腺病毒介导HSV-tk/GCV系统诱导ScaBER细胞凋亡的实验研究

目的 HSV-tk／GCV系统体外诱导膀胱癌队ScaBER细胞凋亡的作用，探讨其治疗膀胱癌的机制。方法 采用了带有CMV(人巨细胞病毒早期蛋白启动子)启动子驱动HSV-tk基因的重组腺病毒载体，体外转染ScaBER细胞后加入不同浓度更昔洛韦(GCV)，MTT法检测对癌细胞生长的抑制作用。应用倒置显微镜、荧光显微镜、透射电镜观察细胞凋亡的形态学，流式细胞仪定量检测细胞凋亡的查分率，琼脂糖凝胶电泳检测DNA断裂片断。结果 当MOI＝100时，MTT法表明HSV-tk／QCV系统作用于ScaBER细胞，能明显抑制细胞生长，呈时间、剂量依赖性。ScaBER细胞的IC50为3．23mg/L。透射电镜下可见凋亡小体。DNA琼脂糖凝胶电泳呈梯形条带。结论 HSV-tk／QCV系统对细胞有较强细胞毒作用，并可以诱导ScaBER细胞凋亡，诱导细胞凋亡是其抗肿瘤的主要机制之一。HSV-tk／GCV系统是一种治疗膀胱癌的新方法。

Study of apoptosis induced by adenoviral-mediated gene therapy for bladder cancer cell line ScaBER

Objective To study the induction of apoptosis of human bladder cancer cell line ScaBER treated with HSV-tk/GCV system.Methods We used the replication-defective adenovirus carrying the HSV-tk gene (Ad -tk)under control of the cytomegalovirus(CMV).In vitro experiments,we analysed the GCV inhabitation to ScaBER cell growth by MTT assay,observed morphological aspects of apoptosis by inverted biological microscope,fluorescent microscope and electronmicroscope,assessed apoptosis rate by flow cytometric method,and identified the DNA break by agarose gel electronphoresis.Results When MOI was 100,HSV-tk/GCV system significantly inhibited growth of ScaBER cell,and the inhibition was dose-dependent and timedependent.The IC 50 of GCV for ScaBER was 3 23mg/L.Apoptotic body was observed under electron microscope.Agarose gel electronphoresis of DNA revealed a ladder pattern.FCM not only showed apoptotic peak,but also helped estimate the rate of apoptosis.Conclusion Induction of apoptosis is one of the mechnisms of HSV-tk/GCV system anti-tumor effect.