hTERT启动子调控下HSV-tk/GCV系统对人卵巢癌细胞系Skov3的杀伤作用

Treatment of Ovarian Cancer Cell Line Skov3 with HSV-tk/GCV under the Control of Human Telomerase Reverse Transcriptase Gene Promoter

目的探讨hTERT启动子驱动下的HSV-tk/GCV系统对人卵巢癌细胞系Skov3的体外杀伤作用。方法应用分子生物学方法构建hTERT启动子调控下的tk基因真核表达载体pBTdel-279-tk,并应用脂质体转染法将之转入Skov3、正常人卵巢上皮细胞(NOEC)和人胚肺成纤维细胞中,加入GCV,用MTT法和流式细胞术检测pBTdel-279-tk/GCV系统对各种细胞的杀伤作用;应用RT-PCR法检测pcDNA3-tk和pBTdel-279-tk转染前后卵巢癌细胞和正常细胞中tk基因的表达情况。结果pBTdel-279-tk/GCV对端粒酶阳性的卵巢癌细胞有明显的杀伤作用,而对端粒酶阴性的正常细胞则无;hTERT启动子系统诱导卵巢癌细胞凋亡的效能与CMV启动子近似,两者差别无显著性意义。pcDNA3-tk转染后,在Skov3细胞和NOEC中均可探测到tk基因;而pBTdel-279-tk转染后只有Skov3可探测到tk基因,NOEC中则无。结论hTERT启动子调控下的tk基因治疗是一种新的卵巢癌靶向治疗方法,具有更高的特异性和高效性。

To investigate the in vitro effect of HSV-tk/GCV using a hTERT promoter-driven vector system on Skov3ovarian cancer cells.Methods An expression vector(pBTdel-279-tk)containing tk gene under the hTERT promoter was constructed by molecular biological methods,and then was transfected into Skov3ovarian cancer cells,normal ovarian epithelial cells(NOEC)and human embryonic lung fibroblast by cationic liposome.Following the transfection with tk,GCV was added,and MTT and flow cytometry methods were applied to investigate its antitumor effect.RT-PCR was used to detect the tk gene in ovarian cancer cells and normal cells after the transfection of pcDNA3-tk or pBTdel-279-tk.Results pBTdel-279-tk/GCV system induced apoptosis in hTERT-positive ovarian cancer cells,but not in hTERT-negative normal ovarian epithelial cells and fibroblasts.The hTERT promoter system was almost as efficient in inducing cancer cell death as the CMV promoter.tk gene was expressed in Skov3cells and NOEC after pcDNA3-tk transfection,while positive was only in ovarian cancer cells after pBTdel-279-tk transfection.Conclusion The telomerase specific transfer of the tk gene under the hTERT promoter is a novel targeting approach for the treatment of ovarian cancer and may lead to an effective and specific gene therapy.