HSV_1-TK/GCV系统作为肿瘤疫苗“死亡开关”的研究  

Herpes Simplex Virus Type 1 Thymidine Kinase/Ganciclovir (HSV1-TK/GCV) System As an Effective "In Vivo Death Switch" of Live Tumor Vaccines

作　　者：康玉[1] 徐丛剑[1] 刘惜时[1] 吴超群[2] 钟翠平[3] 顾健人[4] 

机构地区：[1]复旦大学附属妇产科医院妇产科,上海200011 [2]复旦大学遗传工程国家重点实验室,上海200433 [3]复旦大学上海医学院组胚解剖系,上海200032 [4]上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032

出　　处：《癌症》2005年第8期909-914,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金项目(No.30070783);上海市重大科技攻关项目(No.03DZ19234)~~

摘　　要：背景与目的:肿瘤活疫苗有高效的抗肿瘤免疫能力,但其体内增殖性限制了进一步的临床应用。本研究将Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶/丙氧鸟苷(HSV1鄄TK/GCV)系统作为肿瘤活疫苗的“死亡开关”,以期控制疫苗在体内的存活状态,并探讨其作用机制。方法:以逆转录病毒为载体将HSV1鄄TK基因转导入大鼠卵巢癌细胞株NuTu鄄19,经G418筛选出阳性克隆后与Fischer344大鼠骨髓来源的树突细胞融合,制成携带有HSV1鄄TK基因的卵巢癌DC活疫苗(FC/TK)。以RT鄄PCR和Westernblot检测FC/TK细胞中的HSV1鄄TK表达。体外以XTT法检测不同浓度GCV对FC/TK的体外杀伤作用;以流式细胞仪和Hoechst染色法检测GCV作用5天后FC/TK的凋亡现象。体内实验中,取大鼠经足垫皮下接种FC/TK后分为两组:FC/TK+GCV组大鼠再予GCV腹腔注射7天;FC/TK+生理盐水组作为对照组。接种FC/TK后观察90天,记录注射部位有无肿瘤形成及各脏器的肿瘤转移情况。结果:FC/TK中有HSV1鄄TK的表达,在体外GCV对FC/TK的杀伤率可达86.25%,其中大于80%的FC/TK发生凋亡,并出现细胞核浓缩、边集等凋亡现象。FC/TK+生理盐水组共有3只(60%)的大鼠注射部位出现低分化腺癌;而FC/TK+GCV组未见局部肿瘤形成及脏器转移。结论:HSV1鄄TK/GCV通过诱导肿瘤活疫苗发生凋亡而起到控制其存活状态的作用。BACKGROUND ＆ OBJECTIVE： Live tumor vaccines could achieve better immunotherapeutic effects than irradiated tumor vaccines；however, the tumorigenicity is the crucial drawback incurred by the current procedures for vaccine preparation. This study was to explore the application of herpes simplex virus type 1 thymidine kinase/ganciclovir （HSV1-TK/GCV）system as “in vivo death switch” to control the survival status of cancer vaccines under certain circumstances. METHODS： Suicide gene HSV1-TK was transferred into ovarian cancer cell line NuTu-19 with a retrovirus vector,followed by G418 selection to obtain HSV1-TK-transfected NuTu-19 cells（NuTu-19/TK）. Dendritic cells （DCs） derived from Fischer344 rat bone marrow were fused with NuTu-19/TK cells to construct live tumor vaccine FC/TK. The expression of HSV1-TK in FC/TK cells was determined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The cytotoxic efficacy of GCV on FC/TK cells was evaluated by XTT assay. The apoptosis of FC/TK cells was analyzed by flow cytometry and Hoechst33258 staining after GCV treatment. Rats were vaccinated with FC/TK cells and divided into 2 groups. GCV group （5 rats） were intraperitoneally treated with GCV for 7 days, control group （5 rats） were treated with normal saline. The tumorigenesis and tumor metastasis in the rats were observed 90 days after inoculation. RESULTS： HSV1-TK was specifically expressed in FC/TK cells.GCV showed in vitro cytotoxicity to FC/TK cells in a dose-dependent manner,and 86.25% of the FC/TK cells were killed by GCV at a concentration of 100μg/ml; the apoptosis rate of FC/TK cells was over 80%, and apoptotic morphology, including cell shrinkage, chromatin condensation, was observed. In the rat models, the tumor was developed at the injection site in 3 rats of control group, while no tumor was observed in the rats of GCV group. CONCLUSION： HSV1-TK/GCV could act as the “death switch” of tumor vaccines by triggering apoptosis of tumor vaccines b

关 键 词：肿瘤 免疫疗法 肿瘤疫苗 树突细胞 自杀基因 细胞融合 HSV1-TK/GCV系统 凋亡 

分 类 号：R730.3[医药卫生—肿瘤]