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

使用内源性抗血管生成因子血管抑制素对肝细胞癌进行基因治疗

基本信息

批准号：
11670513
负责人：
NAKATA Keisuke
金额：
$ 2.05万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670513/
关键词：
Hepatoma Gene therapy Angiostatin Anti-neoantigogenesis α-Fetoprotein Adenovines vector 肝癌培養細胞

项目摘要

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.

肝细胞癌（HCC）的特征是富血管肿瘤。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，通过将血管抑制素基因转染到人肝癌细胞系中，评估了有效的内源性抗新生血管生成因子血管抑制素对HCC生长和转移潜力的抑制作用。从Hep G2人肝癌细胞中分离人纤溶酶原cDNA，将对应于kringle结构域1至4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。将血管抑制素基因表达质粒和新霉素抗性基因质粒共转染HuH7和HepG2人肝癌细胞系，G418筛选3周筛选细胞。然而，无法建立表达足够水平的血管抑制素的稳定转染子。相反，当HuH7和HepG2细胞感染AV-S-K1-4时，两种细胞系中均表达大量血管抑制素。 AV-S-K1-4感染的细胞中的细胞生长几乎与亲代细胞相似，而在双室模型中，AV-S-K1-4感染的细胞明显抑制牛毛细血管内皮细胞的细胞增殖，尽管无法鉴定内皮细胞的凋亡。在动物实验中，将亲代HuH7细胞皮下接种到无胸腺小鼠体内。两周后，将AV-S-K1-4或对照腺病毒载体直接注射到皮下肿瘤中。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，将血管抑制素基因转染至肝癌细胞的基因治疗可能通过阻断肿瘤血管生成来抑制肿瘤生长。