Mechanism of Parathyroid hormone-Related Protein Production and Paracrine Function on Bovine Mammary Epithelial Cells

甲状旁腺激素相关蛋白(PTHrP)具有甲状旁腺激素样的功能和结构，1987年被鉴定为恶性肿瘤体液高钙血症的主要因素。甲状旁腺素受体在包括哺乳期乳腺在内的许多正常组织中都有表达，但其作用机制尚不清楚。取产奶后1周荷斯坦奶牛的乳腺组织。乳腺组织被酶消化以释放腺泡。将细胞接种于24孔胶原蛋白涂层塑料培养板中。在第6天融合后，在培养液中添加不同浓度的莫能菌素、秋水仙素和细胞松弛素B，以观察PTHrP产量的变化。莫能菌素和细胞松弛素B可使甲状旁腺素rP的生成量呈剂量依赖性下降，而秋水仙素刺激细胞的甲状旁腺素生成量略有增加。用逆转录聚合酶链式反应和原位杂交技术研究莫能菌素处理细胞后PTHrP基因的表达。提示乳腺上皮细胞PTHrP的产生可能与细胞质内肌动蛋白细丝的形成有关，而与微管的形成无关，提示PTHrP可能是通过结构性途径而非调控途径从乳腺上皮细胞中分泌出来的。在形态上，莫能菌素处理的乳腺上皮细胞肿胀。这一发现还表明，PT1JrP的产生受到细胞内小泡运输的抑制。

Parathyroid hormone-related protein (PTHrP) that had parathyroid hormone-like function and Structure was identified in 1987 as a major factor of humoral hypercalcemia of malignancy. PTHrP is produced in many normal tissues including the lactating mammary gland, however the role of PTHrP is still unclear. Mammary tissues were collected from lactating Holstein dairy cows at 1 week after calving. The mammary tissues were enzymatically digested to release glandular acini. The cells were plated in 24-well collagen-coated plastic culture plates. After confluent at day 6, various concentrations of monensin, colchicine, and cytochalasin B were supplemented to the medium to investigate the change of PTHrP production. The PTHrP production decreased in the cells supplemented with monensin and cytochalasin B in a dose-dependent manner, whereas its production slightly increased by the stimulation with colehicine. Expression of PTHrP mRNA investigated by using reverse transcription-polymerase chain reaction and in situ hybridization techniques decreased when the cells were treated with monensin. This study indicated that PTHrP production from mammary epithelial cells might associated with the formation of actin filaments in the cytoplasm but not with that of microtubules, suggesting that PTHrP might secrete from mammary epithelial cells by the constitutive pathway but not the regulated pathway. Morphologically, mammary epithelial cells treated with monensin were swollen. This finding also suggested that PT1JrP production was inhibited by the inhibition of intracellular vesicle transport.