海马胶质细胞内大麻素受体2（CB2R）参与正畸牙齿移动所致应激反应的机制研究

Orthodonic tooth movement can result in severe abnormal emotional responses, such as stress, anxiety and depression. In our previous experiment, we modified tooth movement model and found that this model can effectively simulated the tooth movement and cause obvious psychiatric behavioral change and stress reponses compared to control animals.Further study showed that the activation of astrocytes in the hippocampus were reduced in the tooth movement model. However, the pain-related neurons in the central nerve system keep intact. Cannabinoid receptor 2(CB2R) were largely expressed in the astrocytes of the hippocampus And the previous reports indicated that the CB2R played important role in the learning and memory.According to these evidences, we hypothesized that the immune responses induced by CB2R expressed in glial cell may be involved in this process. In the present project, we will focus on the mechanism of the tooth movement-related emotional responses in the hippocampus using method of orthodontics combined with the behavioral pharmacology, immunohistoloty, pre-embedding electronic microscopy and molecular biology, and then observe the function of CB2R on the abnormal emotional and stress responses, the dynamic changes of glial cell in the hippocampus, the ultrastructure relationship between the astrocyte and neuron. And further, we will observe the behavioral changes after up-regulation or down-regulation of CB2R use virus interference method.Finally, we will try to uncover the potential cellular and molecular mechanism. Our experiment will help to elucidate the central adjustment mechanism of psychiatric changes caused by the tooth movement, ultimately offer promising new targets for new drug developing and psychological intervention.

正畸牙齿移动常导致患者产生疼痛以及焦虑、抑郁等应激反应，影响正畸治疗。申请人利用改良的大鼠牙齿移动模型能够有效地移动牙齿，且动物出现明显的应激行为学改变，海马内星形胶质细胞活性降低。大麻素受体2（CB2R）在海马胶质细胞上大量表达，且参与情绪，认知等过程，提示胶质细胞CB2R系统所介导的神经免疫应答可能参与了正畸牙齿移动所致的应激反应。本项目拟应用改良的大鼠牙齿移动模型，以海马等边缘系统为靶区，综合应用行为药理学，免疫组织化学和分子生物学技术，从功能和结构两个方面观察胶质细胞表面CB2R在正畸牙齿移动所引起的大鼠疼痛和应激反应过程中的作用及其机制，在体给予CB2R病毒干扰，改变海马CB2R水平，探讨其对大鼠应激行为的影响，最后观察CB2R系统的下游信号分子机制。本课题旨在阐明、揭示胶质细胞参与正畸牙齿移动所引起的疼痛和应激反应的中枢机制，为开发有效的治疗药物、干预方案提供有针对性的靶点。

本研究通过建立大鼠改良牙齿移动动物模型（TM），采用免疫荧光染色、western blot及行为药理学、包埋前免疫电镜等技术对海马和延髓背角CA1区的星形胶质细胞活化状态进行检测，并初步探讨了这两部位中胶质细胞及其表面大麻素受体在大鼠牙齿移动致咬肌痛觉敏感及焦虑应激过程中的作用。研究结果显示： 1. TM组大鼠的行为学发生了明显变化，与对照组相比，大鼠的咬肌疼痛阈值呈明显下降趋势；旷场和高架十字行为学检测表明随时间延长牙齿移动对大鼠造成了心理应激，表现为旷场中央区停留时间和高架十字开臂进入时间缩短；2. 在心理应激后，对大鼠海马CA1的GFAP的检测结果发现，TM组应激大鼠的GFAP表达量上升，且在实验后期达到峰值，说明海马CA1区星形胶质细胞出现激活。在实验的最后一天给予星形胶质细胞抑制剂FC后，海马CA1区的GFAP的表达得到有效的抑制，但是与焦虑应激有关的旷场和高架十字迷宫的行为学未发现明显变化。因此，FC对牙齿移动引起的焦虑应激反应作用有限，需要进一步探讨牙齿移动应激的反应机制。3. 对MDH中GFAP、c-Fos的表达进行了检测，发现牙齿移动组大鼠的c-Fos和GFAP表达量均上升。但是GFAP峰值时间落后c-Fos的峰值时间，说明星形胶质细胞的活化可能与牙齿移动引起的延迟不适相关而不与急性疼痛相关。并且GFAP的活化与咬肌痛敏的时间相平行，进一步说明星形胶质细胞可能参与了牙齿移动导致的大鼠咬肌痛敏升高过程。当给予星形胶质细胞活化抑制剂后，大鼠焦虑样行为、咬肌痛觉敏感较应激组发生有效逆转。抑制MDH中星形胶质细胞激活能够阻断牙齿移动后后 GFAP、IL-1β和NMDAR的上调。因此我们推断，牙齿移动引起的MDH中星形胶质细胞激活能够释放过量IL-1β，进而促进NMDAR磷酸化，最终导致咬肌痛觉敏感增强，而海马内胶质细胞激活则参与正畸后应激的产生。本研究从口颌面疼痛和应激的中枢调控两方面分别研究了胶质细胞在牙齿移动导致的痛敏和应激增强中的作用，为今后更深入地研究口颌面痛的中枢机制提供了理论支持，并且奠定了有力的实验基础。