Fas基因转导胃癌细胞的表达

Expression of Fas gene in gastric cancer cells transducted with Fas gene

目的将Ｆａｓ基因导入胃癌细胞，建立Ｆａｓ基因表达株，并比较转导前后ｍＲＮＡ与蛋白的表达水平．方法采用分子克隆技术将Ｆａｓ基因插入真核表达载体ｐＢＫＣＭＶ的多克隆克隆位点之间，以脂质体介导法将目的基因导入受体细胞ＳＧＣ７９０１，用Ｇ４１８筛选克隆细胞；以Ｓｏｕｔｈｅｒｎｂｌｏｔ，Ｎｏｒｔｈｅｒｎｂｌｏｔ，Ｗｅｓｔｅｒｎｂｌｏｔ检测Ｆａｓ基因的表达．结果成功地构建了真核表达载体ｐＢＫＦａｓｃＤＮＡ．转导细胞后，从１×１０５细胞中筛选出１００个抗性克隆以上，转导率大于０１％，随机挑选２个克隆扩增培养，获得了１株稳定的抗性细胞，从而有效地建立了Ｆａｓ基因表达株（ＳＧＣ７９０１Ｆａｓｃｅｌｓ）．杂交结果表明，转导株与非转导株均有ｃＤＮＡ的表达，但转导株在ｍＲＮＡ及蛋白水平的表达均明显高于非转导株．结论Ｆａｓ基因在胃癌细胞中处于低表达状态；通过真核表达载体的介导，Ｆａｓ基因导入胃癌细胞后，能有效地表达ＦａｓｍＲＮＡ及其蛋白．

AIM To transduct Fas gene into gastric cancer cell, and to compare the expression level of mRNA and protein before and after. METHODS The full length cDNA of Fas was inserted into the multiple cloning site of the expressing vector pBK CMV by molecular cloning technique. And the reconstructed plasmid with lipofectamine was transducted into gastric cancer cell line SGC7901. the positive clones which contained the reconstructed plasmid were chosen by G418. Finally, the expression level of Fas gene was determined by Southern, Northern and Western blots. RESULTS The expressing plasmid pBK CMV cDNA was successfully constructed. More than 100 positive clones were chosen from 1×10 5 transduced cells, which suggested that the transduction efficiency was more than 0 1%. Two positive clones were randomly chosen amplified,and 1 drug resistance cell strain (SGC7901 Fas cells) was obtained. The blotting results suggested that Fas cDNA was expressed in both gene transducted and non transducted cell strains, but the expression level of Fas mRNA and protein was much higher in the former than in he latter. CONCLUSION The expression level of Fas gene was very low in gastric cancer cells. The gastric cancer cells transducted with reconstructed plasmid of pBK Fas cDNA could express Fas mRNA and protein efficiently.