Fas基因转导大肠癌细胞株的表达

Expression of Fas genes transduced into colorectal cancer cells

目的建立表达外源性Fas基因的大肠癌细胞株,观察Fas基因在转导前后的表达。方法采用分子克隆技术将Fas基因插入真核表达载体pBK-CMV的多克隆位点之间,以脂质体介导法将Fas基因导入受体细胞LoVo,用G418筛选克隆细胞。以dot blotting,Western blotting检测转导细胞Fas基因的表达。结果成功建立了Fas基因表达株。转导株在RNA及其蛋白水平表达均明显高于非转导株,转导细胞增殖速度、倍增时间、对数生长期等均比非转导株更为缓慢,但无显著性差异,在Fas抗体作用下,转导株细胞生长明显受到抑制,有非常显著性差异。结论Fas基因在大肠癌细胞中处于低表达状态;通过真核表达载体介导,Fas基因在RNA及其蛋白水平能有效表达。Fas表达株在Fas抗体作用下可明显抑制体外培养的大肠癌细胞的生长增殖。

Objective To construct colorectal cancer cells expressing exogenous Fas gene and observe the expression level of its mRNA and protein before and after transduction. Methods Fas cDNA was inserted into the multiple cloning site of the ex-pression vector pBK-CMV with molecular cloning technique, and the resultant recombinant plasmid was transduced into col-orectal cancer LoVo cells via lipofectamine. G418 was utilized to screen the positive clones containing the recombinant plas-mid, where Fas mRNA and protein expression was determined with Western blotting and dot blotting. Results pBK-CMV Fas cDNA plasmid was successfully constructed. The transduced colorectal cancer cells were screened by G418 and a resistant cell line (LoVo Fas cells) was obtained. Fas expression was detected in both transduced and non-transducted cell lines, but the expression level of both Fas mRNA and protein was much higher in the former, which showed lowered proliferation rate and lengthened doubling time and logarithm growth period than the non-transducted cells, but the difference was not significant. Treatment of the transduced cells with Fas antibody produced significant difference (P<0.05), manifested by apparently inhib-ited cell growth. Conclusions LoVo cells normally has only very low expression level of Fas gene, while transduction with pBK-Fas cDNA can enhance the efficiency of Fas mRNA and protein expressions. Fas antibody significantly inhibits the growth and proliferation of in vitro cultured Fas-expressing LoVo cells.