Yonemura Y, Endo Y, Takino T, Sakamoto K, Bandou E, Kinoshita K, Fushida S, Miwa K, Sugiyama K, Sasaki T
Second Department of Surgery, School of Medicine, Kanazawa University, Japan.
Clin Exp Metastasis. 2000;18(4):321-7. doi: 10.1023/a:1010887014669.
The mechanisms of the lymph node metastasis remain unclear. We demonstrate the role of MT1-MMP on the lymph node metastasis using in vivo experimental model of lymph node metastasis by orthotopic implantation of MT1-MMP transfected gastric cancer cell line in the stomach of nude rats. TMK-1 cell line without expression of MT1-MMP was transfected with the pcDNA3 plasmid containing a 3.4-kb MT1-MMP cDNA fragment by calcium phosphate method, and the transfected cell line was designated as TMK-MT. Western blot and RT-PCR analyses showed the specific bands corresponding to MT1-MMP in the TMK-MT cells. By gelatin zymography, the activated form (62-kDa) of MMP-2 was identified in the medium of TMK-MT cell line, but was not detected in TMK-1 cells. Six weeks after orthotopic implantation of TMK-1 and TMK-MT xenografts of nude mouse-subcutaneus tumor into the stomach of nude rats, gastric tumors were found in all the animals. Histologically, the lymphatic invasion was found in the submucosa of the TMK-MT gastric tumors. Lymph node metastasis was not detected in nude rats bearing TMK-1 gastric tumor (0/8). In contrast, lymph node metastasis was detected in five out of 8 rats, bearing TMK-MT gastric tumor. MT1-MMP immunoreactivity was found on the cell membrane and cytoplasm of TMK-MT cells not only in the lymph node metastasis but also in the stomach tumor. These results suggest that MT1-MMP overexpression induced by transfection of its gene may promote lymph node metastasis of transformed cells.
淋巴结转移的机制仍不清楚。我们通过在裸鼠胃内原位植入MT1-MMP转染的胃癌细胞系，利用淋巴结转移的体内实验模型，证明了MT1-MMP在淋巴结转移中的作用。采用磷酸钙法将不表达MT1-MMP的TMK-1细胞系用含3.4 kb MT1-MMP cDNA片段的pcDNA3质粒转染，转染后的细胞系命名为TMK-MT。蛋白质免疫印迹法和逆转录-聚合酶链反应分析显示TMK-MT细胞中有与MT1-MMP相对应的特异性条带。通过明胶酶谱法，在TMK-MT细胞系的培养基中鉴定出MMP-2的活化形式（62 kDa），而在TMK-1细胞中未检测到。将裸鼠皮下肿瘤的TMK-1和TMK-MT异种移植物原位植入裸鼠胃内6周后，所有动物均发现胃肿瘤。组织学检查发现，TMK-MT胃肿瘤的黏膜下层有淋巴管浸润。携带TMK-1胃肿瘤的裸鼠未检测到淋巴结转移（0/8）。相比之下，携带TMK-MT胃肿瘤的8只大鼠中有5只检测到淋巴结转移。不仅在淋巴结转移灶中，而且在胃肿瘤中，TMK-MT细胞的细胞膜和细胞质上均发现MT1-MMP免疫反应性。这些结果表明，基因转染诱导的MTl-MMP过表达可能促进转化细胞的淋巴结转移。
