腺病毒介导的HSV-tk/GCV系统在hTERT启动子调控下治疗人膀胱癌的实验研究

Studies on adenovirus-mediated HSV-tk/GCV gene therapy controlled by hTERT promoter for human bladder cancer

目的观察人端粒酶逆转录酶(hTERT)启动子调控的腺病毒介导单纯疱疹病毒胸苷激酶(HSV-tk)基因/丙氧鸟苷(GCV)系统在体外及荷瘤裸鼠体内对人膀胱癌的治疗作用。方法利用hTERT启动子及小鼠巨细胞病毒(CMV)启动子调控的携带HSV-tk基因的重组腺病毒(Ad-hTERT-HSV/tk与Ad-CMV-HSV/tk)分别感染人膀胱癌细胞253 J和人正常肺成纤维细胞MRC-5,加入GCV,MTT法观察受染细胞的存活率。建立人膀胱癌裸鼠移植瘤模型,随机分为4组,A组(Ad-hTERT-HSV/tk+GCV组)、B组(Ad-hTERT-HSV/tk+PBS组)、C组(PBS+GCV组)和D组(PBS对照组),观察各组肿瘤生长状况、重要脏器病理变化及裸鼠存活情况。结果体外实验表明,应用GCV处理后,Ad-CMV-HSV/tk对253 J和MRC-5细胞均有杀伤作用,而Ad-hTERT-HSV/tk只对膀胱癌细胞253 J具有杀伤作用。动物实验显示,A组移植瘤的体积和瘤重低于其他3组(P<0.01)。A组裸鼠平均存活期长于其他3组(P<0.01)。结论 hTERT启动子调控的重组腺病毒介导HSV-tk/GCV系统是一种安全、有效且靶向性高的基因疗法。

Objective To explore the selective killing effect of adenovirus-mediated simplex thymidine kinase（HSV-tk）/GCV gene therapy controlled by human telomerase reverse transcriptase（hTERT） promoter on human bladder cancer in vitro and in vivo.Methods Ad-hTERT-HSV/tk and Ad-CMV-HSV/tk were transfected into 253J and MRC-5 followed by GCV treatment.The relative survival rate of cells in presence of prodrug GCV was measured with MTT method.When the transplanted tumor models of human bladder cancer had been established,nude mouse were divided into 4 groups： group A（Ad-hTERT-HSV/tk＋GCV）,group B（Ad-hTERT-HSV/tk＋PBS）,group C（PBS＋GCV） and group D（control）.The volume and weight of tumor sizes were measured;Average survival time of nude mice was observed.Results In vitro,GCV could inhibit significantly the growth of both cell lines infected with Ad-CMV-HSV/tk and the growth of 253J infected with Ad-hTERT-HSV/tk.In vivo,the growth rate of the tumor in nude mice of group A decreased significantly compared those in nude mice of group B,C and D.Average survival time of nude mice in group A was longer than that in group B,C and D.Conclusion Adenoviral vector containing hTERT promoter for the HSV/tk plus GCV treatment may be a potential and safe protocol for clinical use.