<0.01）。초미결구현시，술후1주화3주，실험조퇴변적세포핵균소우대조조，핵질염색균균。결론 FoxO3a기인siRNA중조만병독재대서체내유교고적전염솔，가유효억제FoxO3a기인적표체。RNAi개도적FoxO3a기인하조가재대서실신경조기연완실신경골격기적위축。
Objective To Explore the effect of delaying atrophy of rat denervted skeletal muscles by RNAi- mediated gene downregulation of FoxO3a.Methods Thitry-six SD rats were randomly divided control group and experimental group.Models of extensor digitorum longus muscle denervation atrophy were established at the right lower limb by cutting sciatic nerve.Denervated extensor digitorum longus muscles of experimental groups were administered with 50 μl of recombinant lentivirs vector carrying FoxO3a.50μl lenti-GFP solution was administered into Denervated extensor digitorum longus muscles of control groups.One and 3 weeks after the transfection.the right intact extensor digitorum longus muscles of 3 rats of each group and timepoint were excised to observe green fluorescent under fluorescent microscope. The right intact extensor digitorum longus muscles of 6 rats of each group and timepoint were excised to observe ultrastructure of muscle cells under the transmission electron microscope and measure the total protein content of extensor digitorum longus,muscle wet weight preservation rate,total protein content of extensor digitorum longus and muscle fiber cross-sectional.Results One and three weeks after transfection strong GFP green fluorescent was observed in the extensor digitorum longus muscles of both Groups. One weeks after transfection,wet weight preservation rate,total protein content and muscle fiber cross-seetional area of the denervated muscles in the experimental group were,(90.87±1.56)%,(93.20±1.33) mg/ml and(937.63.42±17.63) μm2 respectively,being obviously greater than those parameters of the control group,which were(77.73±2.21)%,(74.74±1.45) mg/ml and(721.50±14.40) μm2respectively(allP<0.01). After 1 and 3 weeks RT-PCR showed significantly reduced expression of gene FoxO3a in the experimental group compared with the control group(P<0.01). After 3 weeks,the above-mentioned parameters were (86.69±1.31)%,(80.39±2.34) mg/mland(843.10±16.44) μm2 respectively in the experimental group,being obviously higher than those parameters of the control group which were (53.42±2.01)%,(65.22±2.72) mg/ml and (633.90±12.90) μm2 respectively(allP<0.01).The ultramicrostructure of myocyte show that the regressive cell nucleus of the experimental group was significantly less than those in the control group. Conclusion FoxO3a siRNA recombinant lentivirus can achieve high transfection efficiency in rats and efficaciously inhibit the expression of FoxO3a gene. Gene downregulation of FoxO3a by RNAi can delay atrophy of rat denervted skeletal muscles.