The repair of cartilage disorder with parathyroid hormone related peptide.

甲状旁腺激素相关肽修复软骨损伤。

• 批准号: 08457396
• 负责人: YUTANI Yasutaka
• 金额: $ 0.77万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457396/
• 关键词: parathyroid hormone related peptide; cartilage tissue; chondrocytes; rheumatoid arthritis; osteoarthritis; 副甲状腺ホルモン関連ペプチド; 慢性関節リュウマチ

Parathyroid hormone-related peptide (PTHrP) was originally identified as a causative agent of hypercalcemia associated with malignancy. PTHrP binds to and activates the same receptor as PTH does. Mice missing both copies of the PTHrP or the PTH/PTHrP receptor gene die immediately after birth, and exhibit a multitude of skeletal abnormalities. The chondrodysplastic phenotype in both PTHrP-less, and PTH/PTHrP receptor-less mice clearly demonstrates important physiological roles of PTHrP and its receptor in regulating endochondral bone development. Consistent with the data obtained with these mutant mice, PTH has been shown to exert mitogenic effects on chondrocytes isolated from fetal cartilage. Contrary to the stimulatory effects on the proliferation of fetal chondrocytes, it has been shown that PTH does not have mitogenic effects on chondrocytes isolated from post-natal animals. The goal of this study is to test the hypotheses that PTHrP and its receptor play a role (s) in regulating g … More rowth of growth-plate and articular cartilage in postnatal animals, and that PTHrP is involved in disease processes that affter the cartilage. We examined the effects of various analogues of PTH/PTHrP including C-terminal peptides on the growth of postnatal growth-plate and articular chondrocytes in serum free culture, and then we examined PTHrP expression in cartilage from patients with osteoarthritis and rheumatoid arthritis.Materials and Methods : Rabbits chondrocytes were isolated from growth plates of ribs and articular cartilage of knee joints of 4-wk-old rabbits by trypsin and collagenase digestion. Chondrocytes were seeded at 10,000 per type I collagen coated 96-well plates, and maintained in alpha MEM supplemented with egg lipoprotein (20mug/ml), transferrin (10mug/ml), hydrocortisone (10-8M), bFGF (ing/ml) and insulin (10mug/ml) under 5% CO2 in air at 37*C.When cultures became preconfluent, the cells were preincubated for 24 h in same medium with above factors except insulin. We then measured [3H] thymidine incorporation into DNA after the additions of PTH [1-34] or other fragments. We also measured DNA contents, [35S] sulfate incorporation into proteoglycans (PG) in same condition. Furthermore, cartilages obtained at total knee arthroplasties were examined for localization of PTHrP by immunostaining (n=8).Results : PTH [ 1-34], PTHrP [1-34] and PTH [1-84] dramatically increased DNA synthesis by postnatal articular chondrocytes dose-dependently with an ED 100 of 10-7M.Furthermore, PTH/PTHrP increased the DNA content of articular chondrocytes and [35S] sulfate incorporation into PG by growth plate and articular chondrocytes. Immuno-histochemically PTHrP was detected in chondrocytes in affected cartilage with high ratio (6/8).Discussion : In several reports, PTH/PTHrP showed little effect on DNA synthesis by postnatal chondrocytes in cell cultures, probably because the effect of PTH/PTHrP may be masked by 10% fetal calf serum included in conventional culture systems. In this study we could demonstrate that PTH/PTHrP peptide stimulates the growth of postnatal chondrocytes in chemically defined medium. This mitogenic effect of PTH/PTHrP is more prominent in articular chondrocytes. PTHrP is detected immunohistochemically in cartilage from patients with both osteoarthritis and rheumatoid arthritis. Although several explanations are plausible for these results, more investigations on receptor expression or post receptor signaling pathway will be necessary. Less

甲状旁腺激素相关肽(PTHrP)最初被认为是与恶性肿瘤相关的高钙血症的病原体。PTHrP与PTH结合并激活与PTH相同的受体。缺少PTHrP或PTH/PTHrP受体基因的小鼠在出生后立即死亡，并表现出大量的骨骼异常。PTHrP缺失和PTH/PTHrP受体缺失小鼠的软骨发育异常表型清楚地表明了PTHrP及其受体在调节软骨内骨发育中的重要生理作用。与这些突变小鼠获得的数据一致，甲状旁腺素已被证明对从胎儿软骨分离的软骨细胞具有促有丝分裂作用。与促胎儿软骨细胞增殖作用相反，PTH对新生动物软骨细胞无促分裂作用。本研究的目的是验证甲状旁腺素受体及其受体在调节g-…中起作用的假设(S)。在出生后的动物中，生长板和关节软骨的生长更多，而且PTHrP参与了软骨后的疾病过程。我们在无血清培养条件下检测了PTH/PTHrP的C端肽类似物对生后生长板和关节软骨细胞生长的影响，并检测了骨关节炎和类风湿性关节炎患者软骨中PTHrP的表达。材料和方法：用胰酶和胶原酶消化法从4周龄兔的肋骨生长板和关节软骨分离软骨细胞。软骨细胞接种于10,000个I型胶原包被的96孔板中，在37℃空气中添加卵脂蛋白(20µg/m l)、转铁蛋白(10 m g/m l)、氢化可的松(10-8m)、碱性成纤维细胞生长因子(ing/m l)和胰岛素(10 m g/m l)，在相同的培养液中预培养24 h。然后，我们测量了添加PTH[1-34]或其他片段后[~3H]胸腺嘧啶核苷在DNA中的掺入。在相同的条件下，我们还测定了DNA含量，[35S]硫酸盐掺入蛋白多糖(PG)。结果：PTH[1-34]、PTHrP[1-34]和PTH[1-84]可剂量依赖性地促进生后关节软骨细胞DNA合成，ED100为10-7M。PTH/PTHrP还可增加关节软骨细胞DNA含量，并通过生长板和关节软骨细胞将[35S]硫酸盐掺入PG。免疫组织化学显示，PTH/PTHrP在软骨细胞中的表达比例较高(6/8)。讨论：在一些报道中，PTH/PTHrP对细胞培养的新生软骨细胞的DNA合成影响不大，可能是因为PTH/PTHrP的作用可能被常规培养体系中包含的10%胎牛血清所掩盖。在这项研究中，我们可以证明PTH/PTHrP肽在化学定义的培养液中刺激生后软骨细胞的生长。PTH/PTHrP的这种促有丝分裂作用在关节软骨细胞中更为显著。用免疫组织化学方法检测了骨性关节炎和类风湿性关节炎患者的软骨中甲状旁腺素受体的表达。尽管对这些结果有几种合理的解释，但更多关于受体表达或受体后信号通路的研究仍是必要的。较少

项目成果

Koike, T., Iwamoto, M., et al.: "Potent mitogenic effects of PTH/rP on postnatal rabbit chondrocytes in serum free culture" Orthopaedic Transaction. (1997)

Koike, T.、Iwamoto, M. 等人：“PTH/rP 对无血清培养物中产后兔软骨细胞的有效有丝分裂作用”《骨科汇刊》。