WWOX基因转染抑瘤效应的实验研究

Study on suppressing-tumor effect of WWOX gene transfection

目的建立肿瘤抑制基因WWOX真核表达载体,观察其瞬时转染对肿瘤细胞凋亡的影响。方法采用RT-PCR方法,从正常人胚肾细胞HEK-293中钓取WWOX基因,将其克隆至pMD18-T载体上,测序正确后克隆到真核表达载体pcDNA4/myc-His中;采用脂质体介导法体外瞬时转染人肺腺癌A549细胞;RT-PCR法和Western印迹法分别检测WWOX基因mRNA和蛋白表达水平;流式细胞术检测细胞凋亡率;克隆形成试验观察肿瘤细胞克隆形成能力的变化。结果成功构建了WWOX的真核表达载体,体外转染的A549细胞凋亡率明显高于未转染和空载体转染细胞,且细胞克隆形成能力下降,形成率为35.43%,明显低于未转染细胞和空载体转染细胞。结论所构建的pcDNA4/myc-WWOX真核表达载体转染肿瘤细胞能够抑制肿瘤生长并诱导细胞凋亡。

Objective To construct tumor suppressor gene WWOX eukaryotic expression vector and observe the apoptotic effect of WWOX transient transfer on A549 human lung adenocarcinoma cells. Methods Tumor suppressor gene WWOX was cloned by RT-PCR and then linked into pMD18-T vector. After sequenced, the expression vector pcDNA4/myc-WWOX was constructed and transiently transfected into A549 cells under mediation of KeyGenTrans I in vitro. The expression of WWOX was detected by RT-PCR and Western blot. The apoptosis of transfected cells was analyzed by flow cytometry. The cell survival level was determined through clone formation assay. Results The expression vector pcDNA4/myc-WWOX was successfully constructed. The percentage of apoptotic cells in transfected pcDNA4/myc-WWOX cells was significantly increased compared with that in non-transfected and pcDNA4/myc-His transfected cells. The rate of clone formation of pcDNA4/myc-WWOX was lower than that in non-transfected and pcDNA4/myc-His tansfected cells. Conclusions Transiently transfecting expression vector pcDNA4/myc-WWOX into tumor cells can induce apoptosis and obviously inhibit cell survival.