摘要
目的:研究新型自杀基因亚麻苦甙水解酶/亚麻苦甙(lis/lin)统对人肝癌细胞HepG2的体外杀伤效应。方法:应用分子克隆技术构建热带植物木薯的cDNA文库,并从中扩增出lin基因克隆至pEGFP-N1载体,构建质粒pEGFP-N1-lis。通过电穿孔法将其转染人肝癌细胞株HepG2,同时进行G418筛选,直至出现抗性克隆,扩大培养,命名为HepG2/lis,通过荧光显微镜观察、RT-PCR、Western blot鉴定lis基因的表达;采用MTT法观察不同浓度lin对已转染的HepG2的杀伤作用及旁观者效应。结果:RT-PCR和Western blot证明转染的lis基因能够在HepG2细胞中表达并产生融合蛋白lis-EGFP;低浓度lin对转染了lis基因的HepG2细胞具有明显的细胞毒效应;旁观者效应显示仅10%左右的HepG2/lis细胞与90%的HepG2细胞混合培养于lin浓度为500mg/L的培养基时,就可显示出明显的旁观者效应。结论:lis/lin自杀基因系统在lin浓度为500mg/L时对人原发性肝癌细胞HepG2具有明显的杀伤作用和旁观者效应。
AIM:To investigate the killing effect of linamarase/linamarin(lis/lin)system on hepatocellular carcinoma cell line HepG2 in vitro.METHODS:A cDNA library was built from RNA of cassava by RT-PCR,then the linamarase gene was amplified from it by PCR and cloned into the eukaryotic expression vector plasmid pEGFP-N1,which made up the recombinant plasmid pEGFP-N1-lis.The human HCC cells HepG2 were transfected with the recombinant plasmid mediated by electroporation and screened by G418 to yield the positive clone...
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第3期274-277,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30572147)
