ウシ副甲状腺単層培養細胞を用いた副甲状腺ホルモンに関する研究 (第一報)

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We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3-0.5% collagenase at 37°C for 60 min. Dispersed cells were cultured at 37°C in MEM·Hanks' BSS containing 10% fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1% BSA·MEM·Hanks·HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10-7-10-5M in the former and 10-5-10-3M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3-4.4 mM.