摘要
目的 将Fas基图导入胃癌耐药细胞，建立Fas基因表达耐药株，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，以脂质体介导法将重组表述载体转染受体细胞SGC7901／VCR，G418筛选克隆细胞，Southern blot、Northern blot、Weston blot检测Fas基因和Bel-2基因的表达．结果：成功地构建了真核表述载体pBK-Fas eDNA-转导细胞后．从2×10^5细胞中筛选出大约120个抗性克隆，转导率大于0．5‰，随机挑选2十克隆继续筛选与扩增培养-获得了l株稳定的抗生细胞，从而建立了Fas基困表达耐药株，我们命名为SCXC7901／VCRFascells杂交结果表明,转导株与非转导株均有FaseDNA的表达，但转导株Fas mRNA及其蛋白水平的表过则显著高于非转导株。结论：在胃癌耐药细胞中Fas基镯处于低表达状态;通过脂质体介导的基因转染．Fas基因可成功地导入胃癌耐药细胞，并能有效地增强FasmRNA及其蛋白的表达，为诱导细胞凋亡逆转胃癌耐药细胞的研究奠定了重要基础。
Subject headings Fas gene,gene transduction,multidrug resistance,cancer drug-resistance cells AIM To transduce Fas gene into gastric cancer drug-resistance cells,to compare the expressing level of Fas gene in gene transduced and not transduced gastric cancer drug-resistance cells.METHODS By molecular cloning technique,the full length cDNA of Fas were inserted into the multiple cloning site of the expressing vector pBK-CMV.And the reconstructed plasmids with lipofectamine were transduced into gastric cancer drug-resistance cell strain SGC7901/VCR.Then the positive clones which contained the reconstructed plasmids were choosed by G418.Finally, the expressing levels of Fas gene were determined by the means of Southern blot,Northern blot and Western blot.RESULTS The expressing plasmids pBK-Fas cDNA were successfully con- strueted;More than 120 positive clones were choosed from 2×10~5 gene transduced cells,which suggested that the transducing efficiency was more than 0.5‰;2 positive clones were expanded and passage,then 1 drug-resistance cell strain(SGC7901/VCR Fas cells)was obtained;The blot- ing results suggested,Fas cDNA were expressed in both gene transduced and not transduced cell strains.However,the expressing level of Fas mRNA was very high in gene transduced cell strain than not transduced cell strain.CONCLUSION Normally in gastric cancer drug-resistance cells, the expressing level of Fas gene was very low.The Fas gene were successfully transduced into gastric cancer drug-resistance cells.And in gene transduced cell strains,the expressing of Fas gene was enhanced.
