Mechanisms in metastasis of hepatocellular carcinoma and analysis of stimulative molecules

I）通过使用侵袭室的培养的肝癌细胞的侵袭和转移的体外模型。如下估计肝癌细胞（HuH-7和PLC/PRF/5）的潜力。我们计算了下室中的细胞数，其通过涂覆有50 μ g重构基质凝胶的13 μ m孔径趋化性过滤器。两种肝癌细胞均不能自行通过趋化滤器。相反，小鼠黑色素瘤细胞（B16黑色素瘤细胞系）通过过滤器，我们认识到细胞进入并破坏凝胶。通过在上室的肝癌培养基中加入黑色素瘤培养基，肝癌细胞可以通过过滤器，表明黑色素瘤细胞分泌用于侵袭和转移的分子。II）刺激肝癌细胞侵袭和转移的分子的分析在黑色素瘤和PLC/PRF/5的培养基中均识别出IV型胶原降解酶活性。然而，这种酶被发现是一种被APMA激活的无活性形式。Sephacryl-S 200凝胶过滤表明，该分子的分子量为37 kD，表明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.肝癌的功能诊断为了应用临床样本，我们建立了肝癌的功能诊断方法。吲哚菁绿色（ICG）后的抽吸活检被发现是一种有用的设备，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。

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.