条件复制型腺病毒联合HSVtk/GCV自杀基因系统对视网膜母细胞瘤细胞的杀伤性研究

An in vitro study of a conditionally replicative adenovirus delivering the herpes simplex virus thymidine kinase suicide gene in retinoblastoma

目的 研究条件复制型腺病毒（CRAd）联合单纯疱疹病毒胸苷激酶（HSVtk）/更昔洛韦（GCV）自杀基因系统对人视网膜母细胞瘤（RB）细胞系Y79细胞的杀伤作用.方法 实验研究.将携带HSVtk的由人端粒酶逆转录酶（hTERT）启动子调控腺病毒复制必需基因E1A的CRAd,即Ad-hTERT-E1A-CMV-HSVtk感染Y79细胞和原代培养的人视网膜色素上皮（hRPE）细胞.通过测量Y79和hRPE细胞感染病毒后0 h、24 h、48 h和72 h时的病毒滴度,观察CRAd在Y79细胞中的选择性复制情况.CRAd联合HSVtk/GCV自杀基因系统对Y79细胞的杀伤实验分为5组：分别为细胞未经GCV和腺病毒处理的阴性对照组,感染野生型腺病毒dl309的阳性对照组,单独应用GCV组,单独应用CRAd组及CRAd联合GCV处理组.以1、10、50和100感染复数（MOI）的CRAd或dl309感染Y79细胞.病毒感染48 h后,培养液换成含10 μg/ml的GCV或不含GCV的新鲜培养液.细胞继续培养3 d后分别以生物发光成像（BLI）和CCK-8方法检测各组Y79细胞的生物发光强度和细胞存活率.采用单因素方差分析对各组细胞的生物发光强度和存活率进行比较.结果 在Y79细胞感染CRAd后的72 h内,病毒量呈指数级增加,较感染前增加了 100多倍.CRAd在hRPE细胞中无明显复制.BLI和CCK-8检测均发现,在低剂量（MOI≤10）时,CRAd及其联合HSVtk/GCV自杀基因系统对Y79细胞无明显的杀伤效应.在高剂量（MOI≥50）时,CRAd对Y79细胞有一定的杀伤作用,其联合HSVtk/GCV后,杀伤作用更明显.在50和100 MOI时,CRAd可分别导致大约20%和30%的细胞死亡,其联合GCV后,可分别导致50%和80%的细胞死亡.结论 CRAd可在Y79细胞中选择性大量复制,其联合HSVtk/GCV自杀基因系统对Y79细胞有明显的杀伤作用.

Objective To investigate the cytotoxicity of a conditionally replicative adenovirus （CRAd） combined with the herpes simplex virus thymidine kinase （HSVtk）/ganciclovir （GCV） suicide gene system delivered to Y79 cells. Methods Experimental study. Selective replication of CRAd armed with HSVtk, i.e., Ad-hTERT-E1A/CMV-HSVtk, in Y79 cells was observed after Y79 and human retinal pigment epithelial （hRPE） cells were infected with CRAd at 0 h, 24 h, 48 h and 72 h.Y79 cells were divided into 5 groups, an untreated group, a GCV treatment group, a CRAd treatment group, a dl309 group and a CRAd plus GCV treatment group, for studying the cytotoxicity of CRAd combined with HSVtk/GCV system. At 48 h post infection with the viruses, the medium of the cells was replenished with a culture medium with or without 10 μg/ml of GCV. At 3 days post infection with the viruses, the cytotoxicity of CRAd combined with the HSVtk/GCV suicide gene system delivered to Y79 cells was observed with bioluminescent imaging （BLI） and the Cell Counting Kit-8 （CCK-8）. Results CRAd could selectively replicate in Y79 cells. At 72 h post infection, viral titer increased by more than 100 fold in Y79 cells infected with CRAd. CRAd showed no cytotoxicity to Y79 cells at doses of no more than 10 MOI. It showed cytotoxicity to Y79 cells with about 20%and 30% of cell death with viral doses at 50 and 100 MOI, respectively. When combined with GCV, the cytotoxic effect of CRAd was amplified significantly. CRAd plus GCV could lead to about 50% and 80% of Y79 cell death with viral doses at 50 and 100 MOI, respectively. Conclusion CRAd has the ability of tumor-specific replication in Y79 cells. The combination of CRAd with HSVtk/GCV can effectively lyse Y79 cells in vitro.