TRP/Pannexin1通路介导机械刺激相关性牙本质敏感的机制研究

Odontoblasts are closely related to the process of dentin hypersensitivity. They constitute the outermost cellular layer of the dental pulp and may transduce the external stimuli signals to the dentinal nerve. However, the mechanism involved remained to be fully elucidated. Our studies have found that: 1) the odontoblasts expressed mechanosensitive transient receptor potential (TRP) -TRPA1 and TRPV4; 2) the expression of TRPA1 and TRPV4 in odontoblasts was significantly up-regulated in the mechanical stimulated dentine hypersensitivity rats; 3) mechanical stimulation increases the intracellular Ca2+ concentration. It is known that Ca2+ can activate gap junction hemichannel Pannexin1. Then, the ATP will released from the activated Pannexin1, which will further activate the P2X receptor, and finally cause pain. However, it is unknown whether Pannexin1 is expressed in odontoblasts. It is recently reported that the activation of TRPA1 and TRPV4 channels in odontoblasts can stimulate ATP release. Therefore, we hypothesized that the Pannexin1 may mediate the ATP release of odontoblasts. Based on all of these, we thus put forth the hypothesis: the TRP/Pannexin1 pathway mediate the mechanical stimulated dentin hypersensitivity. In order to testify the hypothesis, this study will conduct the animal model of dentin sensitivity in vivo and cell culture in vitro based on our previous studies. It is believed that clarifying the related mechanism of mechanical stimulated dentin hypersensitivity will provide novel evidence and opportunity for the prevention and treatment of dentin hypersensitivity.

成牙本质细胞与牙本质敏感发生密切相关，其是牙髓感受外界刺激的“前哨战”，但其感受机制仍不清楚。我们前期研究发现：1）成牙本质细胞存在机械敏感性TRP通道TRPA1和TRPV4；2）机械刺激牙本质敏感大鼠的成牙本质细胞TRPA1和TRPV4表达显著上调；3）机械刺激可致胞外Ca2+内流。已知Ca2+内流能激活缝隙连接半通道Pannexin1，促进ATP释放，激活P2X受体，产生疼痛，但未知成牙本质细胞是否存在Pannexin1。最新研究发现采用TRPA1/TRPV4激活剂干预成牙本质细胞后，胞外ATP浓度显著增加，推测Pannexin1可能介导了成牙本质细胞ATP的释放，故提出“TRP/Pannexin1通路介导机械刺激相关性牙本质敏感假说”。我们将在前期基础上，拟采用机械刺激牙本质敏感模型进行动物和细胞实验论证该假说，明确机械刺激相关性牙本质敏感发病机制，为牙本质敏感防治提供新的证据和契机

牙本质敏感是最常见的口腔疾患之一，最新研究发现成牙本质细胞及牙髓与牙本质敏感的发生密切相关，但其感受机制仍不清楚，临床缺乏针对性预防和治疗。本课题组在前期预实验的基础上，提出了“TRP/Pannexin1通路介导机械刺激相关性牙本质敏感假说”。在实验实施过程中，因机械刺激牙本质敏感动物模型制备的难度大，效果不确定，因此课题组将机械刺激调整为漂白剂刺激。主要研究内容：1）动物实验：课题组利用临床常用诊室和家庭牙齿漂白凝胶（40%H2O2, 15%H2O2）对大鼠牙齿进行漂白，观察分析漂白凝胶刺激对大鼠牙髓和成牙本质细胞的影响，TRPA1和Pannexin1及炎症因子表达水平的变化。2）通过体外牙齿漂白模型，检测牙齿漂白凝胶使用前后对人牙髓细胞活性、氧自由基（ROS）、细胞内钙离子浓度、以及TRPA1、Pannexin1和炎症因子表达水平的变化。研究结果：1）免疫组化结果显示大鼠牙齿漂白后，牙髓组织和成牙本质细胞出现部分坏死，血管出现明显的扩张和充血；免疫荧光显示正常大鼠成牙本质细胞和牙髓组织仅表达少量TRPA1、 Pannexin1及IL6和TNFα，牙齿漂白后其表达均显著上升；2）体外漂白模型结果显示，漂白后人牙髓细胞活性显著降低，细胞ROS、细胞内钙离子浓度均显著增加，细胞TRPA1、 Pannexin1及IL6和TNFα表达显著上升；使用ROS清除剂NAC后，ROS对细胞的作用被阻断。这些结果均表明漂白凝胶通过上调ROS，激活成牙本质细胞和牙髓细胞上TRPA1通路，导致细胞内钙离子浓度增加，并进一步激活缝隙连接半通道Pannexin1，进而诱导牙漂白敏感的发生。这些研究结果进一步明确了牙漂白相关性牙本质敏感发病机制，为牙本质敏感防治提供了新的证据和契机。