Wnt5a在炎症微环境下的牙周膜干细胞中的作用研究

The mechanism of Wnt5a in periodontal stem cells under inflammatory microenvironment

目的:研究Wnt5a在炎症微环境作用下的牙周膜干细胞(periodontal stem cells,PDLSCs)中的作用。方法:有限稀释法分离获取PDLSCs并进行干细胞鉴定;实时定量qPCR检测正常PDLSCs组及炎症因子组(10ng/ml TNFα预刺激24h)炎症因子TNFα及IL-1β的mRNA表达水平;实时定量qPCR检测两组成骨诱导前后成骨基因Runx2、ALP的mRNA表达水平、Wnt5a的mRNA表达水平;Western Blot检测两组成骨诱导前后Wnt5a的蛋白表达水平。结果:炎症因子组与正常组的克隆形成率无显著性差异、炎症因子组TNFαmRNA表达水平IL-1βmRNA表达水平显著高于正常组(P<0. 05);常规培养条件下,炎症因子组Runx2、ALP mRNA表达水平与正常组无显著性差异,成骨诱导后成骨基因表达水平正常组显著高于炎症组,(P<0. 05);常规培养条件下,炎症因子组Wnt5a mRNA表达水平高于正常组(P<0. 05),成骨诱导后正常组及炎症因子组Wnt5a mRNA表达水平均显著高于常规培养条件;Western Blot结果示,Wnt5a蛋白表达量正常组diff(成骨诱导后)、炎症因子组undiff(成骨诱导前)、炎症因子组diff分别为正常组undiff的1. 2709倍、1. 8246倍、2. 2176倍。炎症因子组Wnt5a蛋白表达水平增加,成骨诱导后两组Wnt5a蛋白表达水平均增加,炎症因子组高于正常组。结论:在炎症微环境下,PDLSCs的炎症因子表达增加,成骨能力降低,Wnt5a mRNA及蛋白表达水平均增加,Wnt5a非经典通路可能通过与经典Wnt通路的交通等参与PDLSCs炎症及破骨的过程。

Objective:To study the role of Wnt5a in periodontal stem cells(PDLSCs)under the action of inflammatory microenvironment.Methods:PDLSCs were isolated and identified by limited dilution method.The mRNA levels of both the normal PDLSCs group and the inflammatory factors group(10ng/ml TNFαpre-stimulation for 24h)were measured by real-time qPCR.The mRNA expression levels of Runx2、ALP and Wnt5a were measured by real-time qPCR.Protein expression levels of Wnt5a before and after bone induction were detected by Western Blot.Results:There was no sighificant difference in the clonal formation rate between the inflammatory factor group and the normal group,and the mRNA expression level of IL-1 mRNA in the inflammatory factors group were significantly higher than that in the normal group(P﹤0.05).Under conventional culture conditions,the expression levels of Runx2 and ALP mRNA in the inflammatory cytokine group showed no significant difference from those in the normal group,and the expression levels of osteoblast genes in the normal group were significantly higher than those in the inflammatory group(P﹤0.05).Under the conventional culture condition,the mRNA expression level of Wnt5a in the inflammatory factors group was higher than that in the normal group(P﹤0.05),and the mRNA expression level of Wnt5a in the normal group and the inflammatory factors group were significantly higher than that in the conventional culture condition.Western Blot results showed that diff(after osteoblast induction),undiff(before osteoblast induction)and diff(before osteoblast induction)of Wnt5a protein expression were 1.2709,1.8246 and 2.2176 times of undiff of the normal group,respectively.The expression level of Wnt5a protein in the inflammatory factor group increased,and the expression level of Wnt5a protein in both groups increased after osteoblast induction,and the inflammatory factor group was higher than the normal group.Conclusion:In the inflammatory microenvironment,the expression of inflammatory factors in PDLSCs increases,the osteogenic capacity decreases,the expression level of Wnt5a mRNA and protein increases,and the non-classical Wnt5a pathway may be involved in the process of PDLSCs inflammation and osteogenesis through transportation with the classical Wnt pathway.