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

甲状旁腺素相关蛋白（PTHrP）是1987年发现的一种具有甲状旁腺素样功能和结构的蛋白质，是恶性肿瘤体液性高钙血症的主要因素。PTHrP在许多正常组织中产生，包括泌乳乳腺，然而PTHrP的作用仍不清楚。从产犊后1周的泌乳荷斯坦奶牛中收集乳腺组织。将乳腺组织酶促消化以释放腺泡。将细胞接种在24孔胶原包被的塑料培养板中。在第6天融合后，向培养基中添加不同浓度的莫能菌素、秋水仙素和细胞松弛素B以研究PTHrP产生的变化。莫能菌素和细胞松弛素B以剂量依赖性方式抑制PTHrP的产生，而colehicine则使PTHrP的产生略有增加。用逆转录-聚合酶链反应和原位杂交技术研究PTHrP mRNA的表达下降时，用莫能菌素处理的细胞。提示乳腺上皮细胞PTHrP的产生可能与细胞质中肌动蛋白丝的形成有关，而与微管的形成无关，提示乳腺上皮细胞PTHrP可能通过组成性途径而非调控性途径分泌。形态学上，莫能菌素处理的乳腺上皮细胞肿胀。这一发现也表明，PT 1 JrP的生产受到抑制的细胞内囊泡运输的抑制。

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.