LeBoff M S, Rennke H G, Brown E M
Endocrinology. 1983 Jul;113(1):277-84. doi: 10.1210/endo-113-1-277.
We developed a method for establishing primary monolayer cultures of bovine parathyroid cells, characterized the cells morphologically, and studied the effects of calcium on PTH secretion and cellular proliferation. Fresh bovine parathyroid glands were enzymatically and mechanically dispersed, as previously described, using sterile solutions and apparatus. The cells were then plated at a density of 2.5 X 10(5) cells/cm2 into cluster wells and incubated at 37 C in a medium containing Dulbecco's Modified Eagle's and Ham's F-12 medium, 15% newborn calf serum, Hepes, antibiotics, and insulin. In 3-4 days, a near-confluent monolayer of polygonal-shaped cells with less than 10% fibroblasts was achieved. Electron microscopy revealed a homogeneous cell population containing electron-dense secretory granules as well as abundant rough endoplasmic reticulum and mitochondria, with no evidence of organelle swelling. Two parameters of cellular proliferation increased significantly in culture; viable cell number increased from 289,000 +/- 63,000 to 530,000 +/- 60,000 per well (P less than 0.02), and cellular protein increased from 40.00 +/- 1.40 to 177.60 +/- 57.10 micrograms/well (P less than 0.035). On an hourly basis, cultured cells showed a greater secretory rate than that of acutely dispersed cells (9.3 vs. 5.0 ng/10(5) cells X h, respectively). Unlike normal bovine parathyroid cells, however, high calcium concentrations inhibited PTH secretion only slightly (21.2 +/- 4.7%) and had no effect on cellular proliferation. Addition of the divalent cation ionophore A23187, on the other hand, inhibited PTH release by 50% or more, suggesting that the secretory apparatus is responsive to increases in the cytosolic calcium concentration. The present study reveals that bovine parathyroid cells in primary culture proliferate and secrete PTH in vitro. These cells display, however, a generalized decrease in sensitivity to the suppressive effects of extracellular calcium on hormonal secretion and cellular proliferation comparable to that of pathological parathyroid tissue. Thus, this cell culture system may provide a useful model for investigating the relationship between secretion and proliferation in normal and abnormal tissue.
我们开发了一种建立牛甲状旁腺细胞原代单层培养物的方法，对细胞进行了形态学表征，并研究了钙对甲状旁腺激素（PTH）分泌和细胞增殖的影响。如前所述，使用无菌溶液和器械，通过酶解和机械分散新鲜牛甲状旁腺。然后将细胞以2.5×10⁵个细胞/cm²的密度接种到多孔培养板中，在含有杜尔贝科改良伊格尔培养基和哈姆F-12培养基、15%新生牛血清、羟乙基哌嗪乙磺酸（Hepes）、抗生素和胰岛素的培养基中于37℃孵育。3 - 4天后，获得了近汇合的单层多边形细胞，其中成纤维细胞少于10%。电子显微镜显示细胞群体均匀，含有电子致密的分泌颗粒以及丰富的粗面内质网和线粒体，没有细胞器肿胀的迹象。培养过程中细胞增殖的两个参数显著增加；每孔活细胞数从289,000±63,000增加到530,000±60,000（P＜0.02），细胞蛋白从40.00±1.40增加到177.60±57.10微克/孔（P＜0.035）。按小时计算，培养细胞的分泌速率高于急性分散的细胞（分别为9.3对5.0 ng/10⁵个细胞×小时）。然而，与正常牛甲状旁腺细胞不同，高钙浓度仅轻微抑制PTH分泌（21.2±4.7%），对细胞增殖没有影响。另一方面，添加二价阳离子载体A23187可抑制PTH释放50%或更多，这表明分泌装置对胞质钙浓度的增加有反应。本研究表明，原代培养的牛甲状旁腺细胞在体外增殖并分泌PTH。然而，这些细胞对细胞外钙对激素分泌和细胞增殖的抑制作用普遍敏感性降低，与病理性甲状旁腺组织相当。因此，这种细胞培养系统可能为研究正常和异常组织中分泌与增殖之间的关系提供一个有用的模型。
