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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 obtain human VEGF1 65 cDNA and construct its eukaryotic expression vector and investigate the feasibility of using vascular endothelial growth factor (VEGF) gene to treat various diseases of orthopedics. Methods The nested (poly merasechain reaction, PCR) technique was used. The human vascular endothelial growth factor (hVEGF165) cDNA was amplified from HL60 cells and cloned into pcDNA3 eukaryotic expression vector to construct pCD-hVEGF1 65 recombinant plasmid. The cDNA fragments were subjected to restriction analysis and DNA sequencing analysis. The expression vector was transfected into rabbit osteoblasts by liposome-mediated gene transfer and then transiently detected by streptavidin-biotin-enzyme complex (SABC) . Results The results of restriction endonuclease digestion and sequencing confirmed that the cloned gene fragment was human VEGF1 65 cDNA. The expression of VEGF was detected by immunohistochemistry after the recombinant plasmid pCD-hVEGF1 65 was transfected into osteoblasts. Conclusions We successfully cloned the human VEGF1 65 gene, which lays the foundation for the further study of VEGF gene therapy for the treatment of ischemic bone necrosis, bone defect and fracture.