Lee E R, Lamplugh L, Leblond C P, Mordier S, Magny M C, Mort J S
Electron Microscopy Unit, Shriners Hospital for Children, Montreal, Quebec, Canada.
Anat Rec. 1998 Sep;252(1):117-32. doi: 10.1002/(SICI)1097-0185(199809)252:1<117::AID-AR10>3.0.CO;2-R.
In view of the extensive lysis of hyaline cartilage known to take place during endochondral bone formation, the current study was designed to test the hypothesis that metalloproteinases are the agents that mediate this lysis. Since these enzymes have been shown in vitro to cleave the core protein of the major proteoglycan of cartilage, aggrecan, at the Asn341-Phe342 bond, an immunohistochemical method has been developed to find out whether or not there are sites in the growth plate of the rat tibia where cleavage of this bond takes place. The cleavage of aggrecan by metalloproteinases is followed by the retention of the fragment known as G1, for it includes the G1 domain. Since the G1 fragment terminates in the amino acid residues ...FVDIPEN, we prepared an antiserum against FVDIPEN, confirmed its specificity, then applied it to the growth plate of 21-day-old rat tibia in the hope of localizing the G1 fragments. The antiserum specificity was shown by its recognition of the ...FVDIPEN sequence at the C-terminus of peptides and of G1 fragments produced by aggrecan cleavage. When the antiserum was applied to Western blots of guanidinium chloride extracts prepared from epiphyseal growth plate, it recognized two species (56 and 52 kDa), which differed only in the degree of glycosylation. These fragments were comparable in size to the G1 fragments generated by the action of recombinant metalloproteinase in vitro, thus confirming antiserum specificity for these fragments. Applying the antiserum to cryosections of 21-day-old rat tibiae revealed immunostaining at two intensities within the growth plate matrix: a strong staining was observed in a 1-5 microm-wide layer designated "peripheral" matrix, which borders the epiphyseal and metaphyseal marrow spaces as well as the perichondrium, while a weak staining was found in the rest of the plate, designated "central" matrix. The abundance of G1 fragments terminating in ...FVDIPEN in the peripheral matrix indicates that this is where the growth plate is lysed to achieve longitudinal and latitudinal bone growth. The site where metalloproteinases exert their main lytic activity is a thin layer of matrix separating central from peripheral matrix.
鉴于已知在软骨内成骨过程中会发生透明软骨的广泛溶解，本研究旨在验证金属蛋白酶是介导这种溶解的因子这一假说。由于这些酶在体外已被证明能在Asn341 - Phe342键处切割软骨主要蛋白聚糖聚集蛋白聚糖的核心蛋白，因此开发了一种免疫组织化学方法来确定大鼠胫骨生长板中是否存在该键被切割的位点。金属蛋白酶切割聚集蛋白聚糖后会保留称为G1的片段，因为它包含G1结构域。由于G1片段以氨基酸残基...FVDIPEN结尾，我们制备了一种针对FVDIPEN的抗血清，确认其特异性，然后将其应用于21日龄大鼠胫骨的生长板，以期定位G1片段。抗血清的特异性通过其对肽C末端的...FVDIPEN序列以及聚集蛋白聚糖切割产生的G1片段的识别得以体现。当将抗血清应用于从骨骺生长板制备的氯化胍提取物的蛋白质印迹时，它识别出两种蛋白（56和52 kDa），它们仅在糖基化程度上有所不同。这些片段的大小与重组金属蛋白酶在体外作用产生的G1片段相当，从而证实了抗血清对这些片段的特异性。将抗血清应用于21日龄大鼠胫骨的冷冻切片显示，生长板基质内有两种强度的免疫染色：在一个1 - 5微米宽的层（称为“外周”基质）中观察到强染色，该层与骨骺和干骺端骨髓腔以及软骨膜相邻，而在生长板的其余部分（称为“中央”基质）发现弱染色。外周基质中以...FVDIPEN结尾的G1片段的大量存在表明，这就是生长板发生溶解以实现纵向和横向骨生长的部位。金属蛋白酶发挥其主要溶解活性的部位是将中央基质与外周基质分隔开的一层薄基质。
