GRP78-c-Src 信号通路在周期性机械拉伸诱导牙周膜成纤维细胞成骨分化中的作用。

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

机构信息

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.

出版信息

Hua Xi Kou Qiang Yi Xue Za Zhi. 2024 Jun 1;42(3):304-312. doi: 10.7518/hxkq.2024.2023354.

DOI:10.7518/hxkq.2024.2023354

PMID:39049649

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11190863/

Abstract

OBJECTIVES

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 (<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (<0.05) after cyclic mechanical stretch.

CONCLUSIONS

GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.

摘要

目的

本研究旨在探讨葡萄糖调节蛋白（GRP）78 对周期性机械拉伸下牙周膜成纤维细胞（PDLF）中成骨细胞分化的影响，并确定其潜在机制。

方法

应用 FlexCell 5000 细胞力学装置模拟正畸牙的应力环境。通过流式分选获得 GRP78High 和 GRP78Low 亚群。基因转染以敲低 GRP78 和 c-Src 的表达并过表达 c-Src。Western blot 分析用于检测 Runt 相关基因 2（RUNX2）、osterix、骨钙素（OCN）和骨桥蛋白（OPN）的蛋白表达。免疫沉淀试验用于确定 GRP78 与 c-Src 的相互作用。通过茜素红染色测定细胞矿化结节的形成。

结果

GRP78 在 PDLFs 中呈异质性表达，通过流式分选获得 GRP78High 和 GRP78Low 亚群。与 GRP78Low 亚群相比，周期性机械拉伸后 GRP78High 亚群的成骨分化能力和 c-Src 激酶磷酸化水平显著增加（<0.05）。GRP78 在 PDLFs 中与 c-Src 相互作用。过表达 c-Src 组的成骨分化能力明显高于载体组（<0.05），而 sic-Src 组在周期性机械拉伸后成骨分化能力明显降低（<0.05）。