中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
33期
5974-5980
,共7页
丁洁%梁炳生%达志峰%朱志祥%韦建%贾英伟%冯勇
丁潔%樑炳生%達誌峰%硃誌祥%韋建%賈英偉%馮勇
정길%량병생%체지봉%주지상%위건%가영위%풍용
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
組織構建%組織構建細胞學實驗%肌萎縮%FOXO3a%L6%失神經%RNA榦擾%基因錶達%國傢自然科學基金
조직구건%조직구건세포학실험%기위축%FOXO3a%L6%실신경%RNA간우%기인표체%국가자연과학기금
tissue construction%cytology experiment in tissue construction%muscle atrophy%feak-headbox 3a%L6%denervation%RNA interference%gene expression%National Natural Science Foundation of China
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。<br> 目的：探讨RNA干扰技术体外抑制FOXO3a基因表达的效果。<br> 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2μg FOXO3a基因siRNA重组质粒转染L6，转染48 h与72 h。<br> 结果与结论：①pEG FP-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。
揹景：最近的研究髮現，一些因子在失神經骨骼肌萎縮的過程中髮揮關鍵性作用，其中“feak-headbox”(Foxo)轉錄因子是調控骨骼肌萎縮最關鍵的分子。<br> 目的：探討RNA榦擾技術體外抑製FOXO3a基因錶達的效果。<br> 方法：6孔細胞培養闆中培養大鼠成肌細胞繫 L6，使用 pEGFP-N1與 siRNA 重組質粒等比例在Lipofectamine2000介導下轉染，優化與檢測繫統的轉染效率；將2μg FOXO3a基因siRNA重組質粒轉染L6，轉染48 h與72 h。<br> 結果與結論：①pEG FP-N1與siRNA重組質粒轉染後48 h，熒光顯微鏡下可見細胞中有大量明亮的綠色熒光錶達，顯示繫統有較高的轉染效率。②實時定量PCR分析結果顯示，轉染後48 h和72 h，榦擾序列FOXO3a-Ⅰ、FOXO3a-Ⅱ、FOXO3a-Ⅲ、FOXO3a-Ⅳ對FOXO3a mRNA的抑製率與未轉染對照組相比差異均有顯著性意義(P <0.05)，轉染72 h與48 h相比，抑製效應更為明顯，併以FOXO3a-Ⅰ的抑製效果最為明顯。③Western印跡灰度分析結果顯示，轉染後48 h和72 h，榦擾序列FOXO3a-Ⅰ、FOXO3a-Ⅱ、FOXO3a-Ⅲ、FOXO3a-Ⅳ對FOXO3a蛋白錶達的抑製率與對照組相比差異有顯著性意義(P<0.05)，轉染72 h與48 h相比，抑製效應更為明顯，與mRNA水平的影響一緻。結果可見RNA榦擾技術在體外能夠明顯抑製扠頭蛋白轉錄因子FOXO3a基因的錶達，FOXO3a基因siRNA重組質粒轉染其榦擾序列對FOXO3a的mRNA和蛋白抑製效果尚不明確，這可為RNA榦擾介導的失神經骨骼肌萎縮基因治療提供一種新的思路。
배경：최근적연구발현，일사인자재실신경골격기위축적과정중발휘관건성작용，기중“feak-headbox”(Foxo)전록인자시조공골격기위축최관건적분자。<br> 목적：탐토RNA간우기술체외억제FOXO3a기인표체적효과。<br> 방법：6공세포배양판중배양대서성기세포계 L6，사용 pEGFP-N1여 siRNA 중조질립등비례재Lipofectamine2000개도하전염，우화여검측계통적전염효솔；장2μg FOXO3a기인siRNA중조질립전염L6，전염48 h여72 h。<br> 결과여결론：①pEG FP-N1여siRNA중조질립전염후48 h，형광현미경하가견세포중유대량명량적록색형광표체，현시계통유교고적전염효솔。②실시정량PCR분석결과현시，전염후48 h화72 h，간우서렬FOXO3a-Ⅰ、FOXO3a-Ⅱ、FOXO3a-Ⅲ、FOXO3a-Ⅳ대FOXO3a mRNA적억제솔여미전염대조조상비차이균유현저성의의(P <0.05)，전염72 h여48 h상비，억제효응경위명현，병이FOXO3a-Ⅰ적억제효과최위명현。③Western인적회도분석결과현시，전염후48 h화72 h，간우서렬FOXO3a-Ⅰ、FOXO3a-Ⅱ、FOXO3a-Ⅲ、FOXO3a-Ⅳ대FOXO3a단백표체적억제솔여대조조상비차이유현저성의의(P<0.05)，전염72 h여48 h상비，억제효응경위명현，여mRNA수평적영향일치。결과가견RNA간우기술재체외능구명현억제차두단백전록인자FOXO3a기인적표체，FOXO3a기인siRNA중조질립전염기간우서렬대FOXO3a적mRNA화단백억제효과상불명학，저가위RNA간우개도적실신경골격기위축기인치료제공일충신적사로。
BACKGROUND:Recent studies found that some factors play important role in the process of denervated muscle atrophy, especial y the feak-headbox transcription factor, is the key element to regulate the denervated muscle atrophy. <br> OBJECTIVE:To investigate the effect of RNA interference on inhibiting feak-headbox 3a gene expression in vitro. <br> METHODS:The myoblast cel line L6 were cultured in the 6-wel cel culture plates, then pEGFP-N1 and smal <br> interfering RNA recombinant plasmid with the same ratio was transfected under the Lipofectamine2000 mediation to optimize the transfection efficiency of the detection system;2μg smal interfering RNA recombinant plasmid of feak-headbox 3a gene were transfected with myoblast cel line L6 for 48 and 72 hours. <br> RESULTS AND CONCLUSION:At 48 hours after pEGFP-N1 and siRNA recombinant plasmid transfection, a large 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 <br> 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, <br> feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after <br> 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 <br> significantly inhibit the fork-head transcription factor feak-headbox 3a gene expression, and the inhibition effect of feak-headbox 3a gene smal interfering RNA recombinant plasmid transfected with the sequence on the mRNA and protein level of feak-headbox 3a is not clear, which can provide new idea for the gene therapy of RNA mediated denervated skeletal muscle atrophy.
