荧光素酶光成像实时监测诱导凋亡配体基因治疗裸鼠肺癌A549细胞移植瘤的实验研究  

Luciferase bioluminescence imaging monitoring gene therapeutic effect of apoptosis-inducing ligand for lung cancer A549 cells nude mice transplantation tumor in vivo

作　　者：曹宏伟[1] 崔建岭[1] 赵纳[1] 郭志远[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》2012年第9期840-845,共6页Chinese Journal of Radiology

基　　金：河北省自然科学基金《活体基因磁共振成像的技术和机理研究》资助（303473）

摘　　要：目的应用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，活体内监测hTRAIL基因的表达及作用。方法皮下接种法建立肺癌A549细胞裸鼠移植瘤模型，瘤内多点注射腺病毒载体ad—luc．hTRAIL、ad—hTRAIL、ad．luc。对照组注射磷酸盐缓冲液（PBS）。于注射后4、7、10、14、21、28d动态测量各组肿瘤大小，并利用高敏感、超低温（CCD）照相机进行活体生物发光成像检测luc活性，然后处死动物，免疫组织化学（简称免疫组化）法检测各组hTRAIL蛋白表达情况，并进行免疫组化评分（IHS）；TUNEL法检测各组肿瘤细胞凋亡情况，并计算凋亡率。统计分析组间比较采用方差分析，两样本比较采用配对t检验。对ad-luc-hTRAIL组的发光光子数、免疫组化评分、凋亡率3种结果进行线性相关分析。结果ad．1uc—hTRAIL与ad—hTRAIL组肿瘤生长速度明显较PBS组缓慢（t=2．71、2．72，P〈0．05），28d后各组肿瘤体积分别为（208．4±42．3）、（181．5±23．9）、（427．0±59．3）mm2，ad—luc组与PBS组肿瘤生长差异无统计学意义（t=2．07，P〉0．05）。ad—luc—hTRAIL组luc活性于治疗后第7天达高峰（1．37±1．04）后快速下降。ad—luc—hTRAIL与ad．hTRAIL2组在注射载体后第7天hTRAIL表达及肿瘤细胞凋亡率达高峰，IHS峰值分别为6．25±2．06和6．5±2．89，肿瘤细胞凋亡率峰值分别为（60．75±8．06）％和（61．50±8．47）％。ad-luc—hTRAIL组lue表达量（绝对光子数）与IHS、肿瘤细胞凋亡率三者间均呈线性正相关（r光子影IHs=0．942，r光子数／凋亡率=0．842，rIHS/凋亡率=0．887，P值均〈0．05）。结论目的基因可以有效地通过ad—luc—hTRAIL转导入裸鼠肺癌A549细胞移植瘤内并高水平表达，发挥生长抑制和促凋亡作用，体外CCD照相�Objective To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand （hTRAIL） in vivo, by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase （ lue ） gene （ ad -luc-hTRAIL） , in which luc was used as reporter gene. Methods Lung cancer A549 cell xenografts in 16 nude mice models were established in subcutaneous inoculation way, the adenovirus vectors （ ad-luc-hTRAIL, ad-hTRAIL, ad-luc） and phosphate buffer saline （PBS） （n = 4） as control were injected into tumor respectively. The size of the tumor was measured at different time points （4,7,10,14,21,28 d） after injection. The activity of luciferase in surface of the tumor was detected in vivo by using high-sensitivity cooled-charged coupled device （CCD） camera. The expression of hTRAIL was demonstrated by immunohistochemistry staining after sacrificing the animals at different time points, and immunohistochemical scores （IHS） were measured. The apoptosis rate of tumor cells was detected by using TUNEL and calculated. Analysis of variance, the paired t test and linear correlation analysis was used for the statistics. Results The growing speed of tumour xenografts was more slowly in ad-luc-hTRAIL and ad-hTRAIL groups than PBS group （ t = 2. 71,2. 72, P 〈 0. 05 ）. The tumor volumes of ad-luc-hTRAIL, ad-hTRAIL, ad-luc and PBS groups 28 days after injection were （208.4 ± 42. 3 ）, （ 181.5 ± 23.9）, （ 403.1 ± 54. 0 ） and （427. 0 ± 59. 3 ） mm3, respectively. There was no significant difference between ad-luc group and PBS group（t =2. 07, P 〉 0. 05）. The expression of luciferase in ad-luc-hTRAIL group reached its peak at 7th day （ 1.37 ± 1.04） , and then decreased quickly. The IHS and apoptosis rate in ad-luc-hTRAIL and ad-hTRAIL groups reached their peaks at 7th day, the peak values of IHS were 6. 25 ± 2. 06 and 6. 5 ± 2. 89, the peak values of apoptosis rate were （60. 75 ± 8.06 ） % and （ 61.50 ± 8.47 ） %, respec

关 键 词：发光蛋白质类 肿瘤坏死因子类 荧光素类 基因 

分 类 号：R734.2[医药卫生—肿瘤]