人血管内皮生长因子基因的克隆及在人成肌细胞中的表达  

Cloning of human vascular endothelial growth factor gene and its expression in human myoblasts

作　　者：于大海 王代友 邝晓聪[2] 周柳艳 

机构地区：[1]广西医科大学口腔医学院口腔颌面外科,广西南宁520021 [2]广西医科大学生理病理教研室

出　　处：《临床口腔医学杂志》2002年第6期406-408,共3页Journal of Clinical Stomatology

基　　金：国家自然科学基金资助项目 (30 0 60 0 83)

摘　　要：目的　克隆人血管内皮生长因子基因VEGF165片段 ,构建pCDNA3 /VEGF165真核表达载体 ,观察其在人成肌细胞中的表达。方法　采用逆转录聚合酶链式反应 (RT -PCR)技术 ,从人肝癌细胞株SMMC -772 1中扩增出血管内皮生长因子VEGF165基因片段 ,通过DNA重组技术将该基因片段重组于pCDNA3 真核表达载体上 ,构建成pCDNA3 /VEGF165重组质粒 ,分别用酶切电泳分析及DNA测序的方法对重组DNA进行鉴定 ,应用脂质体介导的基因转移技术将这一表达载体导入体外培养的人成肌细胞内 ,并用免疫组化法观察其表达。结果　经酶切电泳分析和DNA测序证实 ,本实验克隆的基因片段为人VEGF165cDNA ,重组质粒pCDNA3 /VEGF165转染人成肌细胞后 ,VEGF表达明显增高。结论　本实验成功地克隆了VEGF165基因 。ObjectiveThe aim of this study is to clone VEGF gene, to construct the expressional plamsid pCDNA3/VEGF 165 and observe its expression in human myoblasts. Methods Human vascular endothelial growth factor gene was amplified by RT-PCR method from human cells SMMC-7721 strains and cloned into the expressional plamsid pCDNA 3. The gene was identified by enzyme digestion and DNA sequencing. Human myoblasts were transfected with the expressional plamsid pCDNA 3/VEGF 165 by lipofectin mediated gene transfer technique. The expression of VEGF gene in human myoblasts was detected by immunohistochemical staining assay.ResultsThe cloned DNA was comfirmed to be VEGF 165 gene. The expression of VEGF 165 gene can be observed distinctly 72h after transfecting. Conclusion In the study we successfully cloned VEGF 165 gene and constructed its expression plamsid pCDNA 3/VEGF 165 , which provided the foundation of using VEGF gene therapy to resume or buildup the angiogenesis of tissues after radiotherapy by muscle transfer in vivo.

关 键 词：血管内皮生长因子 基因克隆 基因扩增 基因表达 成肌细胞 

分 类 号：Q785[生物学—分子生物学]