论文部分内容阅读
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 a recombinant adenovirus vector carrying human telomerase reverse transcriptase promoter (hTERTp) and to evaluate its therapeutic effect on human ovarian cancer. Methods Recombinant adenovirus vector AdHT-rev-casp3 expressing caspase-3 under the control of hTERTp was constructed. Ad-rev-casp3, a recombinant adenovirus carrying active caspase-3 regulated by CMV promoter, was used as a control. Western blotting, Counting kit (CCK-8), flow cytometry and TUNEL detection of AdHT-rev-casp3 role in ovarian cancer cell line AO and normal human umbilical vein endothelial cells HUVEC active caspase-3 protein p17 and poly adenosine diphosphate The expression of p85, cell survival rate and apoptosis rate of the ribose polymerase (PARP) cleavage fragment were determined. The subcutaneous and abdominal xenograft model of human ovarian cancer was established in nude mice. Western blotting was used to detect the tumor and liver tissue of nude mice after AdHT-rev-casp3 treatment In active caspase-3 expression, observed before and after the role of survival and tumor volume changes in nude mice and nude mice liver enzymes ALT and AST levels. Results AO cells express p87 protein of active caspase-3 protein p17 and PARP after infection with AdHT-rev-casp3 (MOI = 70), the cell survival rate was 55% and the apoptosis rate was 26% The expression of p17 and p85 was not significant after infection with -rev-casp3, the cell survival rate and apoptosis rate were not significantly different from the negative control group. The expression of active caspase-3 in the tumor tissues of AdHT-rev-casp3- However, the expression of AdHT-rev-casp3 in tumor-bearing nude mice was significantly prolonged ([177 ± 12] d vs (106 ± 11) d], with a tumor inhibition rate of 60% Rat liver enzyme levels were not significantly increased. Conclusion The recombinant adenovirus AdHT-rev-casp3 of rev-caspase-3 under the regulation of hTERTp system has strong ability of inducing apoptosis and tumor targeting, and can significantly inhibit the growth of ovarian cancer xenografts and prolong the tumor-bearing nude mice Of the survival, and significantly reduce the toxic effects of rev-caspase-3 on the liver.