携带人可溶性TRAIL基因的重组腺病毒在肺癌细胞中的转染效率及表达特性研究  

Study on transfection efficiency and expression of recombinant soluble hTRAIL gene adenovirus in Lung cancer cell

作　　者：朴春姬[1] 田梅 杨巍[3] 李修义[1] 

机构地区：[1]吉林大学公共卫生学院放射损伤教研室,吉林长春130021 [2]中国CDC辐射防护与核安全医学所,北京100088 [3]苏州大学放射医学与公共卫生学院放射生物研究室,苏州215123

出　　处：《中国实验诊断学》2010年第10期1515-1518,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金资助(30170209)

摘　　要：目的研究重组腺病毒感染肺腺癌细胞A549的转染效率及重组腺病毒介导的人可溶性肿瘤坏死因子相关诱导凋亡配体(hTRAIL95-281)基因的体外表达。方法用不同感染滴度(MOI)(0-10 000 VP/cell)的重组腺病毒Ad-CMV-EGFP转染A549细胞后48 h收集细胞,经流式细胞仪检测其转染效率,并在共聚焦荧光显微镜下观察绿色荧光蛋白表达;用MOI为5 000 VP/cell的Ad-CMV-EGFP感染肿瘤细胞后不同时间(24 h、48 h、72 h和96 h)收集细胞,经流式细胞仪检测其转染效率;用重组腺病毒Ad-CMV-hTRAIL95-281感染293T细胞后不同时间收集培养物用ELISA方法检测上清中的TRAIL表达。结果重组腺病毒载体对A549细胞的转染效率和绿色荧光蛋白表达在一定范围内随着MOI的增加而增高,MOI为5 000 VP/cell时转染效率达峰值,MOI为10 000 VP/cell时转染效率反而降低。重组腺病毒转染A549细胞后72 h内转染效率随时间的变化而增高,峰值为98%,96 h有所降低(P<0.05)。转染重组腺病毒Ad-CMV-hTRAIL95-281的293T细胞在转染后24 h表达量最高(P<0.001),达到131.45 pg/ml,48 h时TRAIL蛋白表达量明显降低(P<0.01),72小时也有继续降低的趋势。结论重组腺病毒在适宜的MOI值下,在A549细胞中96小时内均有较高的转染效率,且重组腺病毒介导的可溶性hTRAIL基因在体外能够有效表达。Objective To investigate infection efficiency of recombinant adenovirus to lung cancer cell A549 and the expression of hTRAIL95-281 mediated by recombinant adenovirus in vitro.Methods A549 cell was infected with 0-10 000 VP/cell MOI of Ad-CMV-EGFP virus.48 hours later,green fluorescent positive percentage was detected by flow cytometry as an infection percentage determination method.Green fluorescent protein expression was observed by fluorescent microscope.A549 cell was infected with 5000 VP/cell MOI of Ad-CMV-EGFP virus.Cells were collected in different time（24 h、48 h、72 h和96 h）,green fluorescent positive percentage was detected by flow cytometry.293T cell were infected with Ad-CMV-hTRAIL95-281,culture medium and cell were collected in different time（12 h、24 h、48 h和72 h）,the protein of TRAIL in supernatant were detected by ELISA.Results The results showed that infection efficiency of Ad-CMV-EGFP to A549 was increased due to the increase of MOI.It reached its peak when MOI is 5 000 VP/cell.Transfection efficiency reached its peak in 72 hours,98% cell are green.It decreased in 96 hours（P0.05）.293T cell which was infected with Ad-CMV-hTRAIL95-281,expressed protein of TRAIL during 24-48 hours.Expression reached its peak in 24hours,the value was 131.45 pg/ml.Conclusion There is higher transfection efficiency of Ad-CMV-EGFP in A549 cell within 96 hours with optimal MOI,soluble hTRAIL can be expressed efficiently in vitro mediated by recombinant adenovirus.

关 键 词：重组复制缺陷型腺病毒 A549 转染效率 肿瘤坏死因子相关诱导凋亡配体 

分 类 号：R734.2[医药卫生—肿瘤]