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

我们对破骨细胞和成骨细胞之间的相关性进行了 6 个系列的实验： 1）通过单细胞上 Ca 的荧光标记来确定破骨细胞中 Ca 浓度的变化，并使用 Olympus OSP-3 测定降钙素对 Ca 运动的影响。 CT 检测破骨细胞内的 Ca 运动短暂增强，但随后迅速下降，之后细胞恢复其功能活动。在骨组织上培养的破骨细胞上重复实验，然后使用 Olympus CASARUS 对破骨细胞进行二维分析。成骨细胞内的 Ca 转运途径及其通过 CT 的抑制得到了证实。 2)用Olympus IMT2-21CA3通过观察添加CT后肌动蛋白丝和中间丝的定位变化来评估CT对成骨细胞骨架的诱导变化。 3) 为了阐明破骨细胞多核巨细胞 (MNGC) 的分化，我们在维生素 D3 存在下共培养了成骨细胞和脾细胞。由此形成的 PMNGC 是 TRACPase 阳性并表达 CT 受体，这导致将它们视为骨形成的。此外，我们发现破骨细胞 MNGC 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，VDR密集定位于细胞核中，尤其是异染色质中，并且VDR在细胞质内沿着微管转运到达细胞核。 5）我们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的分化/活化之间的相互作用。 ConA、PNA、WGA、UEA-1和MPA在破骨细胞膜表面以及两类细胞相互接触的破骨细胞的膜表面上反应强烈。接触表面也呈强硫酸乙酰肝素蛋白聚糖阳性。这两项发现表明碳水化合物链参与破骨细胞和成骨细胞之间的细胞间识别，以及生长因子和细胞因子的局部储存。 6）我们试图通过细胞化学阐明破骨细胞和骨细胞之间的相互关系，由此我们发现后者细胞与前者发生细胞-细胞相互作用：骨细胞可以通过这种机制促进骨吸收。较少的

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