【摘要】 目的:构建人血管内皮生长因子165(hVEGF165)的真核表达载体,并在大鼠骨髓基质细胞中进行表达。方法:利用基因克隆技术,将原核克隆载体pSP73中的目的基因VEGF165用 BamH I和Xho I双酶切后,再克隆到真核表达载体pcDNA3.1中,构建重组质粒pcDNA3.1-VEGF165。对重组质粒进行酶切分析和测序鉴定。通过脂质体介导,用重组质粒转染SD大鼠骨髓基质细胞,然后以G418筛选阳性克隆,用免疫细胞化学鉴定。结果:经酶切鉴定及基因测序证实,重组体中已插入目的基因片段VEGF165。免疫细胞化学证实,重组质粒转染的骨髓基质细胞中有VEGF165基因的表达。结论:成功地构建真核表达载体pcDNA3.1-VEGF165,并在骨髓基质细胞中得到表达,为将表达VEGF基因的骨髓基质细胞作为骨组织工程的种子细胞的可能性提供了实验依据。更多还原

【Abstract】 AIM: To construct the eukaryotic expression vector containing human vascular endothelial growth factor 165 (VEGF165) gene and express it in rat bone marrow stroma cells (rMSCs). METHODS: The recombinant plasmid pSP73-VEGF165 was digested with BamH I and Xho I. Then the hVEGF165 gene segment obtained was again cloned into pcDIMA3. 1 to construct recombinant eukaryotic expression vector pcDNA3. 1-VEGF165. Then the recombinant vector was identified by enzyme digestion analysis and sequencing. The rMSCs were transformed by recombinant vector and positive clones were screened with G418. The expression of hVEGF165 gene in the transformed cells was detected by immunocytochemical staining. RESULTS: Enzyme digestion analysis and sequencing showed that target gene had been cloned into recombinant vector. The expression of hVEGF165 gene in the transformed cells had been demonstrated by immunocytochemical staining. CONCLUSION: The recombinant eukaryotic expression vector has been constructed and expressed successfully in the transformed cells. Therefore, it is possible to use the rMSCs expressing hVEGF165 gene as seed cells in the bone tissue-engineering.更多还原