摘要
目的:研究低氧反应元件(HRE)对血管内皮生长因子(VEGF)基因121在原代培养大鼠骨骼肌成肌细胞中转染表达的调控作用。方法:利用分子生物学方法,构建pEGFP-C3-9HRE-CMV-VEGF121载体,通过脂质体介导将其转染到原代培养的大鼠骨骼肌成肌细胞中。在不同低氧浓度及不同缺氧时间下培养,通过RT-PCR、Western-Blot及荧光显微镜检测转基因成肌细胞的基因表达情况。结果:低氧浓度组可见明显目的基因条带,且随着氧浓度的降低及缺氧时间的延长,目的基因表达增强;低氧环境下,转染后的成肌细胞表达VEGF121蛋白产物明显增加;低氧环境下可见报告基因EGFP表达,常氧环境下未见报告基因表达。结论:以多拷贝HRE构建低氧启动子插入VEGF基因上游,可作为控制VEGF基因表达的开关,这对于防止VEGF基因转染的成肌细胞移植后VEGF基因过度表达所引起的安全问题,提高基因治疗的安全性有重要意义。
Objective：To investigate the regulation of hypoxic response elements to the expression of vascular endothelial growth factor gene transfected to primary cultured rat skeletal myoblasts in hypoxic environment. Method：pEGFP-C3-9HRE-CMV-VEGF121 vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene and protein expression of transfected myoblasts was detected by RT-PCR, Western-Blot and fluorescence microscope with different oxygen concentration and different hypoxia time. Result：Transfected myoblasts express high levels of VEGF121 gene and protein in hypoxic environment dependent with oxygen concentration and hypoxia time. EGFP expressed only in hypoxic environment and the expression is unavailable in normoxic environment. Conclusion： HRE could regulate the expression of VEGF gene transfected to primary cultured rat skeletal myoblasts in hypoxic environment, which could enhance the reliability of gene therapy.
出处
《临床心血管病杂志》
CAS
CSCD
北大核心
2007年第6期441-444,共4页
Journal of Clinical Cardiology
基金
国家留学基金资助(No:20035002016-17)
关键词
低氧反应元件
血管内皮生长因子
转染
骨骼肌成肌细胞
Hypoxic response elements
Vascular endothelial growth factor
Transfection
Myoblasts, skeletal
