中文摘要：

目的构建能自我激活的活性半胱氨酸天冬氨酸蛋白酶（caspase）3分子，并探讨其对卵巢上皮性癌（卵巢癌）细胞的致凋亡作用。方法利用基因重组技术构建活性caspase-3分子——rev-caspase-3及其重组腺病毒——Ad-rev-casp3；应用免疫组化方法、细胞计数试剂盒8、流式细胞仪和蛋白印迹法分别检测rev-caspase-3作用后卵巢癌细胞系AO细胞中活性caspae-3蛋白的表达情况、细胞存活率、凋亡率、细胞周期、caspase-3活性亚单位p17和聚（腺苷二磷酸-核糖）多聚酶（PARP）的分解产物p85的表达水平，应用透射电镜观察rev-caspase-3作用后细胞的超微结构，实时PCR技术检测细胞中凋亡相关基因的表达情况；建立卵巢癌裸鼠皮下及腹腔移植瘤模型，观测rev-caspase-3作用后裸鼠生存情况及肿瘤体积的变化。结果Ad-rev-casp3作用后的AO细胞中显著表达活性caspase-3蛋白，细胞质明显棕染；细胞中活性caspase-3亚单位p17和PARP裂解片段p85的表达水平升高。Ad-rev-casp3以感染复数（MOI）为70作用后的AO细胞存活率为30．3％，凋亡率为40．2％。Ad-rev-casp3以MOI=10作用后的细胞凋亡率只为3．4％，但此时S期细胞比例高达56．5％，G1期比例下降至9．8％，与未感染Ad—rev—casp3（MOI=0）的AO细胞的S期和G1期比例显著不同（P均〈0．05）。Ad-rev—casp3作用后的AO细胞呈现显著的凋亡形态改变，电镜下见明显的凋亡小体形成；细胞中活性caspase-3基因的表达水平显著升高，为9．44。Ad—rev—casp3能显著延长荷瘤裸鼠的生存期，平均生存时间为（213±16）d，并显著抑制移植瘤的生长，在第1次注射后53d时抑瘤率为70％。结论表达rev—caspase-3的重组腺病毒载体对卵巢癌细胞有强烈的致凋亡作用，并可显著延长荷瘤裸鼠的生存期，抑制肿瘤生长。

英文摘要：

Objective To construct the autocatalytic caspase-3 and investigate its apoptosis-inducing effect in ovarian cancer in vitro and in vivo. Methods PCR recombination technique was used to construct autocatalytic caspase-3 which is named as rev-caspase-3, and Ad-Max system was used to prepare recombinant adenovirus containing rev-caspase-3, which is named as Ad-rev-casp3. Immunohistochemistry was used to detect active caspase-3 expression. Cell counting kit, flow cytometry and western blot were used to measure cell survival rate, apoptotic rate, cell cycle distribution and the expressions of p17, active subunit of caspase-3, and p85, the poly （adenosine diphosphate-ribose） polymerase （PARP） cleavage segment, respectively. Transmission electron microscope was used to detect cell ultrastructure, and real time PCR was used to detect apoptosis-related gene expression. Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma were established using AO cells in BALB/c nude mice. The mouse survival rates were measured for abdominally spread tumor models, and the volume of tumor nodules were determined for subcutaneous tumor models following the treatments of rev-caspase-3. Results Active caspase-3 protein was significantly expressed, and the expression levels of active subunit of caspase- 3, p17, and the PARP cleavage segment, p85, were significantly elevated in cells treated with rev-caspase- 3. The decrease of cell survival rate and the increase of cell apoptotic rate were detected following Ad-revcasp3 treatment. Treatments with Ad-rev-casp3 [ multiplicity of infection （MOI） was 70 ] resulted in survival rate of 30. 3% and apoptotic rate of 40. 2%. There was a significant increase in cell number of Sphase （56. 5% ）, while there was no significant apoptosis （3.4%） following treatments with Ad-rev-casp3 at a low dosage of MOI = 10. Cells treated with rev-caspase-3 displayed significant apoptotic morphology. The levels of active caspase-3 gene expressions （9.44） signifi