CAJ | 학술논문

目的构建人VEGF121逆转录病毒表达载体,探讨应用血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)基因治疗骨科缺血性疾患的可行性.方法采用分子克隆技术从pCDⅠ/VEGF121质粒获得hVEGF121cDNA, 并克隆到pLXSN逆转录病毒质粒.经酶切及测序鉴定正确后,包装成为pLXSN/VEGF121重组逆转录病毒, 并进行滴度测定.培养小鼠骨髓基质细胞,用重组病毒感染小鼠骨髓基质细胞.经筛选后,用PCR及RT-PCR方法测hVEGF121cDNA在基质细胞中的整合及转录;用免疫印迹及免疫组化检测VEGF121蛋白的表达;用MTT检测转基因细胞培养上清中VEGF的促人内皮细胞增殖活性.结果经酶切鉴定及基因测序证实重组逆转录病毒质粒正确,包装的病毒滴度为6×105.PCR证实基因组DNA有hVEGF121cDNA的整合;RT-PCR证实有hVEGF121mRNA的转录;Western blot及免疫组化检测有VEGF121蛋白的表达;MTT试验显示转基因细胞培养上清中VEGF121能促进人内皮细胞增殖.结论我们成功地构建了pLXSN/VEGF121重组逆转录病毒载体,该载体不仅能够有效地表达VEGF121蛋白,且表达的蛋白具有促进人内皮细胞增殖的生物学活性,这些均为进一步应用该载体研究VEGF基因治疗骨科缺血性疾患打下了基础.
목적구건인VEGF121역전록병독표체재체,탐토응용혈관내피세포생장인자(vascular endothelial growth factor,VEGF)기인치료골과결혈성질환적가행성.방법채용분자극륭기술종pCDⅠ/VEGF121질립획득hVEGF121cDNA, 병극륭도pLXSN역전록병독질립.경매절급측서감정정학후,포장성위pLXSN/VEGF121중조역전록병독, 병진행적도측정.배양소서골수기질세포,용중조병독감염소서골수기질세포.경사선후,용PCR급RT-PCR방법측hVEGF121cDNA재기질세포중적정합급전록;용면역인적급면역조화검측VEGF121단백적표체;용MTT검측전기인세포배양상청중VEGF적촉인내피세포증식활성.결과경매절감정급기인측서증실중조역전록병독질립정학,포장적병독적도위6×105.PCR증실기인조DNA유hVEGF121cDNA적정합;RT-PCR증실유hVEGF121mRNA적전록;Western blot급면역조화검측유VEGF121단백적표체;MTT시험현시전기인세포배양상청중VEGF121능촉진인내피세포증식.결론아문성공지구건료pLXSN/VEGF121중조역전록병독재체,해재체불부능구유효지표체VEGF121단백,차표체적단백구유촉진인내피세포증식적생물학활성,저사균위진일보응용해재체연구VEGF기인치료골과결혈성질환타하료기출.
Objective To construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF121) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.Methods hVEGF121 cDNA was obtained from the plasmid pCDI/VEGF121 and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retrovirus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF121 cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF121 in culture medium were performed.Results Recombinant pLXSN/VEGF121 was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF121 gene was integrated into MSC genomic DNA after transfection, and the VEGF121 protein was expressed. Proliferation assays showed VEGF121 in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.Conclusions Recombinant retroviral vector carrying hVEGF121 cDNA was successfully constructed. VEGF121 protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.