【摘要】 目的探讨以抑癌基因p16为靶基因,腺病毒(Ad)为载体的重组基因治疗药物Ad5CMV-p16对人涎腺腺样囊性癌细胞系(SACC-83)的作用。方法含有增强型绿色荧光蛋白基因(EGFP)的腺病毒载体(Ad5CMV-EGFP)转染SACC-83细胞,确定重组腺病毒载体转染效率。Ad5CMV-EGFP为对照组,Ad5CMV-p16和Ad5CMV-EGFP共转染为实验组分别感染SACC-83细胞,采用RT-PCR方法检测p16基因的表达。应用倒置显微镜观察细胞形态,MTT检测细胞的增殖率,软琼脂克隆形成实验检测细胞克隆形成,流式细胞仪检测细胞周期变化。结果Ad5CMV-EGFP在1000 viral particles[vp]/cell时转染效率达95%以上。RT-PCR实验组可检测到p16基因表达,而对照组未见表达。细胞单纯转染Ad5CMV-EGFP或共同转染Ad5CMV-p16后,随时间增长细胞增殖率逐渐下降,共同转染组较单纯转染Ad5CMV-EGFP组比变化更明显。软琼脂克隆形成实验显示,共转染组较对照组有更强的抑制细胞克隆形成能力。流式细胞仪检测细胞周期变化显示Ad5CMV-EGFP和Ad5CMV-p16共转染组G0-G1期细胞比例比单独转染Ad5CMV-EGFP组大。结论Ad5CMV-p16在体外能明显抑制SACC-83细胞增殖能力和活性,有望成为一种有效的基因治疗药物。更多还原

【Abstract】 Objective To study the effect Ad5CMV-p16 on the adenoid cystic carcinoma cell (SACC-83) mediated by adenovirus vector containing p16 gene.Methods SACC-83 cells were infected with different titre of Ad5CMV-EGFP containing enhanced green fluorescence protein gene (EGFP),to obtain the definite of this kind of adenovirus vector.SACC-83 cells were infected with Ad5CMV-p16 and Ad5CMV-EGFP.Ad5CMV-EGFP served as control.p16 gene was then detected by RT-PCR; the activity of SACC-83 cells was evaluated by MTT; the cell cycles were detected by flow cytometer.Results The transfecting rate of Ad5CMV-EGFP was more than 95% at 1000 viral particles [vp]/cell.p16 gene was found in Ad5CMV-p16 and Ad5CMV-EGFP group by RT-PCR,but wasn′t in Ad5CMV-EGFP group.The activity of SACC-83 decreased in both groups,but the Ad5CMV-EGFP and Ad5CMV-p16 group decreased more significantly than Ad5CMV-EGFP group.There was more cloning inhibition in soft agar assay,and more G0-G1 stage cells were found in experimental group than in control group.Conclusion Ad5CMV-p16 could effectively inhibit the activity of SACC-83 cells.更多还原