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

我们从乳牙中分离出人破牙本质细胞，在含有α-MEM和10%FCS的培养皿或牙本质片上进行培养。人破牙本质细胞呈耐酸酸性磷酸酶阳性，在牙本质片上形成吸收陷窝。我们观察了5mU/ml降钙素对破牙细胞形态的影响，但没有观察到形态变化。由于破牙本质细胞分离的困难和样本(乳牙)的缺乏，我们无法从培养的人破牙本质细胞中获得大量的数据。我们还检测了组织蛋白酶L、I型胶原和大鼠唾液胱抑素-3在E-作用下大鼠破骨细胞免疫细胞化学定位的变化。E-处理的破骨细胞胞外组织蛋白酶L免疫反应很弱，细胞内组织蛋白酶免疫反应较强。E-处理组破骨细胞的I型胶原免疫反应阳性细胞位于空泡内，而对照组破骨细胞的I型胶原免疫反应不在空泡内。E-组RSC-3免疫反应显示，皱纹边缘胞浆呈阳性，RSC-3阳性-皱纹边缘尖端似乎深入到骨基质中。在细胞内，RSC-3的颗粒反应产物见于空泡中。这些结果提示，E-处理的破骨细胞胞外释放的组织蛋白酶L不足以抑制胶原的降解，而RSC-3可能与抑制破骨细胞内溶酶体中的组织蛋白酶L有关。

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.