核因子κB受体活化因子配体和肿瘤坏死因子α经炎性牙周膜干细胞外泌体促进破骨细胞分化

doi:10.7518/hxkq.2022.04.002

华西口腔医学杂志 ›› 2022, Vol. 40 ›› Issue (4): 377-385.doi: 10.7518/hxkq.2022.04.002

核因子κB受体活化因子配体和肿瘤坏死因子α经炎性牙周膜干细胞外泌体促进破骨细胞分化

戴振宁¹(), 郑蔚晗², 利时雨¹^,²()

^1.广东省第二中医院口腔科，广州 510095
^2.南方医科大学第三附属医院，广东省医学3D打印应用转化创新平台，广州 510630

收稿日期:2021-11-10 修回日期:2022-05-08 出版日期:2022-07-25 发布日期:2022-07-27
通讯作者: 利时雨 E-mail:409556338@qq.com;li421@126.com
作者简介:戴振宁，主治医师，硕士，E-mail：409556338@qq.com
基金资助:
国家自然科学基金面上项目(82073747);中国博士后科学基金面上资助(2021M701620);南方医科大学第三附属医院院长基金青年启动项目(YQ2021001);广州市开发区英才项目(2019);广东省区域联合基金青年基金项目(2021A1515111074)

Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells

Dai Zhenning¹(), Zheng Weihan², Li Shiyu¹^,²()

^1.Dept. of Stomatology, Guangdong Second Traditional Chinese Medicine Hospital, Guangzhou 510095, China
^2.The Third Affiliated Hospital of Southern Medical University, Guangdong Medical Innovation Platform for Translation of 3D Printing Application, Guangzhou 510630, China

Received:2021-11-10 Revised:2022-05-08 Online:2022-07-25 Published:2022-07-27
Contact: Li Shiyu E-mail:409556338@qq.com;li421@126.com
Supported by:
The National Natural Science Foundation of China(82073747);The Fellowship of China Postdoctoral Science Foundation(2021M701620);The President Foundation of The Third Affiliated Hospital of Southern Medical University(YQ2021001);Yingcai Project in Guangzhou Development District (2019);Guangdong Regional Joint Youth Fund Project(2021A1515111074)

摘要/Abstract

摘要：

目的　慢性牙周炎表现的病理性骨吸收非常常见，牙周膜干细胞（PDLSCs）的外泌体（Exo）对骨吸收的作用和影响尚不明确，本研究分析了该Exo蛋白组分对破骨细胞分化的影响。方法　分别从正畸前拔牙患者和慢性牙周炎患者的牙周膜组织中提取PDLSCs，通过流式细胞术检测表面标记物；通过差速离心分别提取2种细胞的Exo，即Exo-WT和Exo-CP，并通过Western blot、透射电子显微镜（TEM）、蛋白浓度检测、纳米粒径追踪检测Exo特征；通过蛋白质谱检测2种Exo的蛋白组分；通过酶联免疫吸附（ELISA）验证了差异表达蛋白肿瘤坏死因子α（TNF-α）、核因子κB受体活化因子配体（RANKL）、白细胞介素（IL）-1α、转化生长因子β（TGF-β）、骨形态发生蛋白2（BMP-2）表达水平；将10、100、1 000 μg·mL^-1的Exo-CP或Exo-WT加入RAW264.7培养基中并于5 d时通过实时荧光定量聚合酶链反应（RT-qPCR）、Western blot、抗酒石酸酸性磷酸酶（TRAP）染色检测破骨分化相关指标表达情况；采用SPSS 24.0软件对实验数据进行统计学分析。结果　Exo-CP富集的差异表达蛋白主要与破骨细胞分化的TNF信号通路、核转录因子κB（NF-κB）信号通路相关；ELISA实验证实了Exo-CP中高表达TNF-α、RANKL、IL-1α，低表达TGF-β1、BMP-2（P<0.05）；Exo-CP作用于RAW264.7，显著提高了细胞的破骨分化相关基因及蛋白的表达水平，TRAP染色可见分化的破骨细胞，且呈现浓度依赖性，100、1 000 μg·mL^-1浓度Exo-CP对破骨细胞分化的促进作用显著高于10 μg·mL^-1浓度组（P<0.05）。结论　慢性牙周炎的病理性骨吸收可能由炎性PDLSCs所分泌的Exo通过促进破骨细胞分化引起，Exo中主要的作用蛋白可能为RANKL和TNF-α，本研究为慢性牙周炎骨吸收的发病机制提供了新视角。

关键词: 慢性牙周炎, 牙周膜干细胞, 外泌体, 核因子κB受体活化因子配体, 肿瘤坏死因子α, 破骨细胞分化

Abstract:

Objective Pathological bone resorption is common in chronic periodontitis. However, the effect of exosomes (Exo) secreted by periodontal ligament stem cells (PDLSCs) on bone resorption is unclear. This study explored the Exo of inflammatory PDLSCs, their protein components, and their effects on osteoclast differentiation. Methods PDLSCs were isolated from the periodontal ligament tissues of orthodontic patients and those with chronic periodontitis. The surface markers of PDLSCs were detected by flow cytometry. Exo were characterized by Western blot, transmission electron microscope (TEM), bicinchoninic acid assay (BCA), nanosight tracking analysis (NTA). The protein components of Exo were detected by protein profiling. The expression levels of differentially expressed proteins tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB ligand (RANKL), interleukin (IL)-1α, transforming growth factor β (TGF-β), and bone morphogenetic protein 2 (BMP-2) were verified by enzyme linked immunosorbent assay (ELISA). Then, 10, 100, and 1 000 μg·mL^-1 of Exo-CP or Exo-WT were added to RAW264.7 medium, and the expression levels of osteoclast-related indicators were detected by real time quantitative polymerase chain reaction (RT-qPCR), Western blot, and tartrate resistant acid phosphatase (TRAP) staining at 5 days. Experimental data were statistically analyzed using SPSS 24.0 software. Results The differentially expressed proteins enriched in Exo-CP were mainly related to the tumor necrosis factor (TNF) signaling, osteoclast differentiation, and nuclear transcription factor κB (NF-κB) signaling pathways. ELISA experiments confirmed Exo-CP had high expression of TNF-α, RANKL, and IL-1α and low expression of TGF-β1 and BMP-2 (P<0.05). Adding Exo-CP to RAW264.7 significantly increased the expression of mRNA and proteins related to osteoclast differentiation of cells. In a concentration-dependent manner, the effect of Exo-CP on osteoclast differentiation at concentrations of 100 and 1 000 μg·mL^-1 was significantly higher than that on the 10 μg·mL^-1 concentration group (P<0.05). Conclusion Pathological bone resorption of chronic periodontitis may be caused by the activation of Exo-CP to promote osteoclast differentiation. The main protein in Exo may be RANKL and TNF-α. This research provides a new perspective on pathological bone resorption in chronic periodontitis.

Key words: chronic periodontitis, periodontal ligament stem cells, exosomes, receptor activator of nuclear factor-κB ligand, tumor necrosis factor-α, osteoclast differentiation

中图分类号:

Q 254

戴振宁, 郑蔚晗, 利时雨. 核因子κB受体活化因子配体和肿瘤坏死因子α经炎性牙周膜干细胞外泌体促进破骨细胞分化[J]. 华西口腔医学杂志, 2022, 40(4): 377-385.

Dai Zhenning, Zheng Weihan, Li Shiyu. Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells[J]. West China Journal of Stomatology, 2022, 40(4): 377-385.

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ELISA指标	Exo-WT	Exo-CP
TNF-α	1.33±0.2	14.21±0.82**
RANKL	0.75±0.06	6.38±0.13*
IL-1α	1.41±0.68	8.41±0.35**
TGF-β1	12.44±2.08	0.58±0.16***
BMP-2	5.36±1.38	1.61±0.22*

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核因子κB受体活化因子配体和肿瘤坏死因子α经炎性牙周膜干细胞外泌体促进破骨细胞分化

Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells

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