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背景:外源性的血管内皮生长因子能使体外培养的成骨细胞碱性磷酸酶活性及环磷酸腺苷浓度提高4倍,但外源性血管内皮生长因子基因转染对成骨细胞成骨活性影响如何?目的:了解外源性血管内皮生长因子基因转染及表达对成骨细胞成骨活性的影响。设计:随机对照实验。单位:四川大学华西医院移植免疫实验室。对象:新生两三天雄性SD大鼠颅骨成骨细胞。方法:实验于2003-04/12在四川大学华西医院移植免疫实验室完成。分离培养大鼠颅骨成骨细胞用碱性磷酸酶染色鉴定。①用阳离子脂质体转染将pBLAST49-mVEGF基因导入体外培养的成骨细胞为基因转染组。②未转染组加入杀稻瘟菌素为对照。③将转染成功和未转染的成骨细胞进行同步传代培养共5代,对每代细胞进行血管内皮生长因子和Ⅰ型胶原免疫组织化学染色检查和骨钙素的测定。主要观察指标:①观察外源性血管内皮生长因子在成骨细胞内的表达。②对成骨细胞合成分泌骨钙素及Ⅰ型胶原的影响。结果:pBLAST49-mVEGF基因转染组第1～5代细胞合成分泌骨钙素浓度和I型胶原的表达均明显高于对照组,二者间比较差异具有显著性(P<0.05)。结论:pBLAST49-mVEGF质粒转染成功后,成骨细胞合成Ⅰ型胶原明显增强。与对照组相比,通过Mias图像分析,与未转染的对照组比较差异具有显著性,表明pBLAST49-mVEGF质粒转染能明显增加成骨细胞生成骨钙素以及合成Ⅰ型胶原的能力。
BACKGROUND: Exogenous vascular endothelial growth factor (VEGF) can increase the activity of alkaline phosphatase and cyclic adenosine monophosphate in cultured osteoblasts in vitro by 4-fold. However, transfection of exogenous VEGF gene into osteoblasts How is the activity affected? OBJECTIVE: To investigate the effect of exogenous VEGF gene transfection and expression on the osteogenic activity of osteoblasts. Design: Randomized controlled experiment. Unit: West China Hospital of Sichuan University transplant immune laboratory. PARTICIPANTS: Skull osteoblasts in male Sprague Dawley rats for two or three days. Methods: The experiment was performed at the Transplantation Immunology Laboratory of West China Hospital of Sichuan University from April to December in 2003. Isolation and culture of rat skull osteoblasts were identified by alkaline phosphatase staining. ① transfected with cationic liposome pBLAST49-mVEGF gene into cultured in vitro osteoblast gene transfection group. ② untransfected group added blasticidin as a control. ③ The transfected and untransfected osteoblasts were subcultured for 5 generations in synchronism, and the vascular endothelial growth factor and type Ⅰ collagen immunohistochemical staining and osteocalcin determination were performed on each cell line. MAIN OUTCOME MEASURES: ① To observe the expression of exogenous VEGF in osteoblasts. ② on osteoblast synthesis and secretion of osteocalcin and type Ⅰ collagen. Results: The expression of osteocalcin and type I collagen in the first to fifth generation cells transfected with pBLAST49-mVEGF gene were significantly higher than those in the control group, the difference was significant (P <0.05). Conclusion: After successful transfection of plasmid pBLAST49-mVEGF, type Ⅰ collagen synthesis by osteoblasts was significantly enhanced. Compared with the control group, Mias image analysis showed that the difference was significant compared with the untransfected control group, indicating that the transfection of pBLAST49-mVEGF plasmid can significantly increase osteoblast production of osteocalcin and synthesis of type I collagen.