基因成像体外监测人肿瘤坏死因子相关的诱导凋亡配体基因在肺癌A549细胞中表达的实验研究  

In vitro gene imaging by luciferase to detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand in lung cancer A549 cells

作　　者：赵纳[1] 崔建岭[1] 郭志远[1] 郭志萍[1] 孙英彩[1] 刘计存[1] 

机构地区：[1]河北医科大学第三医院CT—MR室,石家庄050051

出　　处：《中华放射学杂志》2009年第1期88-93,共6页Chinese Journal of Radiology

基　　金：基金项目：河北省自然科学基金资助（033473）

摘　　要：目的应用作者构建的同时携带人肿瘤坏死因子相关的诱导凋亡配体（hTRAIL）基因和萤火虫荧光素酶（luc）基因的腺病毒双表达载体（Ad—hTRAIL—luc），以luc为报告基因，体外监测hTRAIL基因的表达及其作用。方法携带增强型绿色荧光蛋白（EGFP）基因的腺病毒载体（Ad—EGFP）以不同感染复数（MOI）感染肺癌A549细胞，48h后检测腺病毒载体的感染效率。Ad—hTRAIL—luc分别以不同MOI感染A549细胞，用流式细胞仪48h后分别检测hTRAIL的表达率及A549细胞的凋亡率；用液闪计数仪检测荧光素酶发光的每分钟计数（cpm）。携带luc基因的腺病毒载体（Ad—luc）以不同MOI感染A549细胞，48h后检测荧光素酶发光的cpm值。Ad—hTRAIL—luc转染A549细胞后，对各组细胞的hTRAIL表达率及细胞凋亡率结果（自然对数转换后）、cpm值结果进行完全随机设计资料的方差分析，并对hTRAIL表达率及细胞凋亡率的2组数据进行直线相关分析。对Ad—EGFP感染后阳性率及Ad—luc感染后cpm值进行完全随机设计多样本比较的秩和检验。结果Ad—hTRAIL—luc转染A549细胞后，各组hTRAIL基因的表达率经自然对数转换后分别为（2．37±0．04）％、（3．16±0．03）％、（3．64±0．03）％、（3．96±0．02）％、（4．24±0．02）％、（4．34±0．02）％，各组间差异具有统计学意义（F=7364，P〈0．01），两两比较差异均有统计学意义（P〈0．01）；将各组细胞凋亡率进行自然对数转换后分别为（1．52±0．04）％、（2．93±0．02）％、（3．39±0．02）％、（3．64±0．02）％、（3．86±0．02）％、（4．08±0．02）％、（4．20±0．02）％，各组间差异具有统计学意义（F=12456．7，P〈0．01），两两比较差异均有统计学意义（P〈0．01）；各组细胞检测到的cpm值分别为4655614-26801、10385764-29417、9376554-23197、7864324-20028、5242884-16338、4015664-15Objective To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand（hTRAIL） in vitro by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase （luc） gene （Ad-hTRAIL-luc）, in which luc was used as reporter gene. Methods A549 cells were transduced with the adenoviral vector encoding enhanced green fluorescent protein （EGFP） gene （Ad-EGFP） at variable muhiplicity of infection （ MOI）. Adenoviral transduction efficiency was determined 48 h later. A549 ceils were transduced with Ad-hTRAIL-luc at variable MOI, and the following tests were performed 48h later, respectively： the expressive ratio of hTRAIL and the apoptotic ratio of A549 cells were measured by flow cytometer; counts per minute （cpm） of luminescence were measured by scintillation counters. A549 cells were transduced with Ad-luc at variable MOI, and cpm of luminescence was measured by scintillation counters 48 h later. After A549 ceils were transduced with Ad- hTRAIL-luc, the expressive ratio of hTRAIL, the apoptotic ratio of A549 cells and cpm of luminescence were analyzed by one-way ANOVA. The positive ratio of EGFP and cpm of luminescence （Ad-luc） were analyzed by nonparametric ANOVA. Results After A549 cells were trausfected with Ad-hTRAIL-luc, the expressive ratio of hTRAIL on the cell membrane of the groups were （2. 37 ± 0. 04） %, （ 3.16 ±0. 03 ） %, （3.64± 0. 03 ） %, （3.96 ± 0. 02 ） %, （ 4. 24 ± 0.02 ） %, （ 4. 34 ± 0. 02 ） % respectively, which showed significant difference between each other （P 〈0. 01 ）; and the apoptotic ratio of A549 cells were （1.52 ±0. 04）%, （2.93±0.02）%,（3.39±0.02）%,（3.64±0.02）%,（3.86±0.02）%,（4.08±0.02）%,（4.20± 0.02）%, respectively, and it showed significant difference between each other （P 〈 0.01 ）; epm of were 465 561 ±26 801, 1 038 576±29 417, 937 655 ±23 197, 786 432 ±20 028, 524 288 ± 16 338,401 566±15 961, respectively, and it also s

关 键 词：基因表达调控 受体 肿瘤坏死因子 基因疗法 诊断显像 

分 类 号：R686[医药卫生—骨科学]