延髓小胶质细胞极化参与实验性牙髓炎疼痛的研究  

Involvement of microglia polarization in experimental pulpitis-induced pain

作　　者：寇雯雯 孙书恺 薛飞 路宇馨 高磊 张静 吴礼安 Kou Wenwen;Sun Shukai;Xue Fei;Lu Yuxin;Gao Lei;Zhang Jing;Wu Lian(National Key Laboratory of Military Stomatology,Department of Pediatrie Dentistry,School of Stomaology,Air Force Medical University,Xi'an 710032;Lintong Rehabilitation and Convalescent Centre of PLA,Xi'an 710600,China)

机构地区：[1]空军军医大学口腔医学院儿童口腔科,军事口腔医学国家重点实验室,国家口腔疾病临床医学研究中心,西安710032 [2]中国人民解放军联勤保障部队临潼康复疗养中心,西安710600

出　　处：《神经解剖学杂志》2022年第3期259-266,共8页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81771095);陕西省重点研发计划-国际合作重点项目(2021KWZ-26)。

摘　　要：目的:检测小鼠实验性牙髓炎发生、发展过程中,延髓背角小胶质细胞的极化状态及其变化,探究延髓背角小胶质细胞不同极化状态与牙髓炎疼痛的相关性。方法:随机抽样法将C57小鼠分为对照组(Control)和实验性牙髓炎症模型组(Pulpitis)。牙髓暴露法建立实验性牙髓炎症模型,HE染色观察不同时间点牙髓炎症的变化情况;擦面实验观察小鼠牙髓炎性痛程度;Western Blot检测小胶质细胞特异性标志物离子钙结合衔接分子1(Iba-1)、M1型小胶质细胞极化标记物诱导型一氧化氮合酶(iNOS)及M2型小胶质细胞极化标记物精氨酸酶1(Arg-1)在延髓背角的表达及变化;免疫荧光组织化学染色法观察Iba-1、iNOS、Arg-1的表达及定位情况;real time RT-PCR检测IL-1β和IL-4 mRNA的表达变化。结果:HE染色结果显示,牙髓暴露1 d小鼠牙髓组织出现明显的炎性反应,3 d炎性发展至整个冠髓,7 d炎症扩散至根髓,14 d牙髓几乎全部坏死。痛行为学检测结果显示,牙髓暴露后1 d疼痛最为明显,随后开始逐渐减弱,7 d后恢复至正常水平。Western Blot、免疫荧光组织化学染色结果显示牙髓炎发生后,小胶质细胞活化且3 d活化量最高,牙髓暴露1 d延髓背角浅层iNOS表达最高,随后逐渐降低;1 d时延髓背角浅层内Arg-1表达水平有所升高,14 d时达到最高。real time RT-PCR结果显示,延髓背角IL-1βmRNA在牙髓暴露后1 d表达最高,随后逐渐下降;同时IL-4 mRNA表达逐渐升高,14 d时达到最高。结论:小鼠牙髓炎疼痛程度与牙髓炎症程度具有一定的相关性;牙髓炎性疼痛发生后,延髓背角小胶质细胞发生极化,初期以M1极化为主;后期以M2极化为主,疼痛状态与极化方向相关。Objective:To investigate the correlation between pulpitis-induced pain and different polarization states of microglia in the medullary dorsal horn(MDH) during the occurrence and development of experimental pulpitis in mice. Methods: C57 mice were randomly divided into control group and experimental pulpitis model group. The pulpitis model was established by pulp exposure method, and hematoxylin-eosin(HE) staining method was used to observe the changes of pulp inflammation at different time points. The pulpitis pain behavior was performed by face grooming test. The expression changes of microglial activation marker ionized calcium binding adaptor molecule-1(Iba-1), M1 type marker inducible nitric oxide synthase(iNOS) and M2 type marker arginase-1(Arg-1) were examined by immunohistochemical experiments and Western Blot, and the expression changes of IL-1β and IL-4 mRNA were detected by real time RT-PCR method. Results: HE staining results showed that 1 day after pulp exposure, there was obvious inflammatory cell infiltration of the pulp tissue in mice, and the pulp gradually began to necrosis at 3 d, and nearly all the pulp necrosis and inflammatory cell infiltration occurred at the root cannel after 14 days. Pain behavioral results showed that the pain was most pronounced 1 day after pulp exposure, gradually decreased at 3 d and 7 d, and returned to normal levels at 14 d, which was related to the inflammatory changes of pulp in HE staining. Immunohistochemical and Western Blot results showed that the expression of the M1 microglia polarization marker iNOS in the medullary dorsal horn increased 1 day after pulp exposure, and gradually decreased after reaching its highest level at 3 d, while the expression of Arg-1, a polarizing marker of M2 microglia reached the highest at 14 d of pulp exposure. Real time RT-PCR results showed that IL-1β mRNA expression increased at 1 d after pulp exposure and then decreased gradually, while IL-4 mRNA expression increased in the medullary dorsal horn, reaching a highest level at

关 键 词：小胶质细胞 极化 牙髓炎 疼痛 延髓背角 小鼠 

分 类 号：R781.31[医药卫生—口腔医学]