PHYSIOLOGY AND IMMUNOHISTOCHEMISTRY OF DENTAL PULP

了解成牙本质细胞的生理特性是一个重要的 进一步了解细胞机制，占 牙髓中的代谢和转导过程。间接证据 表明这种过程是由跨膜蛋白的改变介导的。 通过成牙本质细胞膜中的离子通道的电导。的最 这些离子途径的关键是控制钙周转的那些。 钙调节对于成牙本质细胞尤其重要，因为 细胞内和细胞外的钙水平都可能在 牙本质形成，这些细胞可能直接参与运输 钙的矿化基质。除了成矿作用， 成牙本质细胞离子通道也可能参与躯体感觉， 介导流体静压的变化和/或渗透压的变化 由机械或流体动力学扰动产生的梯度。主 拟议研究的目的是检查离子机制 作为这些细胞对以下因素的反应的基础， 牙髓中的牙本质发生和躯体感觉传导。在这些 研究我将使用膜片钳记录技术来监测单- 通道和全细胞电流在成牙本质细胞膜使用两者， 完整哺乳动物成牙本质细胞的体外和原位制备。在 初步研究，我已经确定了L型钙通道， 阳离子传导机械敏感（“拉伸激活”）通道， 这些细胞。拟议的研究将针对一个定量的 分析这些和其他传导，并将包括一个检查 牙本质营养因子、神经源性因子和炎症因子 与牙髓中的生理或病理生理反应有关。 在初步研究中，我还发现了神经样非成牙本质细胞 卫星细胞在牙髓细胞培养物中。具体地说， 培养的牙髓细胞含有电压门控钠电导， 动力学性质类似于神经胶质细胞或许旺细胞，神经元 在外周和中枢神经系统中发现的卫星细胞。 此外，牙髓细胞对抗体反应，这些抗体通常标记 再生神经系统中的细胞，以及发育中的牙髓细胞 牙齿。因此，这些培养物可以代表损伤、恢复， 和/或牙髓中非成牙本质细胞的成熟。在神经 系统，有人建议，这种卫星细胞可能有助于 邻近初级传入神经的敏感化，通过改变 它的钠电流的密度、分布或动力学性质。因此，在本发明中， 拟议研究的第二个目标是确定 牙髓细胞钠电流在牙本质细胞中可能具有类似的功能作用， 纸浆，通过监测其生理和动力学特性， 分布和密度的基础钠通道， 牙髓细胞培养物的成熟。在这些实验中，培养物将 用已知诱导分化的试剂处理，例如，生长因子， 地塞米松或DMSO，并与之并行进行膜片钳记录 免疫组织化学研究使用单克隆抗体的钠 频道本身

Understanding the physiological properties of odontoblasts is an important step towards understanding the cellular mechanisms that account for metabolic and transductive processes in dental pulp. Indirect evidence now suggests that such processes are mediated by alterations in transmembrane conductances via ion channels in the odontoblast membrane. Among the most critical of these ionic pathways are those that govern calcium turnover. Calcium regulation is especially important for odontoblast cells, since both intra- and extracellular calcium levels may fluctuate during dentinogenesis, and these cells may be directly involved in transporting calcium to the mineralizing matrix. In addition to mineralogenesis, odontoblast ion channels may also be involved in somatosensation, mediating changes in hydrostatic pressure and/or changes in the osmotic gradient produced by mechanical or hydrodynamic perturbation. The primary objective of the proposed research is to examine the ionic mechanisms underlying the response of these cells to factors associated with dentinogenesis and somatosensory transduction in dental pulp. In these studies I will use patch-clamp recording techniques to monitor single- channel and whole-cell current in the odontoblast membrane using both in vitro and in situ preparations of intact mammalian odontoblast cells. In preliminary studies I have identified an L-type calcium channel as well as a cation-conducting mechanosensitive ("stretch-activated") channel in these cells. The proposed studies will be directed towards a quantitative analysis of these and other conductances, and will include an examination of the effects of dentinotrophic, neurogenic, and inflammatory factors linked to physiological or pathophysiological responses in dental pulp. In preliminary studies l have also identified neural-like non-odontoblast satellite cells in explant-derived pulp cell cultures. Specifically, cultured pulp cells contained a voltage-gated sodium conductance with kinetic properties similar to those of glial or Schwann cells, neuronal satellite cells found in both the peripheral and central nervous systems. In addition, pulp cells were reactive to antibodies that typically label regenerating cells in the nervous system, as well as developing pulp cells in teeth. Thus, these cultures may represent a model for injury, recovery, and/or maturation of non-odontoblast cells in dental pulp. In the nervous system, it has been suggested that such satellite cells may contribute to sensitization of adjacent primary afferents, by alterations in the density, distribution or kinetic properties of it's sodium current. Thus, a second objective of the proposed research is to determine whether the pulp cell sodium current may have a similar functional role in dental pulp, by monitoring its physiological and kinetic properties, and the distribution and density of the underlying sodium channels during maturation of pulp cell cultures. In these experiments, cultures will be treated with agents known to induce differentiation, e.g., growth factors, dexamethasone, or DMSO, and patch-clamp recordings made in parallel with immunohistochemical studies using a monoclonal antibody to the sodium channel itself.