CAJ | 학술논문

背景:研究报道外源性酸性成纤维细胞生长因子既可调节肌卫星细胞增殖和分化,又具有预防运动终板退变及肌萎缩的作用. 目的:通过酸性成纤维细胞因子基因转染大鼠骨骼肌卫星细胞,检测目的基因转染肌卫星细胞效果及基因表达情况,探讨建立有效预防运动终板退变及肌萎缩种子细胞可行性. 方法:取 Wistar 成年大鼠后肢肌肉,差速贴壁培养法分离纯化肌卫星细胞,观察细胞生长特性并做免疫组织化学鉴定;取第2代细胞,LipofectamineTM2000 Reagent转染试剂介导,将重组真核表达质粒pEGFP-N1-aFGF转染肌卫星细胞为实验组;阴性对照组转染空载质粒pEGFP-N1;空白对照组仅加入转染试剂.转染后24-72 h和传代后分别用倒置荧光显微镜观察细胞绿色荧光蛋白表达情况,计算转染效率.转染细胞行Western Blot检测酸性成纤维细胞因子表达.提取转染后72 h 细胞总RNA,实时荧光定量PCR检测酸性成纤维细胞因子基因mRNA表达. 结果与结论:分离纯化细胞经免疫组织化学鉴定为肌卫星细胞.荧光显微镜观察到细胞转染6 h后即有绿色荧光发出,荧光强度和表达细胞总数在72 h达到高峰,传代后仍可观察到绿色荧光蛋白表达.实时荧光定量PCR证实目的基因mRNA表达水平远远高于对照组,Western Blot检测实验组有大量酸性成纤维细胞因子产生.提示酸性成纤维细胞基因转染肌卫星细胞可表达基因产物,有望作为基因工程种子细胞预防失神经支配后运动终板退变及肌萎缩.
배경:연구보도외원성산성성섬유세포생장인자기가조절기위성세포증식화분화,우구유예방운동종판퇴변급기위축적작용. 목적:통과산성성섬유세포인자기인전염대서골격기위성세포,검측목적기인전염기위성세포효과급기인표체정황,탐토건립유효예방운동종판퇴변급기위축충자세포가행성. 방법:취 Wistar 성년대서후지기육,차속첩벽배양법분리순화기위성세포,관찰세포생장특성병주면역조직화학감정;취제2대세포,LipofectamineTM2000 Reagent전염시제개도,장중조진핵표체질립pEGFP-N1-aFGF전염기위성세포위실험조;음성대조조전염공재질립pEGFP-N1;공백대조조부가입전염시제.전염후24-72 h화전대후분별용도치형광현미경관찰세포록색형광단백표체정황,계산전염효솔.전염세포행Western Blot검측산성성섬유세포인자표체.제취전염후72 h 세포총RNA,실시형광정량PCR검측산성성섬유세포인자기인mRNA표체. 결과여결론:분리순화세포경면역조직화학감정위기위성세포.형광현미경관찰도세포전염6 h후즉유록색형광발출,형광강도화표체세포총수재72 h체도고봉,전대후잉가관찰도록색형광단백표체.실시형광정량PCR증실목적기인mRNA표체수평원원고우대조조,Western Blot검측실험조유대량산성성섬유세포인자산생.제시산성성섬유세포기인전염기위성세포가표체기인산물,유망작위기인공정충자세포예방실신경지배후운동종판퇴변급기위축.
@@@@BACKGROUND:It has been reported that acidic fibroblast growth factor (aFGF) not only can mediate cel division and differentiation, but also can prevent motor endplate degeneration and muscular atrophy. @@@@OBJECTIVE:To calculate gene transfection efficiency and detect the target protein expression of muscle satel ite cel s which were transfected with aFGF gene, in purpose to further study the method to set up cel bank for preventing motor endplate degeneration and muscular atrophy. @@@@METHODS:Muscle satel ite cel s were extracted from adult Wistar rat, purified by difference-speed adherence method and identified by immunohistochemical assay. The recombinant eukaryotic expression plasmid pEGFP-N1-aFGF was transfected into cel s by LipofectamineTM2000 Reagent as experimental group. Muscle satel ite cel s transfected with pEGFP-N1 served as negative controls. Blank control group was set by adding transfection reagent. Inverted fluorescent microscope was applied to observe green fluorescent protein expression in the cel s to calculate transfection efficiency at 24-72 hours after transpection and passaging. Western Blot of aFGF was performed to detect the target protein. Total RNA was extracted at 72 hours after transfection. Real-time fluorescent quantitative PCR was employed in order to find out the changes of cel s after transfection on mRNA level. @@@@RESULTS AND CONCLUSION:The immunohistochemical results showed that cultivated cel s were muscle satel ite cel s. The expression of green fluorescent protein appeared as early as 6 hours after transfection, and the amount and intensity peaked at 72 hours. Green fluorescent protein was stil seen in the subculture cel s. Real-time fluorescent quantitative PCR proved stronger aFGF mRNA expression in the transfected cel s with aFGF gene, while a little in the control groups. aFGF protein was highly expressed in the cel s transfected with target gene detected by Western Blot. Al the results indicate that the aFGF gene can be transfected efficiently and safely into muscle satel ite cel s and expressed normal y, which can serve as the new seed cel s for tissue engineering to prevent motor endplate degeneration and muscular atrophy.