过表达NICD对肿瘤坏死因子α干扰的牙周膜干细胞成骨分化的影响

Effects of overexpression of NICD on osteogenic differentiation of tumor necrosis factor-α induced periodontal ligament stem cells

目的:研究过表达Notch胞内结构域NICD(Notch intracellular domain,NICD)对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)干扰的牙周膜干细胞(periodontal ligament stem cells,PDLSCs)成骨分化的影响,为临床治疗牙周病提供实验依据。方法:选取课题组前期构建的含NICD基因的PDLSCs细胞株(PDLSCs/NICD)及含空载体的PDLSCs细胞株(PDLSCs/K),每种细胞随机分为两组,一组加入浓度为10 ng/ml的TNF-α模拟炎症环境,另一组不加TNF-α,即TNF-α+PDLSCs/K组、TNF-α+PDLSCs/NICD组、PDLSCs/NICD组和PDLSCs/K组。采用茜素红染色、即时定量聚合酶链连锁反应(real-time quantitative polymerase chain reaction,rt-qPCR)及蛋白质印迹法(western blot,WB)检测过表达NICD对TNF-α干扰的牙周膜干细胞成骨分化的影响。结果:茜素红染色结果显示,TNF-α+PDLSCs/NICD组矿化结节数量最少且面积最小,明显少于其他3组;rt-qPCR结果显示,TNF-α+PDLSCs/NICD组成骨标志基因牙骨质附着蛋白(cementum attachment protein,CAP)、骨钙蛋白(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)表达量明显低于其他3组(P<0.01);WB结果显示TNF-α+PDLSCs/NICD组RUNX2蛋白含量明显少于其他3组(P<0.01)。结论:过表达NICD对TNF-α干扰的PDLSCs的成骨分化能力有双重抑制作用。

Objective: To study the effect of Notch intracellular domain (NICD) on bone differentiation of periodontal ligament stem cells (PDLSCs) interfered by tumor necrosis factor-α(TNF-α),and to provide experimental basis for clinical treatment of periodontal disease. Methods: The PDLSCs with NICD gene (PDLSCs/NICD) and the PDLSCs with empty vector (PDLSCs/K) were selected and randomly divided into four groups:TNF-α+PDLSCs/K group,TNF-α+PDLSCs/NICD group,PDLSCs/NICD group and PDLSCs/K group.PDLSCs/K + TNF-α group and PDLSCs/NICD+TNF-α group were added with TNF-α factor at a concentration of 10 ng/ml to simulate the inflammatory environment for experiments ,and the other groups were not added with TNF-α.The effects of over expression of NICD on osteogenic differentiation of TNF-α induced PDLSCs were detected by alizarin red staining,real-time quantitative polymerase chain reaction (rt-qPCR) and western blot(WB). Results: Alizarin red staining showed that TNF-α+ PDLSCs/NICD group had the least number and area of mineralized nodules,which was significantly less than the other three groups.The rt-qPCR results showed that the expression levels of cementum attachment protein (CAP),osteocalcin(OCN),osteopontin(OPN),Runt-related transcription factor 2(RUNX2) in TNF-α+ PDLSCs/NICD group were significantly lower than those of the other three groups ( P <0.01).WB results showed that the RUNX2 protein content in TNF-α+ PDLSCs/NICD group was significantly lower than that in the other three groups( P <0.01). Conclusion: Overexpression of NICD has double inhibitory effect on osteogenic differentiation of PDLSCs interfered by TNF-α.