应力刺激通过Wnt/β-catenin通路调控牙骨质细胞的矿化

doi:10.13591/j.cnki.kqyx.2023.07.004

摘要/Abstract

摘要：

目的牙骨质再矿化对牙根吸收后牙骨质修复至关重要,本文探究应力下Wnt/β-catenin通路对牙骨质细胞矿化的调控机制。方法对矿化诱导21 d的牙骨质细胞系IDG-CM6细胞加载轻力6 h,通过茜素红染色观察IDG-CM6细胞矿化程度,通过实时荧光定量聚合酶链反应(RT-qPCR)、蛋白质印迹(Western blot)及免疫荧光检测β-catenin及Runx2、Osterix的表达水平。采用生信技术筛选差异表达基因后,通过敲除IDG-CM6细胞内β-catenin检测矿化程度及Runx2、Osterix表达水平变化。结果应力下,IDG-CM6细胞矿化增加,Runx2、Osterix表达水平升高;生信分析及RT-qPCR、Western blot、免疫荧光染色表明应力促进β-catenin的表达,下调β-catenin能逆转应力刺激下的矿化效应。结论应力条件下,Wnt/β-catenin通路参与了牙骨质矿化的调控,可能是牙根吸收后牙骨质修复的关键通路。

关键词: 牙骨质细胞, 牙骨质稳态, Wnt/β-catenin通路, 矿化

Abstract:

Objective The cementum mineralization is important for cementum repair after root resorption. This study aims to investigate the role of Wnt/β-catenin signaling pathway in regulating the mineralization of cementocyes under mechanical stress. Methods The IDG-CM6 cells were subjected to osteogenic-induced medium for 21 days and then harvested for establishing in vitro mechanical loading model for 6 h. RNA-seq technique was used to screen the differentially expressed genes in IDG-CM6 cells under stress. Alizarin red staining was applied to observe the mineralization of IDG-CM6 cells, and the expression levels of β-catenin and osteogenic factors, Runx2 and Osterix, were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot. The expressions of β-catenin and Runx2 were also observed by immunofluorescence staining. After knockdown of β-catenin in IDG-CM6 cells by siRNA technology, the mineralization of IDG-CM6 cells and the expression levels of Runx2 and Osterix were detected under mechanical stress. Results The mineralization of IDG-CM6 cells and the expression levels of Runx2 and Osterix were increased under mechanical stress. The bioinformatic analysis, RT-qPCR, Western blot and immunofluorescence staining showed that mechanical stress could promote β-catenin expression and knockdown of β-catenin could reverse the mineralization promotion effect produced by mechanical stress. Conclusion Wnt/β-catenin pathway is involved in the regulation of cementum mineralization and plays an important role in cementum mineralization after root resorption.

Key words: cementocyte, cementum homeostasis, Wnt/β-catenin signaling pathway, mineralization

中图分类号:

R783.5

王涵, 李天成, 陈硕, 曾心怡, 杨宽, 邹淑娟, 段沛沛. 应力刺激通过Wnt/β-catenin通路调控牙骨质细胞的矿化[J]. 口腔医学, 2023, 43(7): 600-607.

WANG Han, LI Tiancheng, CHEN Shuo, ZENG Xinyi, YANG Kuan, ZOU Shujuan, DUAN Peipei. Mechanical stress regulates the mineralization of cementocytes through Wnt/β-catenin signaling pathway[J]. Stomatology, 2023, 43(7): 600-607.

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基因	上游引物序列 (5'-3')	下游引物序列 (5'-3')
Ctnnb1	GCTGCTGTCCTATTCCGAATGTCTG	GGCACCAATGTCCAGTCCAAGATC
Runx2	CCTTCAAGGTTGTAGCCCTC	GGAGTAGTTCTCATCATTCCCG
Sp7	GACTACCCACCCTTCCCTCACTC	TAGACACTAGGCAGGCAGTCAGAC
GAPDH	AGGTGAAGGTCGGAGTCAAC	CGCTCCTGGAAGATGGTGAT
Stat3	GCTTCTCGTTCTGGGTCTG	GTCTTGCCACTGAT-GTCCTT
Acer2	CCTCTGGTGGACTCTGGCTCTC	CAGGTACGAAGCAAGGCAGATGAG
Sost	GCGTTCAAGAATGATGCCACG	GCTGTACTCGGACACGTCTTTGG
Col1a1	CCCGGGGGATCCATGAGAGATTCTGTTACAGACG	CGGTGGCGGCCGTCAGCAGATCAGGATCAGC
Bmp1	TGGATGAGGAGGACTTGAGG	GTGCTGTCTTGGAGGGTCTC