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

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

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，7d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6h 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6h 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1周接种5×10^6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Hepal-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。

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 mutine 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 theAd 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.