活性半胱氨酸天冬氨酸蛋白酶3分子的构建及其致卵巢上皮性癌细胞凋亡的实验研究

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53 d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.

Construction of autocatalytic caspase-3 and its effects of inducing apoptosis in human ovarian carcinoma

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-rev-casp3 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 S-phase (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) significantly increased. Rev-caspase-3 treatment significantly prolonged survival, the mean survival duration was (213 +/- 16) days, and suppressed tumor growth (tumor growth suppression rate was 70%), when compared with treatment with phosphate buffered saline (PBS). CONCLUSION: Recombinant adenovirus containing rev-caspase-3 can significantly induce apoptosis of ovarian carcinoma cells, suppress tumor growth and prolong the mouse survival duration.