人VEGF在NIH3T3细胞中的基因转移和表达  

Expression of Human Vascular Endothelial Growth Factor in Cultured NIH3T3 Cells

作　　者：王雅梅[1] 李慎涛[2,3] 温铭杰[3] 孙丽翠[1] 司杨[1] 闫豫东[2] 祁雅慧[2] 

机构地区：[1]首都医科大学基础医学院生化与分子生物学系 [2]首都医科大学医学实验测试中心 [3]首都医科大学基础医学院免疫学系

出　　处：《首都医科大学学报》2008年第6期737-740,共4页Journal of Capital Medical University

基　　金：北京市科委科技合同项目(954024800)资助~~

摘　　要：目的研究人血管内皮细胞生长因子(human VEGF)在体外培养的小鼠成纤维细胞(NIH3T3细胞)中的基因转移及表达,为血管化组织工程、缺血性疾病的治疗奠定实验基础。方法应用脂质体介导的基因转移技术,将含有人VEGF165编码序列的真核表达载体pcDNA/V转染至体外培养的NIH3T3细胞系中。G418筛选出稳定表达重组质粒的细胞克隆,转接于细胞瓶中,培养72 h,同时以G418筛选的pcDNA3.1(+)阳性细胞克隆和未转染的NIH3T3细胞为阴性对照,取细胞培养上清及细胞裂解液样品进行Western blotting检测。用细胞免疫组织化学染色检测人VEGF165基因在NIH3T3细胞中的表达情况,不着色者为阴性,细胞胞质着色呈棕黄(褐)色者为阳性。结果Western blotting结果显示在转染pcDNA/V质粒的NIH3T3细胞培养上清中,可以检测到相对分子质量为22 000的反应条带,与预计的人VEGF165单体蛋白的相对分子质量相符。细胞免疫组织化学染色检测结果显示,转染pcDNA/V质粒的NIH3T3细胞呈阳性。结论人VEGF165能够在NIH3T3细胞中有效表达,并获得了稳定表达人VEGF165基因的NIH3T3细胞株。Objective To study human vascular endothelial growth factor （hVEGF） gene transfer and expression in NIH3T3 fibroblast cell line, and establish the experimental foundation of angiogenesis tissue engineering organization and the treatment of ischemic disorders. Methods pcDNA/V eukaryotic expression vector and pcDNA3. 1 （ ＋ ） were transfected into NIH3T3 cells mediated by liposome. The transfected cells were grown in DMEM medium containing G418 for 72 hours, and the clones of cells were selected and continued to grow in G418 medium. The cells stably expressing hVEGF were selected under the pressure of G418. The selected cells were grown in DMEM medium containing G418 for 72 hours, and then the supernatant was collected. The expressed hVEGF protein was analysed by using Western blot and immunohistochemical methods. Results Western blotting showed that hVEGF of 22 000 was detected in culture medium of NIH3T3 ceils transfected with pcDNA/V. Immunohistochemistry demonstrated that the NIH3T3 cells transfected with pcDNA/V showed positive reaction to VEGF in cytoplasm, whereas faint reaction was observed in non-transfected cells and pcDNA3.1 （ ＋ ）-transfected cells. Conclusion Human VEGF gene was successfully transfected and its protein could be expressed in NIH3T3 cells.

关 键 词：血管内皮细胞生长因子 真核表达载体 NIH3T3细胞系 基因表达 

分 类 号：Q785[生物学—分子生物学]