Mechanisms in metastasis of hepatocellular carcinoma and analysis of stimulative molecules

1)利用侵袭室建立体外培养肝癌细胞侵袭转移模型。肝癌细胞（HuH-7和PLC/PRF/5）的潜能估计如下。我们计算了下腔的细胞数量，这些细胞通过13um孔径的趋化过滤器，并涂上50ug重组基质凝胶。两种肝癌细胞均未自行通过趋化过滤。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。在上腔的肝癌培养基中加入黑色素瘤培养液，肝癌细胞可以通过过滤，说明黑色素瘤细胞分泌了侵袭转移的分子。II)刺激肝癌细胞侵袭转移的分子分析在黑色素瘤和PLC/PRF/5培养基中均发现了IV型胶原降解酶活性。然而，这种酶被发现是一种被APMA激活的非活性形式。Sephacryl-S 200凝胶过滤显示该分子分子量为37kD，表明该分子不同于自分泌运动因子、IV型胶原降解酶和碱性FGF。3)肝细胞癌的功能诊断为了临床使用样本，我们建立了肝癌的功能诊断方法。吲哚菁绿（ICG）后抽吸活检是一种有用的方法，95%以上的ICG阴性组织为恶性细胞。我们明确了ICG丢失的部分机制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.