GRP78-c-Src信号通路介导周期性牵张力作用下牙周膜成纤维细胞成骨分化的机制研究  

Role of the GRP78-c-Src signaling pathway on osteoblast differentiation of periodontal ligament fibroblasts induced by cyclic mechanical stretch

作　　者：胡静 崔智华 黄克强 苏荣健 赵颂 Hu Jing;Cui Zhihua;Huang Keqiang;Su Rongjian;Zhao Song(Public Experimental Platform,College of Life Science Institute,Jinzhou Medical University,Jinzhou 121001,China;Center of Oral Implantology,School and Hospital of Stomatology,China Medical University,Shenyang 110002,China;Dept.of Orthodontics,Affiliated Stomatological Hospital,Jinzhou Medical University,Jinzhou 121001,China;College of Basic Medical Science,Jinzhou Medical University,Jinzhou 121001,China)

机构地区：[1]锦州医科大学生命科学研究院公共实验平台,锦州121001 [2]中国医科大学口腔医学院附属口腔医院种植中心,沈阳110002 [3]锦州医科大学附属口腔医院正畸科,锦州121001 [4]锦州医科大学基础医学院,锦州121001

出　　处：《华西口腔医学杂志》2024年第3期304-312,共9页West China Journal of Stomatology

基　　金：辽宁省教育厅基础研究项目(JYTJCZR2020073);辽宁省教育厅面上项目(LJKZ0803)。

摘　　要：目的探讨在周期性牵张力作用下葡萄糖调节蛋白78(GRP78)对牙周膜成纤维细胞成骨分化的影响,并阐述其机制。方法应用FlexCell 5000细胞应力装置模拟牙齿正畸受力环境;应用流式细胞术细胞分选技术获得牙周膜成纤维细胞GRP78高表达细胞和GRP78低表达细胞;采用基因转染技术敲减GRP78、c-Src的表达以及过表达c-Src;蛋白质印迹实验检测成骨转录因子Runt相关基因2(RUNX2)、锌指结构转录因子(Osterix)以及成骨标志蛋白骨钙蛋白(OCN)、骨桥蛋白(OPN)的表达;免疫共沉淀实验检测GRP78与c-Src激酶的相互作用;茜素红染色实验检测细胞矿化结节的形成。结果GRP78在牙周膜成纤维细胞呈异质性表达,流式分选实验获得GRP78高表达和GRP78低表达细胞。周期性牵张力处理后,与GRP78低表达细胞相比,GRP78高表达细胞成骨分化能力及c-Src激酶磷酸化水平均升高,差异具有统计学意义(P<0.05);GRP78与c-Src激酶存在相互作用;与对照组相比,过表达c-Src组细胞成骨分化能力升高(P<0.05),sic-Src组细胞成骨分化能力降低(P<0.05)。结论GRP78通过与c-Src激酶相互作用并上调其表达,进而促进周期性牵张力诱导的牙周膜成纤维细胞成骨分化。Objective This study aims to investigate the influence of glucose regulated protein(GRP)78 on osteoblast differentiation in periodontal ligament fibroblasts(PDLFs)under cyclic mechanical stretch and determine the underlying mechanism.Methods FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth.GRP78High and GRP78Low subpopulation were obtained by flow sorting.Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src.Western blot analysis was used to detect the protein expression of Runt-related gene 2(RUNX2),Osterix,osteocalcin(OCN),and osteopontin(OPN).Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src.The formation of cellular mineralized nodules was determined by alizarin red staining.Results GRP78 was heterogeneously expressed in PDLFs,and GRP78High and GRP78Low subpopulations were obtained by flow sorting.The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch(P<0.05).GRP78 interacted with c-Src in PDLFs.The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group(P<0.05),and the sic-Src group showed significantly decreased osteogenic differentiation ability(P<0.05)after cyclic mechanical stretch.Conclusion GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.

关 键 词：周期性牵张力 成骨分化 牙周膜成纤维细胞 葡萄糖调节蛋白78 

分 类 号：R78[医药卫生—口腔医学]