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  

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

作　　者：张雷[1] 刘彤华[1] 崔全才[1] 高洁[1] 

机构地区：[1]中国医学科学院病理科

出　　处：《中华病理学杂志》1999年第6期440-444,共5页Chinese Journal of Pathology

基　　金：卫生部科学研究基金!( 编号 981010)

摘　　要：目的　探讨用单纯疱疹病毒胸腺嘧啶核苷激酶（ＨＳＶＴＫ）／丙氧鸟苷（ＧＣＶ） 系统进行基因治疗时发生的旁观者效应机制。方法　构建表达ＨＳＶＴＫ和β半乳糖苷酶（Ｌａｃ Ｚ） 基因的重组逆转录病毒载体，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，噻唑蓝法检测旁观者效应，并通过上清移换实验、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抑制率达９２．１％ ，明显高于未转化细胞和对照转化细胞的４ ．９ ％ 和３ ．２％ （ 均Ｐ＜ ０．０１） ；混合细胞中只含１０％ 的ＨＳＶＴＫ转化细胞时，生长抑制率已达３９．０％ ，即存在明显的旁观者效应；ＧＣＶ 作用的ＰＣ２／ＮＴＫ 细胞上清对ＰＣ２细胞生长无影响，维拉帕米则对旁观者效应具有抑制作用，提示该效应需要细胞间接触；电镜观察可见细胞间存在缝隙连接。结论　ＧＣＶ代谢产物通过细胞间缝隙连接进行转运可能是ＨＳＶＴＫ／ＧＣＶ系统旁观者效应的主要机制。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 β 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.

关 键 词：胰腺肿瘤 基因治疗 HSV-TK 丙氧鸟苷 

分 类 号：R735.905[医药卫生—肿瘤]