电穿孔转染酸性成纤维细胞生长因子基因对骨骼肌卫星细胞的影响

Effects of transfection with acidic fibroblast growth factor by electroporation on skeletal muscle satellite cells

背景:课题组早期研究表明体外一定剂量酸性成纤维细胞生长因子对骨骼肌卫星细胞增殖有促进作用。目的:进一步验证电穿孔转染酸性成纤维细胞生长因子基因对骨骼肌卫星细胞生长、增殖及分化的影响。方法:原代培养、纯化骨骼肌卫星细胞,将带有酸性成纤维细胞生长因子基因的质粒p Sectag-GFP-a FGF通过电转染的方法转染大鼠骨骼肌卫星细胞,荧光显微镜观察绿色荧光蛋白的表达情况并计算转染率,以流式细胞仪分析转染后细胞周期,绘制细胞生长曲线,观察转染后肌管形成情况,Western Bloting检测酸性成纤维细胞生长因子基因的表达。结果与结论:1免疫细胞化学检测:骨骼肌肌动蛋白呈阳性表达。2转染效率:p Sectag-a FGF质粒电转染12 h后即可看见散在发绿色荧光的卫星细胞,72-96 h达高峰,阳性表达率约90%。3细胞周期检测:电转染后S期所占的百分比明显多于未转染对照组(P<0.05)。4细胞生长曲线检测:电转染细胞接种后第3天进入对数生长期,第5天后开始减少。5分化能力观察:电转染组肌管较未转染对照组明显减少,老化细胞较少。6Western-blot:酸性成纤维细胞生长因子基因在转染骨骼肌卫星细胞中表达。结果表明,通过电穿孔法可以将酸性成纤维细胞生长因子基因转染进骨骼肌卫星细胞并获得高效持久的表达,并有促进骨骼肌卫星细胞增殖及抑制分化为肌管的作用。

BACKGROUND：Previous studies have shown that a certain dose of acidic fibroblast growth factor can promote skeletal muscle satelite cel proliferationin vitro. OBJECTIVE：To investigate the effects of transfection with acidic fibroblast growth factor by electroporation on growth, proliferation and differentiation of skeletal muscle satelite cels. METHODS： Skeletal muscle satelite cels were cultured and purified, and then transfected with plasmid pSectag-GFP-aFGF by electroporation. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was calculated. After transfection, cel cycle was analyzed by flow cytometry to draw the growth curve of skeletal muscle satelite cels. Western blot assay was employed to measure protein level of acidic fibroblast growth factor. RESULTS AND CONCLUSION： （1） Immunocytochemistry detection： The skeletal muscle satelite cels were positive for a-sarcomeric actin. （2） Transfection efficiency： At 12 hours after transfection with pSectag-aFGF, several cels showed green fluorescence, and the green fluorescent expression reached the peak at 72-96 hours after transfection with a positive rate of about 90%. （3） Cel cycle： After electrotransfection, the proportion of cels at S phase in the electroporation group was higher than that in the control group （P〈 0.05）. （4） Cel growth curve： At 3 days after electrotransfection, the cels entered logarithmic growth phase but the proliferation slowed down at 5 days. （5） Differentiation capacity： There were fewer myotubes and aging cels in the electroporation＆amp;nbsp;group than the control group. （6） Western blot assay： Acidic fibroblast growth factor protein was highly expressed in the cels transfected with target gene detected by western blot assay. These findings indicate that by using electroporation method, acidic fibroblast growth factor can be transferred into skeletal muscle satelite cels and have a high-efficiency and long-term expression, which can promote the proliferation of skeletal muscle satelite cels and inhibit formation of myotubes.