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目的构建含绿色荧光蛋白(GFP)基因的重组慢病毒,导入宫颈癌细胞系,建立稳定表达GFP细胞系。方法 2007年9月至2010年6月将含GTP基因载体质粒pGC-FU、包装质粒pHelper1.0及包膜质粒pHelper2.0,用磷酸钙共沉淀法共转染包装细胞系293T,收集病毒颗粒感染人宫颈癌Siha细胞,以流式细胞仪筛选出荧光亮度较强GFP阳性的细胞继续培养。结果利用慢病毒载体可稳定转入GFP基因,获得稳定表达GFP的新宫颈癌细胞系S3,此细胞系中GFP阳性率达99%以上,比较Siha和S3两者生长曲线、细胞周期分布、HPV-16E7的表达量、裸鼠皮下成瘤能力差异均无统计学意义。结论慢病毒高效转入GFP基因,成功建立了表达GFP的S3细胞系,为宫颈癌基因研究提供了便利。
Objective To construct recombinant lentivirus containing green fluorescent protein (GFP) gene and introduce it into cervical cancer cell lines and establish a stable GFP cell line. METHODS: From September 2007 to June 2010, the packaging vector pGC-FU, the packaging plasmid pHelper1.0 and the envelope plasmid pHelper2.0 were co-transfected into the packaging cell line 293T by calcium phosphate co-precipitation method to collect the virus particles Infection of human cervical cancer Siha cells, flow cytometry screening of GFP-positive cells with strong fluorescence intensity continued to culture. Results The lentiviral vector could be stably transfected into GFP gene to obtain a new cervical cancer cell line S3 stably expressing GFP. The positive rate of GFP in this cell line was over 99%. Compared with the growth curve of Siha and S3, the cell cycle distribution, HPV -16E7 expression in nude mice subcutaneously tumor-forming ability differences were not statistically significant. Conclusion The lentivirus is efficiently transfected into GFP gene and successfully established S3 cell line expressing GFP, which is convenient for the study of cervical cancer gene.