【摘要】 背景:骨骼肌卫星细胞是一种具有多向分化能力的全能干细胞,存在于骨骼肌间质中,对缺血、缺氧有一定的耐受力,是干细胞工程中重要来源细胞。目的:为联合基因工程细胞心肌成形治疗初步探讨较为简便、经济的骨骼肌卫星细胞体外培养方法,建立一种简单、高效的转染骨骼肌卫星细胞的方法及探讨转染后基因表达的特点。方法:分离、培养兔大腿骨骼肌卫星细胞,用CKK-8法测定其生长曲线。根据质粒和脂质体不同比例分组,用脂质体介导增强型绿色荧光蛋白质粒(plasmid enhanced green fluorescent protein,pEGFP)转染骨骼肌卫星细胞。测定各组转染效率及目标基因表达特点。结果与结论:成功分离培养骨骼肌卫星细胞及转染pEFGF。在合适的质粒和脂质体比例下,转染效率可达35%以上。目标蛋白在转染12 h内开始表达,48-72 h表达最强,1周后逐渐减弱,2周后仍可观察到其表达。阳离子脂质体可介导pEGFP高效转染骨骼肌卫星细胞,转染效率与质粒、脂质体比例密切相关,目标基因表达随时间改变。更多还原

【Abstract】 BACKGROUND: Skeletal muscle satellite cells are totipotential stem cells with multi-directional differentiation potential, locate in skeletal muscle interstitium, have a certain tolerance to ischemia and hypoxia, and are important cells in stem cell engineering. OBJECTIVE: To establish a thrifty, convenient culture procedure and create a simple, efficient method to transfect skeletal muscle satellite cells, and investigate genetic expression after the transfection for cellular cardiomyoplasty. METHODS: Skeletal muscle satellite cells were isolated from rabbit thigh and cultured. Their growth curves were determined by CKK-8 method. Grouped by different proportions of the plasmid and liposome, skeletal muscle satellite cells were transfered by the enhanced green fluorescent protein plasmid based on liposome. Aftertransfection, the efficiency and character of target genetic expression was determined. RESULTS AND CONCLUSION: Satellite cells were isolated, cultured and transfected successfully. In suitable ratio of plasmid and liposomes, the transfection efficiency reached up to above 35%. The target protein was expressed within 12 hours after transfection, reached maximum in 48-72 hours and decreased gradually after one week. The expression still could be observed two weeks latter. The enhanced green fluorescent protein plasmid conducted by cationic liposome could be transfered into skeletal muscle satellite cells efficiently. The transfection efficiency was correlated closely to the ratio of plasmid and lipofectamine. The change of target gene expression depended on time.更多还原