酸性成纤维细胞因子基因体外转染肌卫星细胞

Muscle satellite cells transfected with acidic fibroblast growth factor gene in vitro

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,LipofectamineTM2000Reagent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24-72h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WesternBlot检测酸性成纤维细胞因子表达。提取转染后72h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6h后即有绿色荧光发出,荧光强度和表达细胞总数在72h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,WesternBlot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。

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 satellite cells which were transfected with aFGF gene, in purpose to further study the method to set up call bank for preventing motor endplate degeneration and muscular atrophy. METHODS： Muscle satellite cells were extracted from adult Wistar rat, purified by difference-speed adherence method and identified by immunohistochemical assay. The recombinant eukaryotic expression plasmid pEGFP-NI-aFGF was transfected into calls by Lipofectamine^TM 2000 Reagent as experimental group. Muscle satellite cells 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 cells 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 calls after transfection on mRNA level. RESULTS AND CONCLUSION： The immunohistochemical results showed that cultivated calls were muscle satellite cells. 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 still seen in the subculture calls. Real-time fluorescent quantitative PCR proved stronger aFGF mRNA expression in the transfected cells with aFGF gene, while a little in the control groups, aFGF protein was highly expressed in the calls transfected with target gene detected by Western Blot. All the results indicate that the aFGF gene can be transfected efficiently and safely into muscle satellite calls and expressed normally, which can serve as the new seed calls for tissue engineering to prevent motor endplate degeneration and muscular atrophy.