Everts V, Hoeben K, Beertsen W
Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.
Bone Miner. 1993 Jul;22(1):43-55. doi: 10.1016/s0169-6009(08)80080-3.
Tissue inhibitors of metalloproteinases (TIMPs) play an important role in the regulation of the activity of matrix metalloproteinases (MMPs) such as collagenase, stromelysin and gelatinase. Although it has been shown that upon culturing bone tissue releases relatively large amounts of TIMP, little is known as to the source of the inhibitor. In an attempt to investigate this in more detail calvarial bone explants from young rabbits were cultured in serum-free medium. The explants were cultured with or without adhering periosteum. In some experiments solitary periosteal fragments were maintained in the absence of bone. Media were analyzed for the presence of TIMP by immunoblotting and ELISA as well as for their capacity to inhibit the activity of collagenase. In addition, TIMP was immunolocalized in cryosections of the explants. The data demonstrated that bone-conditioned medium contained significantly more (2-10 times) collagenase inhibitor than periosteum-conditioned medium. Removal of the (convex and/or concave) periosteum from the calvariae did not significantly affect the amount of inhibitor released. Immunoblots and ELISA showed the presence of TIMP in the media, being more in bone- than in periosteum-conditioned medium. In immunolabeled cryosections TIMP appeared to be present in osteoblast-like cells lining both the outer bone surface as well as the endosteal spaces. Label was also found in a number of osteocyte lacunae. The periosteum was almost negative. It is suggested that TIMP contributes to the regulation of MMP-activity involved in the remodeling and turnover of bone.
金属蛋白酶组织抑制剂（TIMPs）在调节基质金属蛋白酶（MMPs）如胶原酶、基质溶解素和明胶酶的活性中起重要作用。尽管已表明骨组织在培养时会释放相对大量的TIMPs，但对于该抑制剂的来源却知之甚少。为了更详细地研究这一问题，将幼兔的颅骨外植体在无血清培养基中培养。外植体在有或没有附着骨膜的情况下进行培养。在一些实验中，单独的骨膜碎片在无骨的情况下进行培养。通过免疫印迹和酶联免疫吸附测定（ELISA）分析培养基中TIMP的存在情况以及它们抑制胶原酶活性的能力。此外，TIMP在冷冻切片的外植体中进行免疫定位。数据表明，骨条件培养基中含有的胶原酶抑制剂比骨膜条件培养基多得多（2至10倍）。从颅骨上去除（凸面和/或凹面）骨膜对释放的抑制剂数量没有显著影响。免疫印迹和ELISA显示培养基中存在TIMP，骨条件培养基中的TIMP比骨膜条件培养基中的更多。在免疫标记的冷冻切片中，TIMP似乎存在于骨外表面和骨内膜间隙的成骨样细胞中。在一些骨细胞陷窝中也发现了标记。骨膜几乎呈阴性。提示TIMP有助于调节参与骨重塑和更新的MMP活性。
