牙龈卟啉单胞菌感染对hPDL细胞中成骨标志基因表达及炎症因子分泌的影响

Effect of Porphyromonas gingivalis infection on the expression of osteoblast marker gene and secretion of inflammatory cytokines in hPDL cells

目的观察牙龈卟啉单胞菌(P.gingivalis)感染对人牙周膜(hPDL)细胞中成骨标志基因表达及炎症因子分泌的影响。方法原代hPDL细胞,分为P.gingivalis感染的P.gingivalis组、常规处理的对照组,成骨诱导后茜素红染色检测矿化结节,PCR法检测成骨标志基因Runt相关转录因子2(RUNX2)、骨钙素(OCN)、骨保护素(OPG)、骨碱性磷酸酶(BALP)的mRNA表达量,ELISA法检测炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6的分泌量,Western blot检测核因子-κB(NF-κB)及NF-κB抑制蛋白(I-κB)的蛋白表达量。结果与对照组比较,P.gingivalis组细胞成骨诱导后茜素红染色的矿化结节明显减少,细胞中RUNX2、OCN、OPG、BALP的mRNA表达量及I-κB的蛋白表达量均明显降低,培养基中TNF-α、IL-1β、IL-6的分泌量及细胞中NF-κB的蛋白表达量均明显增加。结论P.gingivalis感染hPDL细胞后能够抑制成骨分化、激活炎症反应且该作用与NF-κB通路的激活有关。

Objective To observe the effect of Porphyromonas gingivalis(P.gingivalis)infection on osteogenic marker gene expression and inflammatory cytokines secretion in human periodontal ligament(hPDL)cells.Methods Primary hPDL cells were cultured and divided into P.gingivalis infection group or control group.Alizarin red staining was used to detect mineralized nodules after osteogenesis induction;the mRNA expressions of Runt-related transcription factor 2(RUNX2),osteocalcin(OCN),osteoprotegerin(OPG)and bone alkaline phosphatase(BALP)were detected with PCR;the secretions of inflammatory cytokines,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 in culture medium were detected with ELISA method;the protein expression of nuclear factor-κB(NF-κB)and inhibitor of NF-κB(I-κB)protein in cells were detected using western blot.Results Compared with the control group,the mineralized nodules stained with alizarin red significantly reduced in P.gingivalis infection group,the mRNA expressions of RUNX2,OCN,OPG,BALP and the protein expression of I-κB in cells significantly decreased,and the secretions of TNF-α,IL-1βand IL-6 in culture medium and the protein expression of NF-κB in cells significantly decreased.Conclusion P.gingivalis can inhibit osteogenic differentiation and activate inflammatory response after infecting hPDL cells,and this effect is related to the activation of NF-κB pathway.