突变型Rad50腺病毒载体的制备及其在人鼻咽癌细胞株CNE1中的最适感染量测定  

The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected by Ad-Rad50-GFP

作　　者：严睿成[1,2] 黄健聪[1] 朱玲[3] 常利红[1] 黎景佳[1] 吴喜福[1] 叶进[1] 张革化[1] 

机构地区：[1]中山大学附属第三医院耳鼻咽喉头颈外科,广州510630 [2]广州市增城区人民医院耳鼻咽喉科 [3]广东省佛山市南海妇幼保健院耳鼻咽喉科

出　　处：《临床耳鼻咽喉头颈外科杂志》2015年第24期2143-2146,共4页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：广东省对外合作项目(No:2012B050600015)

摘　　要：目的:测定携带突变型Rad50基因的重组腺病毒载体Ad-Rad50-GFP在人鼻咽癌细胞株CNE1中的表达及其对CNE1的最适感染倍率(MOI)。方法:终点稀释法测定Ad-Rad50-GFP生物滴度,细胞生长曲线观察重组腺病毒载体对CNE1生长的影响,荧光显微镜下计算重组腺病毒载体的转染效率;免疫印迹实验检测以最适感染量感染后CNE1细胞内突变型Rad50蛋白的表达。结果:Ad-Rad50-GFP生物滴度为1.26×10^(11)pfu/ml,重组腺病毒载体以不高于50的感染倍率转染时对CNE1生长无明显影响,且MOI=50时转染24h后GFP表达水平最高,可检出高表达的突变型Rad50蛋白,在转染72h后仍维持约70%的转染效率。结论:重组腺病毒载体Ad-Rad50-GFP能有效转染CNE1细胞并使其表达突变型Rad50蛋白,MOI=50为Ad-Rad50-GFP对CNE1的最适感染倍率。Objective：The optimal multiplicity of infection（MOI） of the recombmant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNK1 cell infected by this adenovirus were studied.Method：The biological titer of AdRad50-GFP was measured by end point dilution method.The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve.Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope.The expression f mutant Rad50 in the AdRad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection.Result：The biological titer of Ad-Rad50-GFP was 1.26×10^（11） pfu/ml.CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50.Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection,with the high expression of mutant Rad50,and the efficiency still remained about 70%after 72 hours.Conclusion;Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively.MOI=50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector AdRad50-GFP.

关 键 词：重组腺病毒 鼻咽肿瘤 感染倍率 

分 类 号：R739.63[医药卫生—肿瘤]