CD95基因抑制胃癌细胞生长的体外抑瘤效应

In vitro anti tumor effects of CD95 gene in a gastric cancer cell line

目的 将CD95基因导入胃癌细胞 ,建立CD95基因表达株 ,并比较转导前后mRNA与蛋白的表达水平。观察CD95蛋白对体外培养胃癌细胞的抑制作用。方法 采用分子克隆技术将CD95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SGC790 1,用G418筛选克隆细胞 ;以Northernblot,Westernblot检测CD95基因的表达。MTT法检测转导株对化疗药物的敏感性 ;直接记数法描述转导株的细胞生长曲线 ;软琼脂集落形成实验观察基因转导前后细胞的克隆形成力。结果 成功地构建了真核表达载体pBK CD95cDNA。转导细胞后 ,从 1× 10 5细胞中筛选出 10 0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CD95基因表达株 (SGC790 1CD95cells)。杂交结果表明 ,转导株在mRNA及蛋白水平的表达均明显高于非转导株。转导细胞的细胞倍增时间、对数生长期等均体现了比非转导株更为缓慢和处于抑制状态 ,集落形成能力低下 ,而对VCR、5 FU等化疗药物的敏感性明显增强。结论 CD95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 ,CD95基因导入胃癌细胞后 ,能有效地表达CD95mRNA及其蛋白。

Aim To transduct CD95 gene into gastric cancer cell,and to compare the expression level of CD95 gene in gene transduced and non transduced gastric cancer cells.To probe into the tumor inhibitory effects in gastric cancer cell.Methods The full length of CD95 cDNA was inserted into the multiple cloning site of the expression vector pBK CMB by molecular cloning technique.And the reconstructed plasmid with lipofectamine was transduced into gastric cancer cell line SGC7901.Then the positive clones which contained the reconstructed plasmid were chosen by G418.Finally,the expression leve of CD95 gene was determined by means of Southern blot,Northern blot and Western blot.MTT assay was used to detect drug sensitivity of gene transduced cells.The tumor repression function of the transfected strain were observed through cell growth curve,plating efficiency.Results The expression 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 to expand,then 1 drug resistance cell strain (SGC7901 CD95 cells) was obtained.The bloting results suggested,that CD95 cDNA was expressed in both transduced and non transduced cell strains,but the expression level of CD95 mRNA and protein in transduced cell strain was much higher than that in non transduced cell strain.For the transferred strain,population doubling time was 2.5 days,log phase of growth was 4～7 days and plating efficiency and repression rate were 2.05% and 50% respectively.Results of MTT assay showed increasing sensitivity of gene transduced cells to CDDP,VCR,5 Fu.Conclusions The expression leve of CD95 gene was very low in gastric cancer cells.The gastric cancer cells transduced with reconstructed plasmid of pBK CD95 cDNA could express CD95 mRNA and CD95 protein efficienctly.CD95 gene transgenic expression clearly represses the proliferaton of gastric cancer cells in vitro.