真核表达载体pIRES2-EGFP-p16的构建及其在胶质瘤细胞C6内的表达  

Construction of the eukaryotic expression vector pIRES2-EGFP-p16 and its expression in glioma cells

作　　者：李立宏[1] 秦怀洲[1] 王举磊[1] 梁秦川[1] 高国栋[1] 

机构地区：[1]第四军医大学唐都医院神经外科,陕西西安710038

出　　处：《中风与神经疾病杂志》2009年第2期173-175,共3页Journal of Apoplexy and Nervous Diseases

摘　　要：目的构建真核表达载体pIRES2-EGFP-p16,并在神经胶质瘤细胞系C6中进行表达。方法用PCR的方法扩增p16基因,构建真核表达载体pIRES2-EGFP-p16,经BamHⅠ及XhoⅠ双酶切鉴定并测序。通过脂质体法转染C6细胞,用荧光显微镜检测细胞中增强型绿色荧光蛋白(EGFP)的表达,用免疫组化染色的方法检测细胞中p16的表达。结果成功地构建了真核表达载体pIRES2-EGFP-p16,用脂质体法转染神经胶质瘤细胞C6后,经荧光显微镜和免疫组化染色法检测,可见细胞内有EGFP及p16的表达。结论成功地构建真核表达载体pIRES2-EGFP-p16,并在神经胶质瘤细胞C6中表达,为研究p16对肿瘤的生物学作用以及p16在肿瘤基因治疗中的应用奠定了基础。Objective To construct the eukaryotic expression vector pIRES2-EGFP-p16,and to express p16 in glioma cell C6.Methods The p16 gene was amplified by PCR using pCMV5-HA-p16 as a template,and confirmed by DNA sequencing.The eukaryotic expression vector pIRES2-EGFP-p16 was constructed by introducing p16 DNA fragment into the sites of BamHⅠand XhoⅠof pIRES2-EGFP vector.The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the P16 protein expression was detected by immunostaining using anti-P16 antibody.Results The eukaryotic expression vector pIRES2-EGFP-p16 was constructed and transfected successfully into C6 glioma cells.The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and P16 protein was found in the plasma and nuclear.Conclusion The recombinant expression vector pIRES2-EGFP-p16 was constructed, and the EGFP and p16 gene could be co-expressed in the C6 cells.This study laid a foundation for the further research of the function of p16 in cell differentiation, growth and tumorigenesis.

关 键 词：P16 EGFP 真核表达载体 胶质瘤细胞C6 

分 类 号：R739.264[医药卫生—肿瘤] R512.62[医药卫生—临床医学]