人血管内皮细胞生长因子的cDNA克隆及其在兔成骨细胞中的表达  

Cloning of human vascular endothelial growth factor cDNA and its expression in rabbit osteoblasts

作　　者：杨述华[1] 杨操[1] 许伟华[1] 李进[1] 张银钢 屈伸[2] 

机构地区：[1]同济医科大学附属协和医院骨科,武汉430022 [2]同济医科大学基础医学院生化教研室

出　　处：《中华骨科杂志》2000年第2期99-102,共4页Chinese Journal of Orthopaedics

摘　　要：目的　获得人VEGF1 65cDNA并构建其真核表达载体 ,探讨应用血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)基因治疗骨科各种疾患的可行性。 方法　采用巢式 (poly merasechainreaction ,PCR )技术。从HL60细胞中扩增出人血管内皮生长因子 (hVEGF1 65)cDNA ,并将这一cDNA克隆至 pcDNA3 真核表达载体上 ,构建成为 pCD -hVEGF1 65重组质粒。并对cDNA片段进行了限制性酶切分析及DNA测序分析。应用脂质体介导的基因转移技术将这一表达载体导入兔成骨细胞后 ,用链酶亲合素 -生物素 -酶复合物法 (streptavidin -biotin -enzymecomplex ,SABC)进行瞬时表达的检测。结果　经酶切鉴定及基因测序证实 ,克隆的基因片段为人VEGF1 65cDNA ,重组质粒pCD -hVEGF1 65转染成骨细胞后 ,免疫组化检测有VEGF表达。结论　我们成功地克隆了人VEGF1 65基因 ,为进一步研究VEGF基因治疗骨缺血性坏死、骨缺损及骨折的修复打下了基础。Objective To clone VEGF gene and construct its eukaryotic expression vector for the evaluation of the possability of VEGF gene therapy in orthopedic disease. Methods Human vascular endothelial growth factor(hVEGF) cDNA was amplified by nested PCR method from the HL60 cells and cloned to expression vector pcDNA 3. The cDNA was identified by enzyme digestion and DNA sequencing. Rabbit osteoblasts were transfered with pCD-hVEGF165 plasmid by lipofectin mediated gene transfer method. The transient expression of VEGF were detected by Streptavidin-Biotin-enzyme Complex(SABC). Results The cloned cDNA was confirmed to be VEGF165 cDNA. It was observed that the expression of human VEGF gene was detected distinctly 72 h after transfering. Conclusion We successfully cloned hVEGF 165 gene and construced its eukaryotic expression vector, which provided the further foundation of VEGF gene therapy for ostenecrosis,bone defeat and fracture.

关 键 词：血管 基因扩增 基因表达 CDNA VEGF 成骨细胞 

分 类 号：R346[医药卫生—基础医学]