中国组织工程研究Issue(1)：85-90,6.DOI:10.3969/j.issn.2095-4344.2015.01.015
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
李江华 1董少红 2熊玮 1庞新利 1刘启云 1李文君2
作者信息
- 1. 深圳市人民医院心内科暨南大学第二临床医学院，广东省深圳市 518020
- 2. 厦门大学医学院，福建省省厦门市 361005
- 折叠
摘要
Abstract
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 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.关键词
干细胞/分化/骨骼肌卫星细胞/基因转染/绿色荧光蛋白/电穿孔/原代培养Key words
Satelite Cells/Skeletal Muscle/Genes/Transfection/Green Fluorescent Proteins分类
医药卫生引用本文复制引用
李江华,董少红,熊玮,庞新利,刘启云,李文君..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[J].中国组织工程研究,2015,(1):85-90,6.
