Electorophysiological and biochemistrical study on protecture mechaninism of odontoblasts identified with specific monoclonal antibodies

我们展示了成牙本质细胞(OB)和成牙本质细胞层下其他类型的牙髓细胞(PC)之间的功能联系。分离和体外免疫：从猫和人牙髓中新鲜分离出包括成牙本质细胞在内的外髓细胞。经融合筛选，筛选出标记成牙本质细胞的单抗。膜片钳技术：采用具有单极突起和椭圆形细胞体的细胞进行膜片钳记录。我们成功地记录了单通道活动，然后进行了制霉菌素穿孔的全细胞构型。得到了电流与电压之间的简单线性关系。染料偶联：当荧光黄被注入两种离子导入细胞中的一种时，另一种细胞也被标记。但是未偶联的细胞，即使它们离细胞很近，也没有被标记，这表明荧光黄不会泄漏到细胞外空间并标记细胞膜，而是通过染料偶联扩散到不同类型的细胞。缝隙结：电子显微镜研究表明，上述耦合为缝隙结。在我们确认荧光黄注射到OB或PC中转移到另一种类型的细胞(S)后，固定细胞并用于EM研究。两种细胞之间均可观察到缝隙连接。电耦合的电生理证据：电流可以双向通过上面从形态上证实的结构。这些结果表明OBS和PC在功能上是相连的，并且作为一个网络工作，OBS作为外部刺激的传感器工作。

We showed the functional link between odontoblasts (OB) and other type of pulpal cells (PC) beneath the odontoblastic layer.1. Isolation and in vitro immunization : From cat and human dental pulp, outer pulpal cells including odontoblasts were freshly isolated. After fusion and screening, monoclonal antibodies that labeled odontoblasts were chosen.2. Patch-clamp technique : Cells having a monopolar process and an oval cell body were used for patch-clamp recording. We succeeded to record single-channel activity and then nystatin-perforated whole-cell configuration was performed. Simply linear relation between current and voltage was obtained.3. Dye-coupling : When lucifer yellow was injected into one of two types of cells with iontophoresis, the other type of cell was also labeled. But uncoupled cells, even though they were close to the cells, were not labeled, suggesting that lucifer yellow did not leak to the outer-cellular space and labeled the cell membrane, but spread into different kind of cells through dye-coupling.4. Gap-junction : EM study showed that the couplings mentioned above were gap-junctions. After we confirmed that lucifer yellow injected in an OB or a PC transferred to the other type of cell(s), cells were fixed and used for EM study. Between two types of cells, gap-junctions were always observed.5. Electrophysiological evidence of electrical coupling : Current could pass bidirectionally through the structure confirmed above morphologically. However, asymmetric transduction was observed.These results demonstrate that OBs and PCs are functionally linked and work as a network and that OBs work as a sensor of external stimuli.