骨膜蛋白在小鼠牙周炎进程中的时空表达规律研究

doi:10.7518/hxkq.2024.2023336

华西口腔医学杂志 ›› 2024, Vol. 42 ›› Issue (3): 286-295.doi: 10.7518/hxkq.2024.2023336

骨膜蛋白在小鼠牙周炎进程中的时空表达规律研究

李钺(), 许春梅, 谢旭东, 施培磊, 王骏(), 丁一()

口腔疾病防治全国重点实验室国家口腔医学中心国家口腔疾病临床医学研究中心四川大学华西口腔医院牙周病科，成都 610041

收稿日期:2023-10-10 修回日期:2023-11-09 出版日期:2024-06-01 发布日期:2024-05-24
通讯作者: 王骏,丁一 E-mail:lant59liyue@163.com;junwang@scu.edu.cn;yiding2000@126.com
作者简介:李钺，硕士，E-mail：lant59liyue@163.com
基金资助:
国家自然科学基金(82071127);四川省重点研发计划(2023YFS0032);中央高校基本科研业务费专项基金四川大学华西口腔医院探索与研发项目(RD-02-202403);四川省自然科学基金(2022NSFSC0795)

Temporal and spatial expression analysis of periostin in mice periodontitis model

Li Yue(), Xu Chunmei, Xie Xudong, Shi Peilei, Wang Jun(), Ding Yi()

State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of Periodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China

Received:2023-10-10 Revised:2023-11-09 Online:2024-06-01 Published:2024-05-24
Contact: Wang Jun,Ding Yi E-mail:lant59liyue@163.com;junwang@scu.edu.cn;yiding2000@126.com
Supported by:
The National Natural Science Fundation of China(82071127);Sichuan Key Research and Development Program(2023YFS0032);Fundamental Research Fundation for the Central Universities, Research and Development Program of West China Hospital of Stomatology, Sichuan University(RD-02-202403);Natural Science Foundation of Sichuan Province(2022NSFSC0795)

摘要/Abstract

摘要：

目的研究小鼠牙周炎进程中骨膜蛋白表达的时空变化规律。方法采用丝线结扎法构建小鼠牙周炎模型。设置对照组、栓丝4 d组、栓丝7 d组、栓丝14 d组、自愈组（栓丝14 d再去除丝线14 d），通过Micro-CT、组织学染色观察各组小鼠牙周组织的动态变化特征，利用RNAscope、免疫组织化学染色分析牙周膜内骨膜蛋白在牙周炎进程不同阶段的变化规律。培养人牙周膜细胞并随机分为对照组、脂多糖（LPS）刺激组（LPS处理12 h）、去除LPS刺激组（LPS处理3 h后再用培养基培养9 h），实时定量聚合酶链反应（qRT-PCR）检测骨膜蛋白、转化生长因子β1（TGF-β1）、基质金属蛋白酶2（MMP2）的表达。结果栓丝7 d组可见明显的牙槽骨吸收，栓丝14 d组牙周组织破坏加重，表现为破骨细胞增多、牙周膜间隙增宽、牙槽骨高度降低，而自愈组见一定程度的牙周组织修复。与对照组相比，栓丝4 d组、栓丝7 d组牙周膜内骨膜蛋白表达降低，栓丝14 d组骨膜蛋白表达相较于栓丝4 d组及栓丝7 d组增加，自愈组骨膜蛋白表达与对照组相似。qRT-PCR结果显示，LPS刺激组与对照组相比骨膜蛋白、TGF-β1的表达均下降，而去除LPS刺激组与LPS刺激组相比骨膜蛋白、TGF-β1的表达均升高。结论小鼠牙周膜内骨膜蛋白表达随炎症进展呈先下降后回升的趋势，去除炎症刺激后骨膜蛋白表达的恢复可能与TGF-β1表达上调有关。

关键词: 牙周炎, 骨膜蛋白, 牙周再生, 牙槽骨, 动物模型

Abstract:

Objective This study aimed to investigate the temporal and spatial changes in the expression of periostin during periodontal inflammation in mice. Methods A periodontitis model was constructed using silk thread ligation. Mice were randomly divided into five groups including control group, 4-day ligation group, 7-day ligation group, 14-day ligation group, and self-healing group (thread removal for 14 days after 14-day ligation). Micro-CT and histological staining were performed to characterize the dynamic changes in the mouse periodontal tissue in each group. RNAscope and immunohistochemical staining were used to analyze the pattern of changes in periostin at various stages of periodontitis. The cell experiment was divided into three groups: control group, lipopolysaccharide (LPS) stimulation group (treated with LPS for 12 h), and LPS stimulation removal group (treated with LPS for 3 h followed by incubation with medium for 9 h). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of periostin, transforming growth factor-β1 (TGF-β1), and matrix metalloproteinase 2 (MMP2). Results Significant alveolar bone resorption was observed 7 days after ligation. With increasing duration of ligation, the damage to the mouse periodontal tissue was aggravated, which manifested as increased osteoclasts, widening of the periodontal membrane space, and decreased alveolar bone height. Some degree of periodontal tissue repair was observed in the self-healing group. Periostin expression decreased at 4 and 7 days compared with the control group and increased at 14 days compared with 4 and 7 days. A significant recovery was found in the self-healing group. The qRT-PCR results showed that the expression of periostin and TGF-β1 in the LPS stimulation group decreased compared with that in the control group but significantly recovered in the LPS removal group. Conclusion Periostin expression in the PDL of mice showed a downward and upward trend with inflammation progression. The significant recovery of periostin expression after removing inflammatory stimuli may be related to TGF-β1, which is crucial to maintain the integrity of the PDL.

Key words: periodontitis, periostin, periodontal regeneration, alveolar bone, animal model

中图分类号:

R781.4

李钺, 许春梅, 谢旭东, 施培磊, 王骏, 丁一. 骨膜蛋白在小鼠牙周炎进程中的时空表达规律研究[J]. 华西口腔医学杂志, 2024, 42(3): 286-295.

Li Yue, Xu Chunmei, Xie Xudong, Shi Peilei, Wang Jun, Ding Yi. Temporal and spatial expression analysis of periostin in mice periodontitis model[J]. West China Journal of Stomatology, 2024, 42(3): 286-295.

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基因	引物序列
GAPDH	Forward：5’-GCACCGTCAAGGCTGAGAAC-3’
GAPDH	Reverse：5’-TGGTGAAGACGCCAGTGGA-3’
TGF-β1	Forward：5’-GCCTTTCCTGCTTCTCATGG-3’
TGF-β1	Reverse：5’-TCCTTGCGGAAGTCAATGTAC-3’
MMP2	Forward：5’-ACCCATTTACACCTACACCAAG-3’
MMP2	Reverse：5’-TGTTTGCAGATCTCAGGAGTG-3’
骨膜蛋白	Forward：5’-CAACGGGCAAATACTGGAAAC-3’
骨膜蛋白	Reverse：5’-TCTCGCGGAATATGTGAATCG-3’

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骨膜蛋白在小鼠牙周炎进程中的时空表达规律研究

Temporal and spatial expression analysis of periostin in mice periodontitis model

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