Effect of hepatocyte growth factor on liver regeneration and damage of gepatocytes

我们先前发现，在四氯化碳处理的小鼠肝再生之前，血清和肝脏中的小鼠肝细胞生长因子(MHGF)显著增加。然后，从经肝毒素处理的小鼠的肝脏上清液中纯化了约28万倍的mHGF，并对其进行了鉴定。它由两条分子质量分别为62和31 kDa的多肽链组成，并以二硫键相连(S)。其理化和生物学特性与人肝细胞生长因子非常相似。在本研究中，我们发现，用1.7M氯化钠(pH 7.4)在磷酸盐缓冲盐水(PBS)中匀浆损伤的肝脏，提取的mHGF比用0.25M蔗糖加20 mM Hepes(pH 7.4)匀浆的肝脏多5-10倍。用改进的方法提取的人肝细胞生长因子用我们的四步纯化方法进行纯化。接下来，我们研究了在原代培养的大鼠肝细胞中加入mHGF或hHGF是否可以防止D-氨基半乳糖对大鼠肝细胞的损伤。MHGF或hHGF均不能减弱D-氨基半乳糖10-30 ng/ml诱导肝细胞DNA合成的肝毒性。接下来研究了hHGF对不同培养细胞生长的影响。对人肝母细胞瘤细胞株HLE、HH-7和PLC/PRF/5的增殖无明显促进作用。HHGF对另一种人肝母细胞瘤细胞株Hep G2的生长有抑制作用。需要对其他肝癌和肝母细胞瘤细胞株进行研究，以确定肝母细胞瘤而不是肝癌细胞是否对hHGF有反应。目前还不清楚hHGF是否影响成纤维细胞的生长，但在本研究中，我们证明了hHGF对人皮肤成纤维细胞具有显著的促有丝分裂活性。我们之前曾报道，成纤维细胞分泌少量但可检测到的hHGF。HHGF是否以自分泌的方式刺激人皮肤成纤维细胞的增殖，这一问题还需要进一步的研究来回答。

We found previously that mouse hepatocyte growth factor (mHGF) in serum and liver markedly increased prior to liver regeneration in carbon tetrachloride-treated mice. mHGF was then purified about 280,000-fold from the liver supernatant of mice treated with the hepatotoxin and was characterized. It is composed of two peptide chains with molecular masses of 62 and 31 kDa, linked by a disulfide bond(s). Its physicochemical and biological properties are quite similar to those of hHGF. In this study, we found that homogenizing the damaged liver with 1.7 M NaCl (pH 7.4) in phosphate-buffered saline (PBS) extracted 5-10-fold more mHGF than homogenization of the liver with 0.25 M sucrose containing 20 mM Hepes (pH 7.4). mHGF, extracted by this modified method, was purified by our four-step purification method. We next examined whether D-galactosamine-induced damage of rat hepatocytes in primary culture is prevented by adding mHGF or hHGF to the hepatocyte cultures. mHGF or hHGF did not diminish the hepatotoxicity of D-galactosamine at doses of 10-30 ng/ml, which induce DNA synthesis in hepatocytes. Effect of hHGF on the growth of various cultured cells was next investigated. It stimulated proliferation of HuH-6 Clone 5 human hepatoblasstoma cells, but did not that of HLE, HuH-7, and PLC/PRF/5 human hepatoma cell lines. The growth of Hep G2, another human hepatoblastoma cell line, was inhibited by hHGF. Studies on other hepatoma and hepatoblastoma cell lines are required to determine whether hepatoblastoma, and not hepatoma cells are responsive to hHGF. It has not been clear whether hHGF affect the growth of fibroblasts, but in this study we demonstrated significant mitogenic activity of hHGF for human skin fibroblasts. We reported previously that the fibroblasts secreted a small but detectable amount of hHGF. Furhter studies are required to answer the question of whether proliferation of human skin fibroblasts is stimulated by hHGF in an autocrine manner.