Cell biological studies on cell-cell, cell-matrix interaction in bone metabolism.

我们对破骨细胞和成骨细胞的相关性进行了6个系列的实验：1)通过荧光标记单个细胞上的钙来测定破骨细胞中钙浓度的变化，并使用Olympus sp -3观察降钙素对钙运动的影响。CT可短暂增强破骨细胞内钙离子的运动，但很快减弱，之后细胞恢复功能活动。在骨组织培养的破骨细胞上重复实验，并用Olympus CASARUS对破骨细胞进行二维分析。CT证实了钙在破骨细胞内的转运途径及其抑制作用。2)采用Olympus IMT2-21CA3，通过观察加入CT后肌动蛋白丝和中间丝的定位变化，评估CT对成骨细胞骨架的影响。3)为了明确破骨多核巨细胞（MNGCs）的分化，我们将成骨细胞和脾细胞在维生素D3存在下共培养。由此形成的PMNGCs是tracpase阳性并表达CT受体，因此认为它们具有成骨性。此外，我们发现破骨MNGCs的形成需要与成骨细胞的直接细胞间接触。4)我们还通过体外和体内免疫细胞化学证明了VDR密集地定位于细胞核，特别是在异染色质中，并且VDR在细胞质内通过微管和沿微管运输到达细胞核。5)通过凝集素组织化学和免疫细胞化学研究了破骨细胞和成骨细胞分化/活化的相互作用。ConA、PNA、WGA、UEA-1和MPA在破骨细胞和破骨细胞接触处的膜表面反应强烈。接触表面也呈强烈的硫酸肝素蛋白聚糖阳性。这两项发现表明，碳水化合物链参与了破骨细胞和成骨细胞之间的细胞间识别，以及局部生长因子和细胞因子的储存。6)我们试图通过细胞化学的方法阐明破骨细胞和骨细胞之间的相互关系，发现后者与破骨细胞之间存在细胞间相互作用，骨细胞通过这一机制促进骨吸收。少

We conducted 6 series of experiments on the correlation between osteoclasts and osteoblasts : 1) Changes in Ca concentration in osteoclasts was determined by fluorescence-labeling of Ca on single cells, and the effect of calcitonin on Ca movement using an Olympus OSP-3. Ca movement within osteoclasts was transiently enhanced by CT but rapidly decreased, after which the cells recovered their functional activities. The experiment was repeated on osteoclasts cultured on bone tissue, then the osteociasts were also submitted to a 2-dhnentional analysis using an Olympus CASARUS. Ca transport pathway within osteociasts and its inhibition by CT was evidenced. 2) Changes induced by CT on osteociast cytoskeleton was assessed with an Olympus IMT2-21CA3, by observing the changes in the localization of actin filametns and intermediate filaments occurring after addition of CT. 3) To clarify the differentiation of osteoclastic multinuclear giant cells (MNGCs), we cocultured osteoblasts and spleen cel … More ls in the presence of vitamin D3. The PMNGCs thus formed were TRACPasepositive and expressed CT receptors, which lead to consider them as osteociastic. Moreover, we found that a direct cell-to-cell contact with osteoblasts was required in the formation of osteoclastic MNGCs. 4) We also demonstrated by immunocytochemistry in vitro and in vivo that VDR was densely localized in the nucleus, especially in the heterochromatin, and that VDR was transported within the cytoplasm over and along microtubules to reach the nucleus. 5) We studied the interaction between differentiation/activation of osteoclasts and osteoblasts by lectin histochemistry and immunocytochemistry. Reaction of ConA, PNA, WGA, UEA-1 and MPA was intense on the membrane surface of osteoclast and osteoclasts where the two types of cells were in contact each other. The contact surface was also strongly heparan sulphate proteoglycan-positive. These two findings suggest an involvement of carbohydrate chains in the intercellular recognition between osteoclasts and osteoblasts, and to the local reservoir of growth factors and cytokines. 6) We attempted to clarify the inter-relationship between osteoclasts and osteocytes by cytochemistry, whereby we found that the latter cells enters in a cell-ceil interaction with the former : Osteocytes could promote bone resorption by this mechanism. Less