Isolated primary osteocytes express functional gap junctions in vitro
Isolated primary osteocytes express functional gap junctions in vitro
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DOI：
10.1007/s00441-005-0066-3
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发表时间：
2006-02-01
影响因子：
3.6
通讯作者：
Väänänen, H
中科院分区：
文献类型：
--
作者：
Gu, GL;Nars, M;Väänänen, H
骨细胞是骨骼中最丰富的细胞类型，嵌入矿化骨基质中。由于骨细胞的位置和缺乏主要的骨细胞分离方法，骨细胞的特征仍然很差。关于骨细胞的细胞生物学的数据尤其有限。我们通过反复酶消化和脱钙从大鼠皮质骨中分离出原代骨细胞。培养1天后，分离的骨细胞表现出典型的骨细胞形态，细胞与细胞通过长突起接触。这些细胞的碱性磷酸酶呈阴性或弱阳性，但表达高水平的骨钙素、PHEX（与 X 染色体上的内肽酶同源的磷酸盐调节基因）和 DMP1（牙本质基质蛋白 1）。这些细胞还显示出连接蛋白 43 的斑片膜染色。为了研究离体骨细胞间隙连接的功能，我们显微注射了罗丹明标记的右旋糖酐（MW：10,000）和荧光黄（MW：457），发现荧光黄迅速传播到周围的几个细胞，而右旋糖酐保留在注射的细胞中。庚醇和18α-甘草次酸抑制荧光黄的转移。这清楚地表明培养的骨细胞中存在功能性间隙连接。有包膜病毒，如水泡性口炎病毒和甲型流感病毒，被用于研究细胞极性。我们无法证明培养物中分离的原代骨细胞中包膜病毒的质膜极化。我们的结果表明，骨细胞不像成骨细胞（它们的前体）那样具有顶端和基底外侧质膜结构域。
The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18 alpha-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.