半胱天冬酶3前酶增强胞嘧啶脱氨酶-胸苷激酶融合双自杀基因系统治疗人卵巢癌的体外研究  

Procaspase-3 enhances in vitro effect of cytosine deaminase-thymidine kinase fusion disuicide gene therapy system on human ovarian carcinoma

作　　者：宋悦[1] 孔北华[1] 马道新[2] 曲迅[1] 江森[1] 

机构地区：[1]山东大学齐鲁医院妇产科 [2]山东大学齐鲁医院肿瘤防治中心血液病研究室

出　　处：《中华妇产科杂志》2005年第6期411-416,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金资助项目(30271361)

摘　　要：目的探讨半胱天冬酶3前酶(procaspase3)能否增强胞嘧啶脱氨酶胸苷激酶/5氟胞嘧啶+更昔洛韦(CDTK/5FC+GCV)系统对人卵巢癌细胞的体外杀伤作用。方法构建procaspase3的表达载体pcDNA3casp3和人端粒酶逆转录酶(hTERT)启动子调控下的CDTK融合双自杀基因表达载体pBTdel279CDTK。应用蛋白印迹法检测pcDNA3casp3和pcDNA3质粒转染后的3AO细胞(3AOpcDNA3casp3、3AOpcDNA3细胞)中procaspase3蛋白的表达情况;应用RTPCR技术检测pBTdel279CDTK和pBTdel279分别转染的3AOpcDNA3casp3和3AOpcDNA3细胞中CD和TK基因的表达情况;应用四甲基偶氮唑蓝(MTT)法、流式细胞仪技术、蛋白印迹法和荧光底物分析法分别检测4种细胞在CDTK/5FC+GCV系统作用后的细胞存活率、细胞周期及半胱天冬酶3(caspase3)活性。结果3AOpcDNA3casp3细胞有明显的procaspase3蛋白表达,而3AOpcDNA3细胞则无表达。3AOpcDNA3casp3和3AOpcDNA3细胞在pBTdel279CDTK转染后均可检测到CD和TK基因,但在pBTdel279质粒转染后则CD和TK基因表达均为阴性。在5FC+GCV作用下,3AOpcDNA3casp3+pBTdel279CDTK细胞的存活率显著低于3AOpcDNA3+pBTdel279CDTK细胞。在5FC(2mmol/L)+GCV(10μg/ml)作用48h后,3AOpcDNA3casp3+pBTdel279CDTK细胞的凋亡率为37.98%,S期比例为49.67%,分别高于3AOpcDNA3+pBTdel279CDTK细胞的21.34%和35.76%,两者分别比较,差异有统计学意义(P<0.05)。在5FC(1mmol/L)+GCV(1μg/ml)作用48h后,3AOpcDNA3casp3+pBTdel279CDTK细胞中的caspase3活性值为189.7,高于3AOpcDNA3+pBTdel279CDTK细胞的44.9,两者比较,差异有统计学意义(P<0.05)。结论procaspase3可显著增强CDTK/5FC+GCV系统对人卵巢癌细胞的杀伤作用。Objective To investigate whether procaspase-3 could enhance the in vitro effect of cytosine deaminase-thymidine kinase/5-fluorocytosine+ganciclovir (CD-TK/5-FC+GCV) system in human ovarian cancer cells. Methods Eukaryotic expression vectors containing procaspase-3 gene (pcDNA3-casp3) and CD-TK fusion disuicide genes regulated by human telomerase reverse transcriptase (hTERT) promoter (pBTdel-279-CD-TK) were constructed. Western blot was used to detect the expression of procaspase-3 protein in 3AO cells with transfection of pcDNA3-casp3 (3AO-pcDNA3-casp3) or pcDNA3 (3AO-pcDNA3). RT-PCR was used to detect the expression of CD and TK genes in 3AO-pcDNA3-casp3 or 3AO-pcDNA3 cells after transfection with pBTdel-279-CD-TK or pBTdel-279. Following the treatment with 5-FC+GCV, the cell survival rate and cell cycle distribution were detected by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM). Fluorogenic substrate assay and western blot were used to detect caspase-3 activity in these cells. Results Procaspase-3 protein was expressed in 3AO-pcDNA3-casp3 cells, while not in 3AO-pcDNA3. The expressions of CD and TK genes were observed in both 3AO-pcDNA3-casp3 and 3AO-pcDNA3 after transfection with pBTdel-279-CD-TK. After treatment with 5-FC+GCV, the survival rate of 3AO-pcDNA3-casp3+pBTdel-279-CD-TK was significantly lower than that of 3AO-pcDNA3+pBTdel-279-CD-TK cells. After treatment with 5-FC (2 mmol/L) + GCV (10 μg/ml), there was a higher apoptotic ratio (37.98%) and S-phase block (49.67%) in 3AO-pcDNA3-casp3+pBTdel-279-CD-TK than in 3AO-pcDNA3+pBTdel-279-CD-TK cells (21.34% and 35.76%, respectively) (P<0.05). Following the action of 5-FC (1 mmol/L) + GCV (1μg/ml) for 48 h,the relative activity of caspase-3 in 3AO-pcDNA3-casp3+pBTdel-279-CD-TK cells was 189.7, significantly higher than 44.9 in 3AO-pcDNA3 +pBTdel-279-CD-TK cells (P<0.05). Conclusion Co-expression of procaspase-3 may lead to a significant enhancement of the efficacy of CD-TK fusion gene therapy.

关 键 词：胞嘧啶脱氨酶 半胱天冬酶3 胸苷激酶 双自杀基因 体外研究 系统治疗 caspase-3蛋白 融合 人端粒酶逆转录酶 人卵巢癌细胞 CD-TK PCR技术检测 四甲基偶氮唑蓝 流式细胞仪技术 蛋白印迹法 GCV系统 体外杀伤作用 5-氟胞嘧啶 

分 类 号：R737.31[医药卫生—肿瘤]