ヒト正常腎に由来する培養上皮様細胞の性質について

Abstract

Normal human renal cortex surgically obtained from eight patients were dispersed by trypsin and cultured in vitro using Eagle's minimum essential medium supplemented with antibiotics and heat inactivated fetal calf serum (20%) under humidified air with 5% CO2 at 37℃. The results of these procedures and morphological or biochemical characterization of propagated cells were summarized as followings: 1. Monolayer sheets mainly composed with epithelial cells were successfully obtained from all of 8 cases, and the cells grew rapidly for about 7 weeks. Under the light microscopy, the monolayer cells showed marked morphological heterogeneity. In the course of maintenence, transformation of the cellular appearance was recognized, which included the swelling of cells, development of vacuoles or granules in the cytoplasm and loosed intercellular junction. Electron microscopic observation on the cells in the 3rd generations revealed cells with surface structures resembling microvilli, or desmosome-like structure between neighboring cells. 2. Population doubling time was estimated about 46 hours with the cells in the 3rd weeks and about 90 hours at the 6th week of cultivation. 3. Alkaline phosphatase activity, which was detected highly in normal renal proximal tubular cells, was markedly decreased in cultivated renal cells and only detectable in multilayered fibroblastic cells histochemically. 4. The activity of γ-glutamyl transpeptidase in cultured cells was also decreased to a fifth in comparison with that of the normal kidney cortex. The electrophoretic mobility of this enzyme was different from that of normal renal tissues, but not the same with that of a novel isozyme detected in renal cell carcinoma tissues. 5. Lactate dehydrogenase activity was preserved in monolayer cells, but the isozyme pattern showed predominance of LDH-4 and LDH-5, which was a completely inverted pattern of normal kidney enzyme. 6. After the treatment with 10 ng/ml parathyroid hormone, the intracellular cyclic AMP level of cultivated cells was increased 33 folds within 2 minutes. This finding strongly suggested that the monolayers included cells derived from proximal tubular cells, and also implyed the preservation of differentiated biochemical function in the cultivated cells. 7. The detection of a estrogen binding protein in cultivated cells was unsuccessful, which might indicate a disappearance of a biochemical character in normal renal tissues. With brief review of literatures concerning the cultivation of renal cells derived from human or experimental animals, the significance of the morphological and biochemical transformation noted in cultivated cells was discussed, and it was emphasised that the biological process of this transformation did not mean a simple "fetalism" nor "dedifferentiation", but a rather complicated phenomenon.