人端粒酶逆转录酶启动子调控下活性半胱氨酸蛋白酶-3治疗人卵巢癌  

The efficacy of autocatalytic casapse-3 driven by human telomerase reverse transcriptase promoter on human ovarian carcinoma

作　　者：宋悦[1] 沈铿[1] 于靖蓉[1] 

机构地区：[1]中国医学科学院北京协和医院妇产科

出　　处：《中华医学杂志》2007年第41期2919-2924,共6页National Medical Journal of China

摘　　要：目的构建人端粒酶逆转录酶启动子(hTERTp)调控下的活性半胱氨酸蛋白酶-3(caspase-3)重组腺病毒载体并检测其对人卵巢癌的治疗作用。方法构建表达 hTERTp 调控下活性caspase-3的重组腺病毒载体 AdHT-rev-casp3;以 CMV 启动子调控下活性 caspase-3的重组腺病毒载体Ad-rev-casp3为对照,分别应用 Western 印迹、细胞计数试剂盒(CCK-8)法、流式细胞术和 TUNEL 检测AdHT-rev-casp3作用后卵巢癌细胞系 AO 及正常人脐静脉内皮细胞 HUVEC 中活性 caspae-3蛋白 p17和聚腺苷二磷酸-核糖多聚酶(PARP)裂解片段 p85的表达水平、细胞存活率和凋亡率;建立裸鼠人卵巢癌皮下及腹腔移植瘤模型,Western 印迹检测 AdHT-rev-casp3作用后裸鼠肿瘤及肝脏组织中活性caspase-3的表达情况,观测作用前后裸鼠生存率及肿瘤体积的变化以及裸鼠体内肝酶 ALT 和 AST 水平。结果 AO 细胞在 AdHT-rev-casp3(MOI=70)感染后明显表达活性 caspase-3蛋白 p17和 PARP 的裂解片段 p85蛋白,细胞存活率为55%,凋亡率为26%,而 HUVEC 在 AdHT-rev-casp3感染后无明显p17和 p85表达,细胞存活率和凋亡率与阴性对照组差异无统计学意义;AdHT-rev-casp3作用后的裸鼠肿瘤组织中明显表达活性 caspase-3,而肝脏组织中则无表达;AdHT-rev-casp3能明显延长荷瘤裸鼠的生存期[(177±12)d vs(106±11)d],抑瘤率为60%,其作用后裸鼠体内的肝酶水平无明显增高。结论 hTERTp 系统调控下 rev-caspase-3的重组腺病毒 AdHT-rev-casp3同时具有较强的致细胞凋亡能力和肿瘤靶向性,能明显抑制卵巢癌移植瘤的生长,延长荷瘤裸鼠的生存期,并明显降低 rev-caspase-3对肝脏的毒性作用。Objective To construct recombinant adenoviral vector expressing autocatalysis caspase-3 driven by human telomerase reverse transcriptase promoter （ hTERTp）, and investigate its antitumor effect on ovarian cancer in vitro and in vivo. Methods Recombinant adenovirus expressing autocatalytic caspase- 3 （rev-csapase-3） driven by hTERTp, AdHT-rev-casp3, was constructed. Ad-rev-casp3 expressing revcaspase-3 driven by cytomegalovirus promoter （CMVp） was used as a positive control, hTERT positive human ovarian cancer cells of the line AO and hTERT-negative human umbilical venous endothelial cells （HUVECs） were cultured and transfected with AdHT-rev-casp3 , Ad-rev-casp3, or Ad-EGFG expressing enhanced green fluorescent protein as control group. Western blotting, Cell Counting Kit （ CCK-8 ）, flow cytometry, and TUNEL were used to detect the expression of p17, active subunit of caspase-3, and p85, a poly ADP-ribcse polymerase （PARP） cleavage fragment, and they were also used to measure the cell survival rate and apoptotic rate. Western blotting was used to detect the expression of active caspase-3 and its substrate PARP in the AO cells and HUVECs. Twenty nude BALB/c mice were inoculated subcutaneously with AO cells to establish subcutaneous tumor models, when the tumor grew to the volume of 150 mm^3 the rats were divided into 4 equal groups to undergo intra-tumor injection of AdHT-rev-casp3, Ad-rev-casp3, Ad-EGFG, and phosphate-buffered saline （PBS） respectively, the survival rate tumor inhibition rate was observed, 72 days later the mice were killed with their livers and tumors taken out, and Western blotting was used to detect the expression of active caspase-3. Another 40 mice underwent intraperitoneal injection of AO cells to establish intraperitoneal transplanted tumor models, 21 days later the rats were divided into 4 equal groups to be injected intraperitoneally with AdHT-rev-casp3, Ad-rev-casp3, Ad-EGFG, or PBS, the survival rate was observed, and the blood levels of alanine transaminase

关 键 词：端粒 末端转移酶 启动区(遗传学) CASPASE类 卵巢肿瘤 

分 类 号：R737.31[医药卫生—肿瘤]