Ultrastructural investigation of the membrane modifications of osteoclasts

本研究的主要目的是观察骨细胞超微结构的变化，特别是骨吸收破骨细胞的变化。目前，快速冷冻被认为是最好的，但困难的方法，以保持细胞结构在其天然状态尽可能。冷冻复制品（FR）清楚地显示了膜内部。膜颗粒（MP）的FR显示不同的破骨细胞的膜域的大小，形状和分布模式。特定的杆状MP定位于皱褶缘（RB）膜内。这种类型的MP讨论有关的质子泵ATP酶。RB拥有最密集的MP。在透明带膜上，MP呈簇状分布，这可能是细胞粘附的原因。另一个我们的实验使用快速冷冻，然后冷冻置换显示了一些有趣的发现破骨细胞的结构。RB膜由细胞骨架成分支撑。在RB内，观察到一些封闭的接触，如反射性间隙连接，其功能作用尚不清楚。与常规固定的样品相比，快速冻存的破骨细胞表现出明显的差异，各种细胞器、膜结构的保存都有明显的改善。在细胞质内，管泡状结构占据在细胞器之间，这种结构通常不能通过固定保存下来。来自吸收骨基质的碎片羟基磷灰石晶体位于管状泡状结构内。这一发现表明了钙摄入的可能途径。我们计划在以后发表我们在目前的科学研究资助金支持下工作期间获得的各种数据。

Main purpose of our study is to investigate the morenative state of bone cell ultrastructure, especially bone resorbing osteoclasts. At the present, quick freezing is believed to be the best but difficult way to preserve cell structure in its native state as possible. Freeze replicas (FR) showed clearly the membrane interior. Membrane particles (MP) by FR showed a different size, shape and distribution pattern of various membrane domains of osteoclasts. Specific rodshaped MP were localized within the ruffled border (RB) membrane. This type of MP is discussed in relation to the proton pump ATPase. RB had the most intensive MP. In the attaching clear zone membrane, MP showed a cluster fashion which may be responsible for cell attachment. Another our experiment using quick freezing followed by freeze-substitution showed several interesting findings about osteoclast structures. RB membrane were supported by cytoskeletal components. Within RB, some closed contacts were observed like the reflexive gap junction which its functional role is obscure. Comparing with the routinely fixed samples, quick frozen osteoclasts showed remarkable differences; the preservation of various organelles, membrane structures were dramatically improved. Within the cytoplasm, tuburovesicular structure which was never preserved by routinely fixation occupied among the cell organelles in the cytoplasm. Fragmented hydroxyapatite crystals derived from resorbed bone matrix were located within the tubulovesicular structures. This finding indicates the possible pathway for up-take calcium.We are planning, hereafter, to publish various data which were obtained during our work supported by the present Grant-in-Aid for Scientific Research.