目的：扩增出单纯疱疹病毒Ｉ型（ＨＳＶ－Ｉ）Ｓｔｏｃｋｅｒ株胸苷激酶（ｔｋ）基因，并将其克隆到真核表达质粒中，构建一个含有ｔｋ基因片段的高效真核表达载体。方法：根据已发表的ＨＳＶ－Ｉ ＣＬ１０１株ｔｋ基因的核苷酸序列，设计并合成了一对引物，以ＨＳＶ－Ｉ Ｓｔｏｃｋｅｒ株酸为模板，进行ＰＣＲ扩增，并将扩增产物连接到ｐＵＣ１１９中，进行序列分析，将此基因进一步克隆到含巨细胞病毒极早期启动子的真核表达质粒

Objective: To investigate the therapeutic effects of the murine IL-12 retrovirus packaging cell line on hep-atoma injected locally. Methods: The retrovirus vector encoding mIL-12 gene was constructed and transfected into packaging cell line PA317. The cells were then used to treat the rats with experimental orthotopic hepatoma at different time. The therapeutic effects, immune functions of the hosts, pathological and toxicological responses were documented. Results: The results showed that the mIL-12 retrovirus packaging cell line could significantly inhibit the growth of the hepatoma cells injected locally to the hepatoma. The early treatment made the rats survive long, while the medium or late stage treatment could prolong the life time of the rats compared with the bland control group or bland vector control group, though the rats did not survive. The number of NK cells and T cells increased significantly in the treatment group. The effects of the early treatment were superior to those of the medium and late stage treatment. Moreover, the transfection of IL-12 gene locally in the hepatoma tissue could make the hepatoma disappear from other liver lobe. This phenomenon demonstrated that IL-12 could activate the immune cells of the host to kill the untransfected tumor cells. This is very important for IL-12 to be used in gene therapy clinically. Meanwhile, the hepatoma would not recur in the rats that had survived more than 2 months from the early treatment after being rechallenged with tumor cells. Conclusion: The results showed that IL-12 gene injected locally in the hepatoma tissue could enhance the anti-tumor immunity of the host.

R735.7

国家自然科学基金重点项目(批准号39730440)，上海市现代生物与新药产业发展基金(批准号984319119)，国家自然科学基金(批准号39870760)资助项目