An immuno-electron microscopic study of the localization of cathepsins B and L and collagenase in cultured human odontoclasts

组织蛋白酶 B 和 L 以及胶原酶在培养的人破牙细胞中定位的免疫电子显微镜研究

基本信息

批准号：
07671975
负责人：
TANAKA Teruo
金额：
$ 1.54万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

We isolated human odontoclasts from primary teeth and cultured them on the culture dishes or dentine slices with alpha-MEM and 10% FCS.Human odontoclasts were tertrate-resistant acid phosphatase positive and formed resorption lacunae on the dentine slices. We examined the effect ot 5 mU/ml calcitonin on the morphological changes of odontoclasts, but no morphological changes were observed. Because of the difficulty of the isolation of odontoclasts and lack of the supply of the samples (primary teeth), we could not obtain many data from cultured human odontoclasts. Further investigations are needed for finding the efficient-isolation techniques and an optimal condition for human odontoclasts.We also examined the changes in the immunocytochemical localization of cathepsin L,type I collagen and rat salivary cystatin-3 (RSC-3) in rat osteoclasts treated with E-64. The immunoreactivity for cathepsin L was extracellularly very weak, but was strong intracellurlarly in the E-64-treated osteoclasts. The intracellular immuno-reactivity for type I collagen was found inthe vacuoles in the E-64-treated osteoclasts but not in any vacuoles in the control osteoclasts. In the immunoreaction for RSC-3 in the E-64-treated, the cytoplasm of the ruffled border was positive, and also the tips of the RSC-3 positive-ruffled border appeared to deeply enter into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in vacuoles. These finding suggests that in the E-64-treated osteoclasts, the extracellular release of cathepsin L was insufficient to suppress the degradation of collagen, and that RSC-3 is probably associated wit the inhibition of cathepsin L in the lysosomes in the osteoclasts.

我们从乳牙中分离出人破牙细胞，将其培养在含有α-MEM和10％FCS的培养皿或牙本质切片上。人破牙细胞呈四酸抗性酸性磷酸酶阳性，并在牙本质切片上形成吸收陷窝。我们检测了5 mU/ml降钙素对破牙细胞形态变化的影响，但没有观察到形态变化。由于破牙细胞的分离困难以及样本（乳牙）供应的缺乏，我们无法从培养的人类破牙细胞中获得很多数据。需要进一步的研究来寻找人破牙细胞的有效分离技术和最佳条件。我们还检测了经处理的大鼠破骨细胞中组织蛋白酶 L、I 型胶原和大鼠唾液胱抑素 3 (RSC-3) 免疫细胞化学定位的变化与E-64。 E-64 处理的破骨细胞中组织蛋白酶 L 的免疫反应性在细胞外非常弱，但在细胞内则很强。在 E-64 处理的破骨细胞的液泡中发现了 I 型胶原的细胞内免疫反应性，但在对照破骨细胞的任何液泡中都没有发现。在E-64处理的RSC-3的免疫反应中，皱边的细胞质呈阳性，并且RSC-3阳性皱边的尖端似乎深深地进入骨基质。在细胞内，RSC-3的颗粒反应产物存在于液泡中。这些发现表明，在E-64处理的破骨细胞中，组织蛋白酶L的细胞外释放不足以抑制胶原蛋白的降解，并且RSC-3可能与破骨细胞中溶酶体中的组织蛋白酶L的抑制有关。