DKK1对AGEs/TNF-α微环境牙周膜干细胞骨向分化能力的影响

Effect of DKK1 on osteogenic differentiation of periodontal ligament stem cells in AGEs/TNF-alpha microenvironment

目的研究Wnt糖基化终末产物(AGEs)/肿瘤坏死因子-α(TNF-α)模拟糖尿病伴牙周炎微环境时,Wnt信号通路因子DKK1对牙周膜干细胞(PDLSCs)骨向分化能力的影响。方法采用组织块酶消化法培养人PDLSCs;采用流式细胞仪进行PDLSCs间充质干细胞表面分子鉴定;采用重组外源性AGEs/TNF-α模拟糖尿病伴牙周炎微环境;将实验组分为对照组(PDLSCs组)、AGEs/TNF-α模拟糖尿病伴牙周炎微环境组(AT-PDLSCs组)及DKK1处理AGEs/TNF-α模拟糖尿病伴牙周炎微环境组(D+AT-PDLSCs组);采用逆转录-聚合酶链式反应(RT-PCR)及蛋白质印迹法(Western blotting)检测成骨相关基因m RNA及蛋白的表达量。结果采用组织块酶消化法成功培养出牙周膜来源的细胞。经流式细胞仪检测,细胞阳性表达STRO-1、CD271、CD146、CD44、CD90,阴性表达CD45。碱性磷酸酶(ALP)及茜素红染色表明,AT-PDLSCs组的成骨能力受到抑制。RT-PCR检测结果表明,AT-PDLSCs组Runx2、ALP表达量最低,D+AT-PDLSCs组表达量高于AT-PDLSCs组,PDLSCs组表达量最高。Western blotting检测结果表明,Runx2及Osterix表达水平趋势与RT-PCR结果相一致。结论 AGEs/TNF-α模拟的糖尿病伴牙周炎PDLSCs骨向分化能力明显受损,D+AT-PDLSCs组PDLSCs骨向分化潜能可逆转,DKK1介导的Wnt通路可作为糖尿病伴牙周炎的潜在治疗靶点。

Objective To investigate the effect of Wnt signaling pathway factor DKK1 on the osteogenic differentiation of periodontal ligament stem cells in the microenvironment of diabetic patients with periodontitis after the glycation end prod-ucts(AGEs)/tumor necrosis factor-α(TNF-α).Methods Human periodontal ligament stem cells were cultured by tissue block enzymatic digestion.The surface molecules of periodontal ligament stem cell mesenchymal stem cells were identified by flow cytometry.Recombinant exogenous AGEs/TNF-αwas used to simulate the microenvironment of diabetes with periodontitis.They were devided to the control group(PDLSCs group),AGEs/TNF-αsimulated diabetes with periodontitis microenviron-ment group(AT-PDLSCs group)and DKK1 treatment AGEs/TNF-αsimulated diabetes with periodontitis microenvironment group(D+AT-PDLSCs group).Reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting were used to detect the expression levels of osteogenic genes and proteins.Results The periodontal membrane-derived cells were success-fully cultured by tissue block enzymatic digestion.By flow cytometry,cells positively expressed STRO-1,CD271,CD146,CD44,CD90,and negatively expressed CD45.Alkaline phosphatase(ALP)and alizarin red staining showed that the osteogen-ic capacity of the AT-PDLSCs group was inhibited.The results of RT-PCR showed that the expression of Runx2 and ALP was the lowest in the AT-PDLSCs group,those in the D+AT-PDLSCs group was higher than the AT-PDLSCs group,and those in the PDLSCs group was the highest.Western blotting analysis showed that the trend of Runx2 and Osterix expression levels was con-sistent with RT-PCR results.Conclusion AGEs/TNF-αmimics the ability of diabetic patients with periodontal ligament stem cells to differentiate into bone.The bone differentiation potential of periodontal ligament stem cells in D+AT-PDLSCs group can be reversed.DKK1-mediated Wnt pathway may be a potential therapeutic target for diabetic periodontitis.