自杀性肿瘤疫苗对大鼠卵巢癌的治疗作用及其安全性研究  

Suicidal cancer vaccine enhances anti-tumor immunotherapeutic effect and its safety in the treatment of ovarian cancer

作　　者：康玉[1] 徐丛剑[1] 刘惜时[1] 邵志敏[2] 欧周罗[2] 罗建明[2] 吴超群[3] 钟翠平[4] 顾健人[5] 

机构地区：[1]复旦大学附属妇产科医院妇产科,上海200011 [2]复旦大学肿瘤医院乳腺癌研究所 [3]复旦大学遗传工程国家重点实验室 [4]复旦大学上海医学院组胚解剖系 [5]上海市肿瘤研究所癌基因及相关基因国家重点实验室

出　　处：《中华肿瘤杂志》2006年第9期654-657,共4页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目(30070783);上海市重大科技攻关项目资助项目(03DZ19234)

摘　　要：目的将自杀基因修饰的卵巢癌细胞与树突状细胞(DC)融合制备成自杀性肿瘤疫苗(FC／TK),观察FC／TK对大鼠卵巢癌的免疫治疗作用,并探讨对FC／TK进行体内灭活的可行性。方法以聚乙二醇法,用大鼠骨髓来源的DC与转导有自杀基因(HSV1-TK基因)的大鼠卵巢癌上皮细胞株NuTu-19细胞融合制备成FC／TK,在扫描电镜下观察FC／TK的形态;用3H-TdR渗入法,检测FC／TK刺激T细胞增殖的能力。在大鼠体内的免疫治疗实验中,以FC／TK治疗大鼠卵巢癌皮下瘤,观察肿瘤形成及肿瘤大小,分别设立经60Co照射的FC／TK组、未经自杀基因修饰的卵巢癌细胞与DC融合的疫苗(FC)组和PBS对照组。在体内灭活实验中,将FC／TK以荧光染料标记后进行大鼠体内示踪,腹腔注射羟甲基无环鸟苷(GCV)7 d后,采用末端脱氧核苷酸转移酶(TdT)介导的d-UTP缺口末端标记技术,对大鼠淋巴组织的冰冻切片做原位细胞凋亡检测。结果FC／TK为多形性,扫描电镜下可见不规则的突起。与对照组相比,FC／TK能明显刺激T细胞增殖(P<0．01)。大鼠体内的免疫治疗实验结果显示,FC／TK组与FC组大鼠的成瘤时间晚于经60Co照射的FC／TK组(P<0．05),较PBS组明显延迟(P<0．01);接种NuTu-19细胞90 d后,FC／TK组大鼠的平均肿瘤体积较经60Co照射的FC／TK组和PBS组明显缩小(P<0．01),但FC／TK组与FC组之间,差异无统计学意义(P>0．01)。荧光显微镜下,经红色荧光标记的FC／TK出现在大鼠脾脏;进一步TUNEL染色显示,经GCV处理后,进入大鼠体内的FC／TK已发生凋亡。结论FC／TK对大鼠卵巢癌有免疫治疗作用,并可由自杀基因系统控制其在体内的存活。Objective To study the anti-tumor immunotherapeutic effect induced by the suicidal cancer vaccine FC/TK, and to evaluate the safety of this vaccine. Methods The suicidal cancer vaccine, named FC/TK, was prepared by fusion of suicide gene （ HSV1 -TK gene） -modified ovarian carcinoma NuTu- 19 cells with rat bone marrow-derived dendritic cells （DCs）. The morphology of FC/TK was evaluated by scanning electron microscopy. The stimulatory effect of FC/TK on T cells was determined by T cell proliferation assay. In immunotherapeutic studies in vivo, Fischer344 rats were injected subcutaneously with NuTu-19 cells, followed by treatment of FC/TK on clays 7 and 14, compared to controls treated with irradiated FC/TK, FC or PBS, respectively. Tumor incidence and volume were measured in 90 clays after challenge. To determine the killing effect of FC/TK in vivo, TUNEL assays were applied to detect apoptotic cell death in spleen of vaccinated rats with prodrug ganciclovir administration, Results FC/TK cells were of irregular shape with surface membrane processes. Compared to the control groups, FC/TK significantly promoted T cell proliferation （P 〈0.01 ）. The rats vaccinated with FC/TK and FC significantly inhibited the tumor growth compared to rats vaccinated with irradiated FC/TK （P 〈 0.05 ）or with PBS（ P 〈 0.01 ）. The immunotherapeutic effect induced by FC/TK was similar to that using FC. Fluorescence microscopy showed that fluorescein-stained FC/TK cells migrated into spleen also showed to be TUNEL-positive, suggesting that the FC/TK cells were killed by ganciclovir in vivo. Conclusion Our data indicate that suicidal cancer vaccine is an effective and safe therapy for ovarian carcinoma and may serve as a broadly applicable approach for other cancer vaccines in the future.

关 键 词：树突状细胞 自杀基因 卵巢肿瘤 肿瘤疫苗 融合细胞 

分 类 号：R737.31[医药卫生—肿瘤]