TGFβ1真核表达载体对^(60)Coγ射线照射的人胚肺成纤维细胞的稳定转染方法研究

Studies on stable transfection of TGFβ1 sense and anti-sense gene in human embryolung fibroblasts irradiated by ^(60)Co-rays

选择6 0 Coγ射线 5Gy照射后第 7天的人胚肺成纤维细胞 (HELF) ,采用聚阳离子脂质体LipofectAMINE介导的基因转染法将人TGFβ1正、反义基因表达载体pMAMneo TGFβ1和pMAMneo AntiTGFβ1转入HELF ,转染细胞经G418抗性筛选出来。提取培养细胞的染色体DNA ,用DNA斑点印迹分析表明 ,它们能与neo基因特异杂交。用neo基因特异的PCR引物能从转染细胞扩增出 2 76bp的阳性片段 。

Human embryo lung fibro blasts(HELFs) irradiated with 5 Gy 60Co γ-rays were transfected with the expression vector pMAMneo-TGFβ1 or pMAMneo-anti-TGFβ1 that encode the human TGFβ1 sense or antisense gene.The transfected cells were elected by G418 resistance. The chromosome DNA were extracted. Production of 276bp was amplified from chromosome DNA by a PCR method of neo-specific primer. DNA dot blot analysis showed that cells transfected pMAMneo-TGFβ1 or pMAMneo-antiTGFβ1 hybridized with Dig-labeling neo-specfic probe. Results indicated HELFs after irradiation of 60 Co γ-rays could be transfected stablly using a polycation lipid-mediated method.