腺病毒载体介导人血管内皮细胞生长因子受体-2诱导抗小鼠肝癌免疫

目的 探讨复制缺损型腺病毒载体(Ad)介导人血管内皮细胞生长因子受体-2(hKDR)诱导抗小鼠肝癌血管免疫、打破免疫耐受的效果. 方法 用RT-PCR方法从人脐静脉内皮细胞和小鼠胚胎细胞中分别克隆hKDR和小鼠KDR(mKDR),构建Ad hKDR和Ad mKDR.用Ad hKDR和Ad mKDR分别皮内免疫C57BL/6小鼠,7 d后取脾细胞作为效应细胞,Hepa 1-6/mKDR作为靶细胞,行乳酸脱氢酶释放试验,检测特异性细胞毒性T淋巴细胞(CTL)杀伤率;免疫小鼠接种Hepa 1-6肝癌细胞,观察荷瘤小鼠成活情况. 结果 Ad hKDR和Ad mKDR分别免疫小鼠1周后,在效应细胞:靶细胞为100:1、50:1和25:1时,Ad hKDR诱导的6 h CTL杀伤率分别为84.3%±6.7%、71.5%±5.2%和44.6%±4.7%;Ad mKDR诱导的6 h CTL杀伤率分别为65.2%±6.1%、46.7%±5.0%和22.6%±3.7%.Ad hKDR免疫小鼠后l周接种5×106个Hepal-6肝癌细胞,2个月后仍然有60%的小鼠无瘤生长;而接种2×106个Hepa 1-6细胞至Ad mKDR免疫小鼠,2个月后小鼠成活率为40%.上述CTL效应和肿瘤保护作用在清除CD8+和CD4+T淋巴细胞后消失. 结论 Ad介导异种KDR能有效地打破肿瘤的免疫耐受,诱发强烈的抗原特异性细胞免疫反应,这种特异性细胞免疫反应是CD8+和CD4+T淋巴细胞依赖性的.

Adenovirus vector encoding human KDR elicits immunity against hepatocellular carcinomas in mice

OBJECTIVE: To investigate the effect of adenovirus vector encoding human vascular endothelial growth factor receptor-2 (hVEGFR-2 or hKDR) on breaking the immune tolerance and inducing immunity against murine hepatocellular carcinomas. METHODS: Human and mouse KDR cDNA were cloned from human umbilical vein endothelial cells (HUVEC) and C57BL/6 mouse embryo cells respectively using RT-PCR, and then Ad hKDR and Ad mKDR were constructed. Seven days after immunization of the mice with Ad hKDR or Ad mKDR, an analysis of cytotoxic activity of antigen-specific cytotoxic T lymphocytes (CTL) was made by lactate dehydrogenase (LDH) release assay, in which splenocytes of the immunized mice acted as effectors and Hepa 1-6/mKDR cells as the targets. In addition, the survival of the mice immunized with Hepa 1-6 hepatoma cells was checked. RESULTS: Seven days after immunization, the 6 h killing activities of CTL elicited by the Ad hKDR were 84.3%+/-6.7%, 71.5%+/-5.2%, and 44.6%+/-4.7% at the ratio of the effectors:targets (E:T) of 100:1, 50:1, and 25:1, respectively. Correspondingly, the CTL activities by Ad mKDR were 65.2%+/-6.1%, 46.7%+/-5.0%, and 22.6%+/-3.7%. Sixty percent of the Ad hKDR-immunized mice with 5*10(6) Hepa 1-6 hepatoma cells were still alive two months after the inoculation, whereas just 40% of the Ad mKDR-immunized mice with 2*10(6) Hepa 1-6 cells survived two months. When CD8+ or CD4+ T lymphocytes were deleted in the mice the above mentioned CTL activities and protection of the mice from tumors disappeared. CONCLUSION: Adenovirus vector-mediated xenogeneic KDR can effectively break the immune tolerance to hepatocellular carcinomas in an animal model and induce a strong antigen-specific T cell response, which is dependent on CD8+ and CD4+ T cells.