Caveolin-1真核表达载体的构建及其Hepa1-6细胞中的表达

摘要

Caveolin-1 plays different roles in different kinds of tumors. Caveoiln-1 may act as tumor-suppressor, as well as tumor-promoter. This study aims to analyze the expression of caveolin-1 in mouse hepatoma cell lines and construct stable hepa1-6 cells expressing exogenous caveolin-1. The expression of caveolin-1 in mouse hepatoma cell lines (H22,Hca-F and Hepal-6)was investigated by RT-PCR and Western-blot assays; mouse caveolin-1 cDNA expression vector was constructed by molecular clone, and stable hepa1-6 cells transfected with caveolin-1 gene were obtained through transfection and screening. Results showed that the expression levels of caveolin-1 were undetectable in hepa1-6 cells, and upregulated in H22 and Hca-F cells. Mouse caveolin-1 cDNA expression vector and stable transfected hepa1-6 cells were generated successfully. The stable hepa1-6 cells transfected with caveolin-1 will lay a foundation for further studies of caveolin-1 function.%Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用.旨在分析caveolin-1 在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepa1-6细胞.利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hea-F和Hepa1-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达栽体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepa1-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株.结果显示,caveolin-1在Hepa1-6细胞中表达呈阴性,在H22和Hca-F 中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2/Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepa1-6稳定细胞株C1和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础.