The repair of cartilage disorder with parathyroid hormone related peptide.

甲状旁腺素相关肽（PTHrP）最初被鉴定为与恶性肿瘤相关的高钙血症的病原体。PTHrP与PTH结合并激活相同的受体。缺失PTHrP或PTH/PTHrP受体基因的小鼠在出生后立即死亡，并表现出多种骨骼异常。PTHrP-少和PTH/PTHrP受体-少小鼠的软骨发育不良表型清楚地表明PTHrP及其受体在调节软骨内骨发育中的重要生理作用。与从这些突变小鼠获得的数据一致，已显示PTH对从胎儿软骨分离的软骨细胞发挥促有丝分裂作用。与对胎儿软骨细胞增殖的刺激作用相反，已显示PTH对从出生后动物分离的软骨细胞不具有促有丝分裂作用。本研究的目的是验证PTHrP及其受体在调节细胞增殖中发挥作用的假说。 ...更多信息 生长板和关节软骨的生长在出生后的动物，PTHrP参与的疾病过程后，软骨。我们检测了PTH/PTHrP的各种类似物（包括C-末端肽）对无血清培养的出生后生长板和关节软骨细胞生长的影响，然后我们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：从4周龄的家兔肋骨生长板和膝关节软骨中分离软骨细胞，老年兔胰蛋白酶和胶原酶消化。将软骨细胞以10，000个/I型胶原包被的96孔板接种，并维持在补充有卵脂蛋白的α MEM中（20 μ g/ml），转铁蛋白（10 μ g/ml）、氢化可的松（10- 8 M）、bFGF（ng/ml）和胰岛素（10 μ g/ml）。当培养物达到预融合时，将细胞在具有上述因子（胰岛素除外）的相同培养基中预孵育24小时。然后，我们测量了加入PTH [1-34]或其他片段后[3 H]胸苷掺入DNA的情况。在相同条件下，测定了DNA含量、[35 S]硫酸盐掺入蛋白多糖（PG）的量。此外，在全膝关节置换术中获得的软骨通过免疫染色检查PTHrP的定位（n=8）。PTH [ 1-34]、PTHrP [1-34]和PTH [1-84]剂量依赖性地显著增加出生后关节软骨细胞的DNA合成，艾德100为10- 7 M。PTH/PTHrP可增加关节软骨细胞DNA含量及生长板和关节软骨细胞对PG的[35 S]硫酸参入。免疫组化检测PTHrP在软骨细胞中的比例很高（6/8）。讨论：在一些报告中，PTH/PTHrP显示对DNA合成的影响不大，由出生后的软骨细胞在细胞培养中，可能是因为PTH/PTHrP的效果可能被掩盖的10%胎牛血清包括在传统的培养系统。在这项研究中，我们可以证明，PTH/PTHrP肽刺激出生后软骨细胞在化学成分确定的培养基中的生长。PTH/PTHrP的这种促有丝分裂作用在关节软骨细胞中更为突出。PTHrP在骨关节炎和类风湿性关节炎患者的软骨中检测到。虽然有几种解释是合理的这些结果，更多的研究受体表达或受体后信号通路将是必要的。少

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