自杀基因单纯疱疹病毒胸腺嘧啶激酶对鼻咽癌细胞株杀伤作用机制的研究

目的探讨自杀基因单纯疱疹病毒胸腺嘧啶激酶（HSV-tk）对鼻咽癌（NPC）细胞株的杀伤作用机制.方法将HSV-tk基因通过脂质体法转染NPC细胞株,经G418筛查挑选阳性细胞克隆并经逆转录聚合酶链反应（R-PCR）鉴定,加入前体药物戊环鸟苷（GCV）后,观察其对NPC细胞的杀亡状况.结果MTT法显示转染的NPC细胞株在加入GCV后,成活率明显降低,与未转染基因的NPC细胞株相比,差异有显著性（P＜0.01）.流式细胞仪显示与未加GCV的细胞相比,转染基因的细胞加入GCV后生长受到明显抑制,S期细胞数从占总细胞数的35.71%降至17.41%,凋亡率从1.33%增长至8.15%,并出现明显的凋亡峰.电镜下,转染细胞经GCV作用后大量细胞发生凋亡,可观察到凋亡细胞内特征性的凋亡小体.结论自杀基因HSV-tk对NPC有明显的杀伤作用,其可能性机制是HSV-tk基因联合GCV,对NPC细胞产生选择性细胞毒性作用,抑制肿瘤细胞生长和诱导肿瘤细胞凋亡.

Mechanisms of antitumor effects of herpes simplex virus-thymidine kinase suicide gene on nasopharyngeal carcinoma cells

Objective To appraise the antitumor effects of herpes simplex virus-thymidine kinase(HSV-tk) gene/ganciclovir(GCV) systems on nasopharyngeal carcinoma cells. Methods HSV-tk gene was transfected into nasopharyngeal carcinoma cells. The transfected cells were selected by G418 and the expression of HSV-tk gene was detected by RT-PCR and adding GCV to observe the killing effect. Results MTT method was used to evaluate the antitumor effect of the suicide gene and reveal the survival rate of the transfected cells which was less than that of the untransfected cells, the difference was significant (P<0.01). The cell cycle change and apoptosis ratio were studied by using flow cytometry(FCM). After given GCV, the S phase cell was 17.41% and the ratio of the apoptotic cells was 8.15% with significant apoptotic peak. In the controls without GCV, the S phase cell was 35.71% and the ratio of the apoptotic cells was only 1.33%. Abundant apoptotic bodies were observed in the transfected cells with GCV under electronmicroscope, some apoptotic cells could be seen in apoptotic body. Conclusions Suicide gene HSV/tk has strong antitumor effect on nasopharyngeal carcinoma cells and the selective toxic effects of HSV-tk/GCV with inhibiting tumor cell growth and inducing apoptosis are possible the involved mechanisms.