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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本质细胞，将其培养在含有α-MEM和10%FCS的培养皿或牙本质切片上，结果显示人破牙本质细胞呈抗酒石酸酸性磷酸酶阳性，并在牙本质切片上形成吸收陷窝。我们观察了5 mU/ml降钙素对破牙细胞形态学的影响，但未观察到形态学改变。由于破牙细胞分离的困难和样品（乳牙）的缺乏，我们不能从培养的人破牙细胞中获得很多数据。我们还研究了经E-64处理的大鼠破骨细胞中组织蛋白酶L、I型胶原和大鼠唾液半胱氨酸蛋白酶抑制剂-3（RSC-3）的免疫细胞化学定位变化。组织蛋白酶L的免疫反应是细胞外非常弱，但在E-64处理的破骨细胞胞内强。在E-64处理的破骨细胞的空泡中发现了I型胶原的细胞内免疫反应性，而在对照破骨细胞的任何空泡中均未发现I型胶原的细胞内免疫反应性。在E-64处理的RSC-3免疫反应中，皱褶边缘的细胞质呈阳性，并且RSC-3阳性皱褶边缘的尖端似乎深深地进入骨基质。在细胞内，RSC-3的颗粒状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，细胞外释放的组织蛋白酶L不足以抑制胶原的降解，RSC-3可能与破骨细胞中溶酶体中组织蛋白酶L的抑制有关。