Gene therapy for hepatocellular carcinoma using an endogenous antineoangiogenetic factor, angiostatin

肝细胞癌（HCC）以高血管肿瘤为特征。因此，经导管动脉栓塞是人类HCC的主要治疗方式，但其疗效尚不理想。在本研究中，通过血管抑制素基因转染人肝癌细胞系，研究了一种有效的内源性抗新血管生成因子血管抑制素对肝癌生长和转移潜力的抑制作用。从Hep G2人肝癌细胞中分离到人纤溶酶原cDNA，将kringle结构域1 ~ 4对应的片段与分泌信号序列（SS）和预激活肽（PA）连接，并克隆到腺病毒载体（AV-S-K1-4）中。用血管抑制素基因表达质粒和新霉素耐药基因质粒共转染人肝癌细胞株HuH7和HepG2，用G418筛选3周。然而，稳定的表达足够水平的血管抑制素的转染不能建立。相反，当AV-S-K1-4感染HuH7和HepG2细胞时，两种细胞系都表达了大量的血管抑制素。在av - s - k1 -4感染的细胞中，细胞的生长与亲本细胞几乎相似，而在双室模型中，av - s - k1 -4感染的细胞明显抑制了牛毛细血管内皮细胞的增殖，尽管内皮细胞的凋亡未被发现。在动物实验中，将亲代HuH7细胞接种于胸腺小鼠皮下。2周后，将AV-S-K1-4或对照腺病毒载体直接注射到皮下肿瘤。与接受载体治疗的小鼠相比，注射AV-S-K1-4的小鼠的肿瘤生长明显受到抑制。这些结果表明，血管抑制素基因转染肝癌细胞可能通过阻断肿瘤血管生成来抑制肿瘤生长。

Hepatocellular carcinoma (HCC) is characterized with a hypervascular tumor. Therefore, transcatheter arterial embolization is a major treatment modality for human HCC, but its efficacy is not yet satisfactory. In the present study, the inhibitory effects of a potent endogenous anti-neoangiogenetic factor, angiostatin, on growth and metastatic potentials of HCC were evaluated by means of angiostatin gene transfection into human hepatoma cell lines. Human plasminogen cDNA was isolated from Hep G2 human hepatoma cells, and the fragment corresponding to the kringle domains 1 to 4 was linked to a secretory signal sequence (SS) and preactivation peptide (PA), and cloned into adenovirus vector (AV-S-K1-4). HuH7 and HepG2 human hepatoma cell lines were co-transfected with the angiostatin gene expression plasmid and the neomycinresistance gene plasmid, and the cells were selected by G418 serection for 3 weeks. However, the stable transfectants expressing sufficient levels of angiostatin could not be established. In contrast, when HuH7 and HepG2 cells were infected with AV-S-K1-4, large amounts of angiostatin were expressed in both cell lines. Cell growth in AV-S-K1-4-infected cells was almost similar to that in the parental cells, while, in a double chamber model, AV-S-K1-4-infected cells clearly suppressed cell proliferation of bovine capillary endothelial cells, although apoptosis of the endothelial cells could not be identified. In animal experiments, parental HuH7 cells were inoculated subcutaneously in athymic mice. Two weeks later AV-S-K1-4 or a control adenovirus vector were directly injected into the subcutaneous tumor. Tumor growth in mice injected with AV-S-K1-4 was apparently suppressed, compared with that in mice receiving a vehicle treatment. These results suggest that gene therapy with angiostatin gene transfection into hepatoma cells inhibits tumor growth probably through blocking tumor angiogenesis.