Mechanisms in metastasis of hepatocellular carcinoma and analysis of stimulative molecules

肝细胞癌转移机制及刺激分子分析

• 批准号: 02670307
• 负责人: HIGASHI Toshihiro
• 金额: $ 1.22万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670307/
• 关键词: Hepatocellular carcinoma; Melanoma; Metastasis; Stimulative molecule for metastasis; Type IV collagen degrading enzyme; Invasion chamber; ICG; GST-alpha; メラノ-マ; IV型コラ-ゲン分解酵素; GSTーα

I)In vitro model of invasion and metastasis of cultured Hepatoma cells by using the invasion chamber.The potentiality of hepatoma cells(HuH-7 and PLC/PRF/5) was estimated as follows. We calculated cell numbers in the lower chamber which passed through the 13um pore size chemotaxis filter coated with 50ug of reconstituted matrix gel. The both hepatoma cells did not pass through the chemotaxis filter by themselves. On the contrary, mouse melanoma cells(B16 melanoma cell line) passed through the filter and we recognized that the cells entered into and destroyed the gel. Hepatoma cells could pass through the filter by adding culture medium of melanoma to medium of hepatoma in the upper chamber, indicating that melanoma cells secreted molecules for invasion and metastasis.II)Analysis of the molecules which stimulate invasion and metastasis of hepatoma cellsType IV collagen degrading enzyme activity was recognized in the culture medium of both melanoma and PLC/PRF/5. However, this enzyme was found as an inactive form which was activated by APMA. Sephacryl-S 200 gel filtration revealed that the molecular weight of the molecule was 37kD, suggesting that this molecule was different from autocrine motility factor, type IV collagen degrading enzyme and basic FGF.III)Functional diagnosis of hepatocellular carcinomaIn order to use clinical samples, we established functional diagnostic method for hepatoma. Aspiration biopsy after indocyanine green(ICG) was found as a useful device in which more than 95% of ICG negative tissues were malignant cells. We clarified that the mechanism of ICG loss was partly due to the decrease of cytoplasmic glutathion-S-transferase.

I)利用侵袭室建立培养的肝癌细胞的体外侵袭和转移模型。肝癌细胞(HuH-7和PLC/PRF/5)的潜力评估如下。我们计算了下室中通过涂有 50ug 重构基质凝胶的 13um 孔径趋化过滤器的细胞数。两种肝癌细胞均未自行通过趋化过滤器。相反，小鼠黑色素瘤细胞（B16黑色素瘤细胞系）通过过滤器，我们发现细胞进入并破坏了凝胶。上室肝癌培养基中加入黑色素瘤培养基，肝癌细胞可通过过滤器，说明黑色素瘤细胞分泌侵袭转移分子。二）刺激肝癌细胞侵袭转移分子IV型胶原降解的分析在黑色素瘤和 PLC/PRF/5 的培养基中均发现了酶活性。然而，这种酶被发现是一种非活性形式，可被 APMA 激活。 Sephacryl-S 200凝胶过滤显示该分子的分子量为37kD，提示该分子与自分泌运动因子、IV型胶原降解酶和碱性FGF不同。III)肝细胞癌的功能诊断为了使用临床样本，我们建立了肝癌的功能诊断方法。吲哚菁绿（ICG）后的抽吸活检被发现是一种有用的装置，其中超过95％的ICG阴性组织是恶性细胞。我们阐明ICG损失的机制部分是由于细胞质谷胱甘肽-S-转移酶的减少。