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Analysis of mechanism of HGF antagonist NK4 mediated anti-angiogenesis for cancer and assessment for human clinical trial

HGF拮抗剂NK4介导的抗肿瘤血管生成机制分析及人体临床试验评估

基本信息

批准号：
17591449
负责人：
HIRANO Tadamichi
金额：
$ 2.24万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591449/
关键词：
NK4 angiogenesis of tumor Gene Therapy Hepatocellular carcinoma HGF 肝細胞癌骨髄移植遺伝子導入

项目摘要

As a new strategy for hepatocellular carcinoma, we have reported NK4 mediated gene therapy that could inhibit tumor angiogenesis, metastasis, and invasion. It markedly suppressed tumor growth and invasion not only in vitro but also in vivo. Based on this study, we aimed to elucidate the mechanism of NK4 mediated anti-angiogenesis, and estimated method of gene transfer for applying human clinical trial. (1) Although NK4 inhibits growth and migration of small-vessel endothelial cells, an inhibitory effect of NK4 in BM-derived cells has not been shown. We attempted to assess whether NK4 suppressed recruitment of BM-derived endothelial cells in tumor vessels using lethally irradiated, tumor-bearing athymic nude mice transplanted with LacZ^+ bone marrow (BM) cells. Nude mice were lethally irradiated (4.5 Gray, with cesium-137 as the source). LacZ^+ bone marrow (BM) cells (5x10^6) obtained from transgenic mice were injected into tail veins of irradiated nude mice. After 4 weeks, these mice r … More eceived injections in the flank of 10^7 HUH7 cells. At 14 days post tumor cell implantation ( or when tumor volume reached approximately 500 mm3 ), either Ad.NK4 (10^9 pfu) or PBS (50 μl) was injected directly into the growing tumor. After 5 days the tumor was removed and stained with anti-lacZ rabbit polyclonal antibody and anti-CD31 rat monoclonal antibody. In all mice most of the original splenic cell population was replaced by donor cells. However, no cells were positive for LacZ by immunohistochemical staining in CD31-positive tumor vessels, even in PBS-injected control mice. In NK4-treated tumors, CD31-positive vessels were significantly fewer than in LacZ-treated controls. These results indicate that NK4 suppressed tumor angiogenesis, although any s uppressive effect for BM-derived e ndothelial cells remains unknown in the present animal model. Further study of this issue is required, since it has important implications for future approaches to of anti-angiogenic therapy. (2) To assess the NK4 influence for liver regeneration, Ad.Nk4 was infected t o mice t hat were performed 70 % hepatectomy. Liver regeneration significantly delayed regarding to control that were infected Ad.LacZ, as a result of hepatic weight, PCNA staining and BrDU. (3) Efficacy of hydrodynamics transfection mediated NK4 transduction was assessed instead of adenoviral vector to aid human clinical trial. Suppression of tumor growth was confirmed by this transfection method. Less

作为治疗肝细胞癌的新策略，我们报道了NK4介导的基因治疗，可以抑制肿瘤血管生成、转移和侵袭。它不仅在体外而且在体内均显着抑制肿瘤生长和侵袭。基于本研究，我们旨在阐明NK4介导的抗血管生成的机制，并估计用于人体临床试验的基因转移方法。 (1)虽然NK4抑制小血管内皮细胞的生长和迁移，但尚未显示NK4对BM来源的细胞的抑制作用。我们尝试使用移植了 LacZ^+ 骨髓 (BM) 细胞的经致命辐射、荷瘤无胸腺裸鼠来评估 NK4 是否抑制肿瘤血管中 BM 来源的内皮细胞的募集。裸鼠接受致命辐射（4.5 格雷，以铯 137 为辐射源）。将从转基因小鼠获得的LacZ^+骨髓(BM)细胞(5x10^6)注射到受辐射的裸鼠的尾静脉中。 4 周后，这些小鼠在侧腹接受了 10^7 HUH7 细胞的注射。肿瘤细胞植入后 14 天（或当肿瘤体积达到约 500 mm3 时），将 Ad.NK4 (10^9 pfu) 或 PBS (50 μl) 直接注射到生长的肿瘤中。 5天后，取出肿瘤并用抗lacZ兔多克隆抗体和抗CD31大鼠单克隆抗体染色。在所有小鼠中，大部分原始脾细胞群被供体细胞取代。然而，通过免疫组织化学染色，即使在注射 PBS 的对照小鼠中，CD31 阳性肿瘤血管中也没有细胞呈 LacZ 阳性。在 NK4 处理的肿瘤中，CD31 阳性血管明显少于 LacZ 处理的对照。这些结果表明 NK4 抑制肿瘤血管生成，尽管在目前的动物模型中对 BM 来源的内皮细胞的任何抑制作用仍然未知。需要对该问题进行进一步研究，因为它对未来的抗血管生成治疗方法具有重要意义。 (2)为了评估NK4对肝再生的影响，将Ad.Nk4感染到进行了70％肝切除术的小鼠中。由于肝重量、PCNA染色和BrDU的结果，与感染Ad.LacZ的对照相比，肝再生显着延迟。 (3)评估流体动力学转染介导的NK4转导代替腺病毒载体的功效以辅助人体临床试验。通过该转染方法证实了肿瘤生长的抑制。较少的