Ad介导人AFP和IFN-γ协同诱发抗小鼠肝癌免疫效应

Synergic Effect of Adenoviral Vector-encoding Human Alpha-fetoprotein and Interferon-gamma on Immunity against Hepatocellular Carcinoma in Mice

背景与目的:原发性肝癌(hepatocellular carcinoma,HCC)是常见的恶性肿瘤之一,目前对HCC的治疗尚无行之有效的手段。本研究探讨腺病毒(Adenovirus,Ad)载体介导异种甲胎蛋白(Alpha-fetoprotein,AFP)和酌干扰素(Interferon-gamma,IFN-γ酌)的协同抗肝癌效应。方法:用RT-PCR(reverse transcri-ptase-polymerase chain reaction)方法克隆小鼠IFN-γ酌基因并构建复制缺损型腺病毒编码人AFP和小鼠IFN-γ酌联合表达载体(Ad-hAFP/IFN-γ酌)。皮内免疫C57BL/6小鼠7d后,取脾细胞行51Cr释放实验检测特异性细胞毒T淋巴细胞(Cytotoxic Tlymphocytes,CTLs)杀伤活性;或给免疫小鼠皮下接种Hepa1-6肝癌细胞,观察荷瘤小鼠成活情况。结果:51Cr释放实验显示,Ad-hAFP/IFN-γ酌免疫小鼠1周后其诱导产生的特异性CTL杀伤活性明显强于Ad-hAFP或Ad-IFN-γ酌单独免疫,在效∶靶比(E∶T)为10∶1时,Ad-hAFP/IFN-γ酌、Ad-hAFP和Ad-IFN-γ酌诱发的CTL杀伤率分别(43.8±5.5)%、(28.2±3.2)%和(12.8±1.9)%;30∶1时,为(79.6±6.4)%、(51.9±4.3)%和(15.6±2.3)%以及90∶1时(88.2±6.3)%、(62.5±4.8)%和(26.5±2.4)%。荷瘤试验表明,Ad-hAFP或Ad-IFN-γ酌单独免疫小鼠后1周接种5×106Hepa1-6肝瘤细胞,观察2个月,Ad-hAFP免疫组80%的小鼠荷瘤,Ad-IFN-γ酌免疫组小鼠则100%荷瘤;而Ad-hAFP/IFN-γ酌免疫小鼠在接种同样数量的Hepa1-6细胞,2个月无小鼠荷瘤,小鼠100%存活。结论:腺病毒载体介导异种AFP能有效地诱发针对小鼠AFP的特异性细胞免疫反应,IFN-γ酌能明显增强这种效应。

BACKGROUND ＆ OBJECTIVE： Primary hepatocellular carcinoma （HCC） is a common malignant tumor. Up to date, no effective treatment for HCC is available. This study was to investigate the synergic effect of adenoviral vector-encoding xenogeneic alpha-fetoprotein （AFP） and interferon-gamma （IFN-γ） on the immunity against HCC in mice. METHODS： IFN-γ gene was cloned using reverse transcription-polymerase chain reaction （RT-PCR）. Replication-defective adenovirus encoding both human AFP and murine IFN-γ was constructed. The cytotoxic activity of antigen-specific cytotoxic T lymphocytes （CTLs） was detected 7 days after the intradermal immunization of C57BL/6 mice with Ad-hAFP, Ad-IFN-γ or Ad-hAFP/IFN-γ using standard ^51Cr release assay. The survival of mice after the subcutaneous inoculation of 5×10^6 hepatoma Hepa 1-6 cells was checked. RESULTS： The cytotoxic activity of CTLs elicited by Ad-hAFP/IFN-γ was much stronger than that by Ad-hAFP or Ad-IFN-γ alone. At an effector：target （E：T） ratio of 10：1, the cytotoxic activities of CTLs in Ad-hAFP/IFN-γ, Ad-hAFP, and Ad-IFN-γ groups were （43.8±5.5）%, （28.2±3.2）%, and （12.8±1.9）%, at a ratio of 30：1, were （79.6±6.4）%, （51.9±4.3）%, and （15.6±2.3）%, and at a ratio of 90：1, were （88.2±6.3）%, （62.5±4.8）%, and （26.5±2.4）%, respectively. The tumor formation rates were 80% in Ad-hAFP-immunized mice and 100% in Ad-IFN-γ-immunized mice at 2 months after inoculation with Hepal-6 cells; no tumor formed in Ad-hAFP/IFN-γ-immunized mice, and all mice in this group survived during two-month observation. CONCLUSION： Ad-hAFP can efficiently induce immunity against HCC in mice, and IFN-γ enhances such an effect.