目的:探讨TNF-α对人乳牙脱落干细胞成骨分化的影响。并分析ERK1/2-Runx2信号通路在调控过程中的变化。
方法:从6-8岁健康儿童的正常乳恒牙中分离培养SHED细胞,取第3代SHED细胞,分为对照组(成骨诱导培养),观察组(成骨诱导剂与TNF-α共培养)和激动剂组(成骨诱导剂,TNF-α和ERK途径激动剂共培养)。通过茜素红染色确定成骨分化。Osterix的蛋白质表达水平,OPN,通过Western印迹测定SHED细胞中的ERK1/2、pERK1/2和Runx2。Osterix的表达,OPN,qRT-PCR检测ERK1/2、pERK1/2和Runx2mRNA。采用SPSS26.0软件包进行统计学分析。
结果:三组细胞成骨分化能力的比较显示,三组细胞均可见红棕色矿化结节。三组比较,对照组的矿化结节最多,其次是激活组,观察组矿化结节最少。与对照组相比,观察组和激动剂组Osterix和OPN蛋白及mRNA的表达水平均显著降低,激动剂组Osterix和OPN蛋白及mRNA的表达水平明显高于观察组。ERK1/2蛋白和mRNA的表达水平在三组间差异无统计学意义。而观察组和激动剂组的pERK1/2和Runx2蛋白及mRNA表达水平明显高于对照组,激动剂组pERK1/2和Runx2蛋白及mRNA的表达水平明显高于观察组。
结论:TNF-α可抑制SHED细胞成骨分化,可能与抑制ERK1/2-Runx2信号通路有关。
OBJECTIVE: To investigate the effect of TNF-α on osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED), and to analyze the changes of ERK1/2-Runx2 signaling pathway in the regulation process.
METHODS: SHED cells were isolated and cultured from normal deciduous permanent teeth of healthy children aged 6-8 years old, and the third passage of SHED cells were taken and divided into control group (osteogenic inducer culture), observation group (osteogenic inducer and TNF-α co-culture) and agonist group (osteogenic inducer, TNF-α and ERK pathway agonist co-culture). The osteogenic differentiation was determined by alizarin red staining. The protein expression levels of Osterix, OPN, ERK1/2, pERK1/2 and Runx2 in SHED cells were determined by Western blot. The expressions of Osterix, OPN, ERK1/2, pERK1/2 and Runx2 mRNA were detected by qRT-PCR. Statistical analysis was performed with SPSS 26.0 software package.
RESULTS: Comparison of osteogenic differentiation ability of the three groups of cells showed that red-brown mineralized nodules were observed in the three groups of cells. Compared among the three groups, the control group had the most mineralized nodules, followed by the activation group, and the observation group had the least mineralized nodules. Compared with the control group, the expression levels of Osterix and OPN protein and mRNA in the observation group and the agonist group were significantly decreased, while the expression levels of Osterix and OPN protein and mRNA in the agonist group were significantly higher than those in the observation group. There was no significant difference in the expression levels of ERK1/2 protein and mRNA among the three groups, while the expression levels of pERK1/2 and Runx2 protein and mRNA in the observation group and the agonist group were significantly higher than those in the control group, and the expression levels of pERK1/2 and Runx2 protein and mRNA in the agonist group were significantly higher than those in the observation group.
CONCLUSIONS: TNF-α can inhibit osteogenic differentiation of SHED cells, which may be related to the inhibition of ERK1/2-Runx2 signaling pathway.