应力刺激下牙根吸收和修复过程中相关microRNAs的调控作用与机制研究

Orthodontically induced in?ammatory root resorption (OIIRR), is an unavoidable side effect of orthodontic tooth movement. Cementoblasts locate in periodontal ligament, lining on the root surface. It has been proved that cementoblast play important roles in the process of root resorption and repair. However, the effects of mechanical stress on the expression of related gene and signal transduction pathway in cementoblasts remains unknown. .MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. It has been reported miRNAs are closely related to the biological behavior of metabolism and homeostasis of cementum. The aim of this study is to explore the effect and mechanism of miRNAs and key transcription factors in root resorption and repair process induced by mechanical stress..Cementoblasts OCCM30 are cultured subjected to mechanical strain by four-point bending system with tension and compression stress. The flow cytometry is used to examine the cell cycle and proliferation activity after loading at each time point. The expression of mineral associated proteins, as well as osteoblastic and osteoclastic differentiation markers are quanti?ed by RT-PCR and Western blotting. Meanwhile, gene chip is used to detect the key transcription factors of Wnt and Notch signaling pathways in cementoblasts under mechanical stress. .MicroRNA array hybridization is used to find out the differentially expressed miRNAs in cementoblasts under mechanical stress. To con?rm the microarray data, real-time RT-PCR is performed. Additionally, a bioinformatic analysis of developmentally regulated microRNAs suggests potential mRNA targets. Furthermore, functional evaluation of the key miRNAs in regulation of cementoblasts is carried out, in vivo and in vitro respectively. Retroviral overexpression and inhibition of key miRNAs is done in order to confirm the function of specific miRNAs in regulating cementoblasts in root resorption and repair process. .This is the first time to explore the effect and mechanism of miRNAs and key transcription factors with possible signaling pathways in root resorption and repair induced by mechanical stress. It will be great helpful in orthodontic clinical work in prevention and treatment of orthodontically induced root resorption.

正畸导致的炎性牙根吸收是正畸治疗中不可避免的不良反应，受到正畸学者的广泛关注。成牙骨质细胞是牙骨质形成、牙根修复的关键效应细胞，但其对力学信号反应特性的变化及其调控牙根吸收与修复的具体作用机制和信号通路仍不清楚。MicroRNAs是单链非编码小分子RNA，通过特异性基因沉默导致靶mRNA降解调控转录后基因表达水平，且MicroRNAs与牙骨质代谢的细胞生物学行为密切相关。本课题首次从 MicroRNAs角度探讨Wnt、Notch通路的关键转录因子在应力刺激成牙骨质细胞过程中的分子机制，采用微阵列分析、RNAi、前体MicroRNAs等技术筛选出与调控成牙骨质细胞各转录因子相关的关键 MicroRNAs, 并通过体内、体外实验验证关键MicroRNAs对应力刺激下成牙骨质细胞的调控作用。研究结果将为揭示应力刺激下牙根吸收与修复过程的分子生物学基础以及正畸导致的牙根吸收的防治提供理论依据。

成牙骨质细胞作为牙骨质形成、牙根修复的关键效应细胞，在正畸牙移动，牙根吸收与修复中发挥重要作用，但其对力学信号的反应特性及其调控牙根吸收与修复的具体作用机制仍不清楚。本课题首先从miRNAs角度探讨周期性张应力作用下差异表达的miRNAs，通过生物信息学分析、靶基因验证以及双荧光素酶验证明确了miR-146b-5p可能是力学刺激下成牙骨质细胞增殖和分化成熟的关键miRNAs，针对特定靶基因smad4表达进行调节，通过TGFβ信号通路参与牙根吸收的修复。同时，本课题还利用体内及体外实验证实正畸应力能够激活Notch信号通路，通过调控炎症因子的表达（TNF-a和IL-6）而促进破骨细胞分化，加速骨改建过程，从而提高牙移动效率。此外，体内及体外实验还表明wnt信号通路可促进成牙骨质细胞的分化成熟，从而有利于正畸效果的保持。本研究成果进一步阐明了正畸牙移动、牙根吸收与修复的生物学机制，为将来利用信号通路策略解决临床问题提供了理论依据。