胸腺嘧啶核苷激酶/丙氧鸟苷自杀基因系统旁观者效应机制的探讨

目的 探讨用单纯疱疹病毒胸腺嘧啶核苷激酶／丙氧鸟苷系统进行基因治疗时发生的旁观才效应机制。方法 构建表达ＨＳＶ－ＴＫ和β－半乳糖苷酶基因的重级逆转录病毒载体,将其分别直接导入胰腺癌细胞,细胞计数检测ＧＣＶ对ＨＳＶ－ＴＫ基因转化细胞的抑制作用;以Ｌａｃ Ｚ基因转化细胞为旁观细胞,唑蓝法检测旁观者效应,并通过上清移换实验１维拉帕米抑制实验及电镜观察超微结构。结果 ＧＣＶ对ＨＳＶ－ＴＫ基因转化细胞的生长

The mechanism of bystander effect in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy

OBJECTIVE: To study the mechanism of bystander effect in herpes simplex virus thymidine kinase (HSV-TK)/Ganciclovir (GCV)-mediated gene therapy. METHODS: Recombinant retroviral vectors expressing HSV-TK and beta-Galactosidase (Lac Z) genes were constructed and transferred into pancreatic carcinoma cell line respectively. Cell counting was used to detect the growth inhibition rate of HSV-TK-transduced cells in presence of GCV. Taking Lac Z-transduced cells as bystander cells, the bystander effect was detected by MTT method, and its mechanism was studied by the experiments of supernatant shifting, Verapamil inhibition and ultrastructural observation. RESULTS: The growth inhibition rate of the HSV-TK-transduced cells in the presence of GCV was 92.1%, which was obviously 4.9% and 3.2% higher than of the non-and control vector-transduced cells. Mixed cells containing only 10% of HSV-TK-transduced cells showed 39.0% reduction of the proliferation, which meant there was an obvious bystander effect in the system. However this effect disappeared when transferring GCV-containing supernatant of HSV-TK-transduced cells to the parent cells and could be reduced significantly when verapamil was added in the medium, indicating that this bystander effect requires cell-cell contact. Gap junctions were observed existing between PC-2 cells by electron microscopy. CONCLUSION: The bystander effect in HSV-TK/GCV-mediated gene therapy occurs by transfer of GCV metabolite from cell to cell through gap junction.