中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (35): 5633-5638.doi: 10.12307/2022.889

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

鼻阻塞模型大鼠下颌骨自噬水平的变化

徐怡馨，王一鑫，李永明   

  1. 上海牙组织修复与再生工程技术研究中心，同济大学口腔医学院，同济大学附属口腔医院正畸科，上海市  200072
  • 收稿日期:2021-10-23 接受日期:2021-12-03 出版日期:2022-12-18 发布日期:2022-05-17
  • 通讯作者: 李永明，博士，主任医师，上海牙组织修复与再生工程技术研究中心，同济大学口腔医学院，同济大学附属口腔医院正畸科，上海市 200072
  • 作者简介:徐怡馨，女，1989年生，湖北省咸宁市人，汉族，同济大学附属口腔医院在读博士，主治医师，主要从事低氧、炎症、骨代谢、口呼吸方面的研究。 王一鑫，男，1996年生，云南省曲靖市人，汉族，在读硕士，主要从事低氧、髁突、口呼吸方面的研究。
  • 基金资助:
    国家自然科学基金面上项目(31370943)，项目负责人：李永明

Autophagy level of the mandible in nasal obstruction rats

Xu Yixin, Wang Yixin, Li Yongming   

  1. Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Orthodontics, School and Hospital of Stomatology, Tongji University, Shanghai 200072, China
  • Received:2021-10-23 Accepted:2021-12-03 Online:2022-12-18 Published:2022-05-17
  • Contact: Li Yongming, MD, Chief physician, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Orthodontics, School and Hospital of Stomatology, Tongji University, Shanghai 200072, China
  • About author:Xu Yixin, MD candidate, Attending physician, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Orthodontics, School and Hospital of Stomatology, Tongji University, Shanghai 200072, China Wang Yixin, Master candidate, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Orthodontics, School and Hospital of Stomatology, Tongji University, Shanghai 200072, China Xu Yixin and Wang Yixin contributed equally to this work.
  • Supported by:
    the National Natural Science Foundation of China (General Program), No. 31370943 (to LYM)

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摘要：

文题释义：
阻塞性睡眠呼吸暂停低通气综合征：指上气道部分或完全阻塞所诱发的一系列症状，如睡眠结构紊乱、鼾症、血氧饱和度下降等病征。诱发阻塞性睡眠呼吸暂停低通气综合征的直接病因是上气道的狭窄和阻塞，核心特征是睡眠呼吸暂停、间歇性低氧和高碳酸血症，可以引起冠心病、高血压等并发症甚至夜间猝死，儿童期的阻塞性睡眠呼吸暂停低通气综合征被证实影响颌面部生长发育。
自噬：是真核生物胞对其内部受损的细胞器、错误折叠的蛋白质及入侵的病原体进行降解，并利用降解产物为细胞的再生和修复提供新原料，实现细胞循环再利用的一种生物过程。自噬在细胞应激尤其是低氧、营养匮乏时被大量激活。

背景：低氧状态下细胞自噬状态易被激活，而自噬水平对骨代谢的调控作用已经被证明。睡眠呼吸暂停使机体处于间歇低氧状态，而睡眠呼吸暂停诱发的低氧是否影响下颌牙槽骨及牙周膜的自噬水平未见报道。
目的：观察机体低氧状态下对大鼠下颌自噬水平的影响。
方法：将 30只 1 周龄雄性 Wistar大鼠随机分为3组，每组10只，单侧鼻阻塞组、双侧鼻阻塞组分别构建幼年大鼠单侧及双侧鼻阻塞模型，麻醉30 min后用高频电刀电灼阻塞大鼠的左鼻孔，1周后同样方法阻塞双侧鼻阻塞组右侧鼻孔；对照组不处理鼻孔。单侧鼻阻塞建模成功4周后，模型大鼠5周龄时麻醉处死取下颌骨，制备下颌第一磨牙牙周骨组织切片，进行苏木精-伊红染色和 LC3免疫组化染色，利用 Image-J 图像分析系统对染色后的切片做定位定量分析。Western blot法检测下颌牙槽骨缺氧标志蛋白低氧诱导因子1α、自噬相关蛋白p62的蛋白表达水平，以及自噬标志蛋白LC3亚型LC3-Ⅱ/LC3-Ⅰ的比值。
结果与结论：①苏木精-伊红染色显示，单侧鼻阻塞组、双侧鼻阻塞组模型大鼠下颌牙槽骨骨小梁排列混乱；②免疫组化结果显示，单侧鼻阻塞组、双侧鼻阻塞组下颌第一磨牙牙槽骨细胞及牙周膜细胞胞浆LC3表达升高，提示自噬主要发生在牙周膜及牙槽骨区；③Western blot结果显示，与对照组相比，单侧鼻阻塞组、双侧鼻阻塞组模型大鼠下颌牙槽骨组织低氧诱导因子1α蛋白的表达水平显著升高，p62蛋白的表达水平显著下降(P < 0.05)，LC3-Ⅱ/LC3-Ⅰ比值显著升高(P < 0.05)；而双侧鼻阻塞组LC3-Ⅱ/LC3-Ⅰ比值较单侧鼻阻塞组稍下降(P > 0.05)；④提示大鼠单侧、双侧鼻阻塞导致的低氧使下颌低氧诱导因子1α蛋白的表达水平升高，下颌第一磨牙牙周膜及牙槽骨组织自噬水平升高，但自噬升高的水平并不与鼻阻塞的严重程度呈正相关。
缩略语：阻塞性睡眠呼吸暂停低通气综合征：obstructive sleep apnea hypopnea syndrome，OSAHS；低氧诱导因子1α：hypoxia inducible factor-1α，HIF-1α

https://orcid.org/0000-0002-4672-9177 (徐怡馨)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 阻塞性睡眠呼吸暂停低通气综合征, 牙槽骨, 鼻阻塞, 间歇低氧, 自噬, 大鼠

Abstract: BACKGROUND: Autophagy is easily activated under hypoxia and has been demonstrated to regulate bone metabolism. Sleep apnea keeps the body in a state of intermittent hypoxia, and whether sleep apnea-induced hypoxia affects the autophagy levels of mandibular alveolar bone and periodontal ligament has not been reported.
OBJECTIVE: To observe the autophagy level in the rat mandible under hypoxic conditions. 
METHODS: Thirty 1-week-old male Wistar rats were randomly divided into three groups (n=10 per group). In unilateral and bilateral nasal obstruction groups, the left nostril was blocked with high-frequency electrocautery after 30 minutes of anesthesia, and 1 week later, the right nostril in the bilateral nasal obstruction group was blocked using the same method. Rats in control group were with no nostril blocking. Four weeks after the successful modeling of unilateral nasal obstruction, the model rats at the age of 5 weeks were anesthetized and sacrificed, and the mandibles were removed to prepare periodontal bone tissue sections of the mandibular first molar for hematoxylin-eosin staining and LC3 immunohistochemical staining. Localization of the stained sections was quantified using the image-J image analysis system. Western blot assay was used to detect the expression of hypoxia-inducible factor 1α and autophagy-related protein p62 in mandibular alveolar bone, as well as the ratio of autophagy marker protein LC3 isoforms LC3-II/LC3-I.
RESULTS AND CONCLUSION: Hematoxylin-eosin staining results revealed disordered arrangement of trabecules of the mandibular alveolar bone in the unilateral and bilateral nasal obstruction groups. Immunohistochemical results indicated an increased expression of LC3 in the alveolar bone cells and periodontal ligament cells of mandibular first molars in the unilateral and bilateral nasal obstruction groups, suggesting that autophagy mainly occurred in the periodontal ligament and alveolar bone. Western blot results showed that compared with the control group, the expression level of hypoxia-inducible factor 1α protein in the mandibular alveolar bone tissue was significantly increased, the expression level of p62 protein was significantly decreased (P < 0.05), and the ratio of LC3-II/LC3-I was significantly increased in the unilateral and bilateral nasal obstruction groups (P < 0.05). Moreover, the ratio of LC3-II/LC3-I in the bilateral nasal obstruction group was slightly lower than that in the unilateral nasal obstruction group (P > 0.05). All these findings indicate that hypoxia caused by unilateral and bilateral nasal obstruction increases the expression level of hypoxia-inducible factor 1α protein in the mandible and the level of autophagy in the periodontal ligament and alveolar bone of mandibular first molars in rats. However, the increase in autophagy has no positive correlation with the severity of nasal obstruction.

Key words: obstructive sleep apnea hypopnea syndrome, alveolar bone, nasal obstruction, intermittent hypoxia, autophagy, rat

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