腺病毒介导EGFP共转染p16基因对腺样囊性癌细胞的影响  

Effect of adenovirus-mediated EGFP and p16 gene on adenoid cystic carcinoma cells

作　　者：柯小亮[1] 孙宏晨[1] 臧光祥[1] 刘金钟[1] 苗雷英[1] 乔春燕[1] 

机构地区：[1]吉林大学口腔医学院口腔生物医学工程吉林省重点实验室,长春130041

出　　处：《北京口腔医学》2009年第1期20-23,共4页Beijing Journal of Stomatology

基　　金：国家自然科学基金项目(编号:30672338)

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。Objective To study the effect AdSCMV-pl6 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 AdSCMV-EGFP containing enhanced green fluorescence protein gene （EGFP）, to obtain the definite of this kind of adenovirus vector. SACC-83 cells were infected with AdSCMV-p16 and AdSCMV-EGFP. AdSCMV-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 AdSCMV-EGFP was more than 95% at 1000 viral particles[vp]/cell, p16 gene was found in AdSCMV-p16 and AdSCMV-EGFP group by RT-PCR, but wasn＇t in AdSCMV-EGFP group. The activity of SACC-83 decreased in both groups,but the AdSCMV-EGFP and AdSCMV-p16 group decreased more significantly than AdSCMV-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 AdSCMV-p16 could effectively inhibit the activity of SACC-83 cells.

关 键 词：腺样囊性癌 腺病毒 基因治疗 

分 类 号：R739.8[医药卫生—肿瘤]