牵张应力对人牙周膜成纤维细胞细胞外金属基质蛋白酶诱导剂表达的影响及相关信号通路的研究

Effect of tensile force on expression of extracellular matrix metalloproteinase inducer in human periodontal ligament fibroblasts and study on relevant signal pathways

目的研究人牙周膜成纤维细胞(PDLFs)在循环牵张力下细胞外金属基质蛋白酶诱导剂(EMMPRIN)(CD147/basgin)的表达变化以及对血管内皮生长因子(VEGF)表达的影响,推测EMMPRIN在应力作用下牙周组织改建过程中的可能作用。方法采用荧光免疫组织化学法检测EMMPRIN和VEGF在PDLFs中的表达定位;使用Flexcell System对PDLFs加载6%、12%、20%的循环牵张力0.5、2、6、12、24、36、48及72 h后,采用实时PCR和Western blotting的方法分别观察PDLFs内EMMPRIN及VEGF的基因及蛋白的表达变化;在PDLFs培养液中加入外源性EMMPRIN蛋白6、12、24 h后,分别观察PDLFs内VEGF的基因及蛋白的表达变化。结果 EMMPRIN蛋白主要表达于PDLFs的细胞膜和细胞质,而VEGF则在PDLFs的细胞核和细胞质中有较强表达。对PDLFs分别施加6%、12%及20%的机械牵张力后,细胞内EMMPRIN与VEGF的基因和蛋白表达均特异性上调;外源性的EMMPRIN蛋白可以促进PDLFs中VEGF mRNA的表达。结论机械牵张力可特异性上调PDLFs中EMMPRIN和VEGF的表达;EMMPRIN可能调控VEGF的表达并与之共同参与应力作用下的牙周组织改建的过程。

Objective To investigate changes of the expression of extracellular matrix metalloproteinase inducer（EMMPRIN） in periodontal ligament fibroblasts（PDLFs） under cyclic tensile force and relevant effect on the expression of vascular endothelial growth factor（VEGF） and evaluate possible effect of EMMPRIN during the course of periodontium remodeling under mechanical stress.Methods Immunofluorescent histochemical staining was performed to locate the expressions of EMMPRIN and VEGF in PDLFs.PDLFs were exposed to 6%,12%,and20%cyclic tensile force by the Flexcell System for 0.5,2,6,12,24,36,48,and 72 h.Changes of gene and protein expressions of EMMPRIN and VEGF in PDLFs were observed by real-time PCR and Western blotting.Changes of mRNA and protein expressions of VEGF in PDLFs were observed 6,12,and 24 h after exogenous EMMPRIN protein was added into the PDLFs culture medium.Results The protein expression of EMMPRIN was mainly detected in the cell membrane and cytoplasm of PDLFs,while the expression of VEGF was observed in nuclei as well as cell cytoplasm.The mRNA and protein expressions of both EMMPRIN and VEGF specifically up-regulated after PDLFs were exposed to 6%,12%,and 20%mechanical tensile force.The exogenous EMMPRIN protein enhanced the mRNA expression of VEGF in PDLFs.Conclusion The mechanical tensile force can specifically up-regulate the expressions of EMMPRIN and VEGF in PDLFs.EMMPRIN may regulate the expression of VEGF and involve in the course of periodontal remodeling under stress with VEGF.