摘要：Melanoma, a kind of carcinoma cutaneum, occurs at high frequency in aged gray horses. For the basic study of horse melanoma occurrence, the establishment of melanoma culture cell lines derived from three aged gray horses was attempted. Three types of enzymes, collagenase, hyaluronidase and pronase, were used for the separation of melanoma cells in the primary culture. Those enzyme treatments were highly efficient for the resolution of connective tissue. The culture medium was Iscove's modified Dulbecco's medium (IMDM) + 10% fetal calf serum (FCS; Toyobo) and the culture condition was 37°C temperature, 100% humidity, 5% CO2 and 95% air. After cloning a separated cell, three cell lines term culture were established for long term culture. These culture cell lines were named EMT-1, EMT-2 and EMT-3, respectively. These cell lines had high growth ability and the colony of the cell line was formed by a low density culture. The form of each culture cell, the cell surface and the distribution of the cytoskelton completely agreed with the characteristics observed in cancer cells. The numbers of chromosomes in these culture cell lines showed wide distribution mainly on 2n=64, and the hereditary characteristics were different from normal cell lines. Lactate dehydrogenase (LDH), nucleoside phoshorylase (NP) and glucose-6-phosphate dehydrogenase (G6PD) isozyme patterns of these culture cell lines were compared by starch gel electrophoresis, but there was no difference from the original cells. As a result of the above, it is considered that there was no contamination between the culture cell lines and the culture cells that came from the horse melanoma which showed signs of special hereditary variation.
