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

我们发现成牙细胞（OB）与成牙细胞层下其他类型的牙髓细胞（PC）之间存在功能联系。分离和体外免疫：从猫和人牙髓中新鲜分离出牙髓外细胞，包括成齿细胞。融合筛选后，选择标记成牙本质细胞的单克隆抗体。膜片钳技术：采用单极过程和卵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性，然后进行了nystatin穿孔的全细胞配置。得到了电流与电压之间的简单线性关系。染料偶联：将路西法黄通过离子电泳注射到两种细胞中的一种时，另一种细胞也被标记。但未偶联的细胞，即使离细胞很近，也没有被标记，这表明路西弗黄并没有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种类的细胞中。间隙结：EM研究表明上述联轴器为间隙结。在我们确认将路西法黄注入OB或将PC转移到其他类型的细胞后，将细胞固定并用于EM研究。在两种细胞之间，总能观察到间隙连接。电耦合的电生理证据：电流可以双向通过上述形态学证实的结构。然而，观察到不对称转导。这些结果表明，OBs和pc在功能上相互联系，并作为一个网络工作，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.