HSVtk/GCV介导的旁观者效应对人膀胱癌的作用机理

HSVtk/GCV system-mediated bystander effect on human bladder cancer cells and its potential mechanism

目的 研究单纯疱疹病毒胸腺苷激酶基因 (HSVtk)联合丙氧鸟苷 (ganciclovir,GCV)对人膀胱癌细胞 (BIU87)的旁观杀伤作用 ,探讨旁观者效应产生的机制。方法 将携带HSVtk基因的细胞BIU87 TK与BIU87混合培养 ,观测HSVtk/GCV系统的旁观者效应。染料划痕标记观察细胞间缝隙连接功能 ;RT PCR检测连接蛋白Connexin2 6 (Cx2 6 )mRNA表达。同时观测维甲酸对缝隙连接和旁观者效应的影响。结果 当BIU87 TK占 4 0 %时 ,共培养细胞对GCV的杀伤敏感性接近 6 0 % ,显示出旁观者效应。大量细胞凋亡。划痕实验见染料传至临近细胞 ,RT PCR检测出Cx2 6mRNA表达 (0 .15 )。维甲酸处理后 ,染料传递至更多细胞 ,Cx2 6mRNA表达增加到 0 .34,HSVtk/GCV的旁观者效应随之提高。结论 HSVtk/GCV对人膀胱癌细胞具有旁观杀伤作用 ,其机理与细胞间缝隙连接和凋亡有关。维甲酸能增强细Cx2 6表达和缝隙连接功能 ,提高旁观者效应。

Objective To evaluate the bystander effect mediated by HSVtk/GCV system on the human bladder cancer cell line (BIU87) and the potential mechanisms of bystander effects.Methods HSVtk gene-tranduced BIU87 cells (BIU87-TK) were cocultured with parental BIU87. The bystander tumoricidal effect rate was monitoried. Scrap loading and dye transferring was used to evaluate the Gaq junction intercellular communication (GJIC) of BIU-87 cells. The mRNA of bladder cancer cells were evaluated by semi-quantity- RT-PCR, the effects of all trans retinoic acid (ATRA) on GJIC, expression of Cx26 and bystander effect mediated by HSVtk gene were also observed. Results When the proportion of BIU87-TK cells was about 40%, the co-cultured cells sensitive to GCV was about 60%. This indicated presence of bystander tumoricidal effect mediated by HSVtk/GCV. Many BIU-87 cells showed apoptosis. Scrap loading dye transfer technology demonstrated the presence of GJIC and semi-quantity-RT-PCR revealed that Cx26 of BIU87 cells was 0.15.After ATRA treatment, scrap loading dye transferred to much more cells and the expression of Cx26 increased to 0.34. In additions, ATRA enhanced the bystander effect mediated by HSVtk/GCV on BIU87.Conclusion HSVtk/ GCV system had bystander effect on BIU87. The mechanism of bystander effect was related to GJIC and cell apoptosis. ATRA can improve the GJIC function, increase the expression of Cx26 and enhance bystander effect.