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

特异性单克隆抗体对成牙本质细胞保护机制的电生理生化研究

基本信息

批准号：
10470404
负责人：
IKEDA Hideharu
金额：
$ 8.58万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

项目摘要

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.

我们发现成牙本质细胞（OB）和成牙本质细胞层下的其他类型牙髓细胞（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克隆抗体.膜片钳技术：具有单极突起和椭圆形细胞体的细胞用于膜片钳记录。我们成功地记录了单通道活动，然后进行制霉菌素穿孔的全细胞配置。得到了电流与电压的简单线性关系.染料偶联：当荧光黄被注入两种类型的细胞之一，离子电渗，另一种类型的细胞也被标记。而未偶联的细胞，即使离细胞很近，也没有被标记，说明荧光黄并没有渗漏到细胞外空间标记细胞膜，而是通过染料偶联扩散到不同种类的细胞中。缝隙连接：电镜研究表明上述偶联为缝隙连接。在我们确认注入OB或PC的荧光黄转移到其他类型的细胞后，将细胞固定并用于EM研究。两种细胞之间的缝隙连接总是存在的.电耦合的电生理学证据：电流可以双向通过上述形态学证实的结构。这些结果表明，OBs和PC在功能上是相互联系的，并作为一个网络工作，OBs作为一个传感器的外部刺激。