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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。
BACKGROUND: Skeletal muscle satellite cells are pluripotent differentiating pluripotent stem cells that exist in the skeletal muscle interstitium and have some tolerance to ischemia and hypoxia. They are important cells in stem cell engineering. OBJECTIVE: To establish a simple and efficient method for transfection of skeletal muscle satellite cells in vitro and to explore the gene expression characteristics of the transfected skeletal muscle satellite cells in vitro. Methods: Skeletal muscle satellite cells of rabbit thigh were isolated and cultured, and their growth curves were measured by CKK-8 method. According to the different ratios of plasmids and liposomes, plasmid-mediated green fluorescent protein (pEGFP) was transfected into skeletal muscle satellite cells. The transfection efficiency and target gene expression characteristics of each group were determined. RESULTS AND CONCLUSION: Skeletal muscle satellite cells were successfully isolated and transfected with pEFGF. At appropriate plasmid and liposome ratios, the transfection efficiency can be over 35%. The target protein began to express within 12 h after transfection, the expression was highest at 48-72 h, gradually weakened after 1 week, and was still observed after 2 weeks. Cationic liposomes can efficiently transduce pEGFP into skeletal muscle satellite cells. The transfection efficiency is closely related to the ratio of plasmid and liposome, and the expression of target gene changes with time.