人端粒酶逆转录酶启动子-二步转录增强系统调控下的活性半胱氨酸天冬氨酸蛋白酶3的构建及其对卵巢上皮性癌的治疗作用  

Construction of autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase promoter and its enhanced efficacy of inducing apoptosis in human ovarian carcinoma

作　　者：宋悦[1] 沈铿[1] 何春霞[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院妇产科

出　　处：《中华妇产科杂志》2007年第9期617-622,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金(30271361)

摘　　要：目的构建含人端粒酶逆转录酶启动子-二步转录增强系统(hTERTp-TSTA)调控下的活性半胱氨酸天冬氨酸蛋白酶3(rev-caspase-3)的重组腺病毒 AdHTVP2G5-rev-casp3,并检测其对卵巢上皮性癌(卵巢癌)的治疗作用。方法采用基因重组法构建 AdHTVP2G5-rev-casp3,经 DNA 测序鉴定并证实其序列正确。实验分为4组:分别以 AdHTVP2G5-rev-casp3(AdHTVP2G5-rev-casp3组)、含 hTERTp 调控下的 rev-caspase-3的重组腺病毒 AdHT-rev-casp3(AdHT-rev-casp3组)和含人巨细胞病毒启动子(CMVp)调控下的 rev-caspase-3的重组腺病毒 Ad-rev-casp3(Ad-rev-casp3组)转染人卵巢癌细胞系 AO 细胞及正常人脐静脉内皮细胞系 HUVEC 细胞,以含 hTERTp-TSTA 调控下的增强型绿色荧光蛋白(EGFP)的重组腺病毒 AdHTVP2G5-EGFP(AdHTVP2G5-EGFP 组)为阴性对照。采用蛋白印迹法(western blot)、细胞计数法(CCK-8)、流式细胞术分别检测各组 AO 和 HUVEC 细胞中 caspase-3的活性单位 p17蛋白及其底物聚腺苷二磷酸-核糖多聚酶的裂解片段 p85蛋白的表达强度、细胞存活率、细胞凋亡率和细胞周期的变化;western blot 法检测各组裸鼠移植瘤及肝脏组织中 p17蛋白的表达强度,检测各组裸鼠生存率、肿瘤体积、体内肝酶水平及各器官组织的病理检查结果。结果 AO细胞中 p17蛋白表达强度,AdHTVP2G5-rev-casp3组明显高于 Ad-rev-casp3和 AdHT-rev-casp3组;而HUVEC 细胞中 AdHTVP2G5-rev-casp3组则明显低于 Ad-rev-casp3,与 AdHT-rev-casp3组相似。当病毒感染复数(MOI)为70时,AO 细胞的存活率和凋亡率,AdHTVP2G5-rev-casp3组(分别为17.8%和40.2%)与 AdHT-rev-casp3组(分别为75.2%和16.1%)比较,差异有统计学意义(P<0.01);而HUVEC 细胞的存活率和凋亡率,AdHTVP2G5-rev-casp3组(分别为97.7%和2.1%)与 AdHT-rev-casp3组(分别为98.5%和1.7%)比较,差异则无统计学意义(P>0.05)。荷瘤裸鼠肿瘤组织中 p17蛋白的表达强度,AdHTVP2G5-rev-casp3组最高,Ad-rev-casp3组次之,AdHT-rev-casp3�Objective To construct recombinant adenoviral vector expressing autocatalysis caspase- 3 driven by human telomerase reverse transcriptase promoter amplified by two-step transcription amplification （hTERTp-TSTA）, and investigate its antitumor effect in ovarian cancer in vitro and in vivo. Methods Recombinant adenoviruses expressing autocatalytic caspase-3 （rev-caspase-3） driven by hTERTp-TSTA were prepared, which were named as AdHTVP2G5-rev-casp3. AdHT-rev-casp3, Ad-rev-casp3 and AdHTVP2G5- EGEP, which express rev-caspase-3 driven by hTERTp, cytomegalovirus promoter （CMVp） and enhanced green fluorescent protein （EGFP）, respectively, were used as controls. Western blot, cell counting kit （CCK-8）, flow cytometry （FCM） and TdT-mediated dUTP-biotin nick end labeling （TUNEL） were used to detect the expression of p17, active subunit of caspase-3, and p85, and to measure cell survival rates, apoptotic rates and cell cycle distribution in ovarian cell line AO and normal human umbilical vein endothelial cell line HUVEC, following treatments of AdHTVP2G5-rev-casp3. subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/c nude mice were established. Following treatments of AdHTVP2G5-rev-casp3, western blot was used to detect the expression of active caspase-3 in abdominally spread tumors and liver tissues, respectively, and the mouse survival rates and the volume of tumor nodules were measured, and the serum level of alanine transaminase （ALT） and aspartate transaminase（AST） were analyzed to monitor liver damages and HE staining was used to detect the histopathological changes of various organs. Results The levels of p17 expression in AdHTVP2G5-rev-easp3-treated AO cells were significantly higher than that in Ad-rev-casp3 or AdHT-rev- casp3 treated AO cells, while no expression was observed in AdHTVP2G5-rev-casp3-treated HUVEC. There was strong cell killing of AdHTVP2G5-rev-casp3 of hTERT positive AO cells, but not of the hTERT-negat

关 键 词：卵巢肿瘤 端粒 末端转移酶 启动区(遗传学) 半胱氨酸天冬氨酸蛋白酶 

分 类 号：R73-3[医药卫生—肿瘤]