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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%,G_1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 G_1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。
Objective To construct an active caspase 3 molecule and explore its apoptosis-inducing effect on ovarian epithelial carcinoma (ovarian cancer) cells. Methods The active caspase-3 molecule -rev-caspase-3 and its recombinant adenovirus-Ad-rev-casp3 were constructed by gene recombination technique. The expression of caspase-3 was detected by immunohistochemistry, cell counting kit 8, flow cytometry and Western blot Method were used to detect the expression of active caspase-3 protein in ovarian cancer cell line AO cells after rev-caspase-3 treatment, cell survival rate, apoptosis rate, cell cycle, caspase-3 activity subunit p17 and poly Phospho-ribose) polymerase (PARP). The expression of p85 was detected by transmission electron microscopy. The expression of apoptosis-related genes was detected by real-time PCR. The establishment of ovarian cancer Nude mice model of subcutaneously and intraperitoneally transplanted tumor was observed after rev-caspase-3 the survival of nude mice and tumor volume changes. Results The activated caspase-3 protein was significantly expressed in AO cells after Ad-rev-casp3 treatment. The cytoplasm was obviously brown stained. The expression of active caspase-3 subunit p17 and PARP cleavage fragment p85 in cells increased. The survival rate of AO cells treated with Ad-rev-casp3 at a multiplicity of infection (MOI) of 70 was 30.3% and the apoptosis rate was 40.2%. The apoptotic rate of Ad-rev-casp3 treated with MOI = 10 was only 3.4%, but the percentage of cells in S phase reached 56.5% and the proportion of G1 phase decreased to 9.8% = 0), the proportion of S phase and G_1 phase in AO cells were significantly different (all P <0.05). AO cells treated with Ad-rev-casp3 exhibited significant apoptotic morphological changes and obvious apoptotic bodies formed under electron microscopy. The expression of active caspase-3 gene in cells was significantly increased to 9.44. Ad-rev-casp3 could significantly prolong the survival of tumor-bearing nude mice, with an average survival time of (213 ± 16) d and significantly inhibited the growth of transplanted tumor. The tumor inhibition rate was 53% at the first injection . Conclusion The recombinant adenovirus vector expressing rev-caspase-3 has a strong apoptotic effect on ovarian cancer cells, and can significantly prolong the survival of tumor-bearing nude mice and inhibit tumor growth.