三碘甲腺原氨酸对根尖牙乳头干细胞成牙/骨向分化的影响及其机制的初步探究

Effect of Triiodothyronine on the odonto/ostegenic differentiation of stem cells from apical papilla and the preliminary study of its mechanism

目的探讨三碘甲腺原氨酸(3,5,3′-triiodothyronine,T3)对根尖牙乳头干细胞(stem cell from apical papilla,SCAPs)成骨向分化的影响。方法酶消化法分离培养SCAPs。制备含不同浓度T3的条件培养基,培养5 d,检测各组碱性磷酸酶表达和活性,选取差异最显著的浓度作为实验浓度。CCK-8法检测增殖能力,real-time RT-PCR、Western Blot、碱性磷酸酶染色检测牙/骨向分化能力。Western Blot检测MAPK通路相关蛋白表达情况。结果1×10^-9 mol/L组ALP活性最高,选作最佳刺激浓度进行后续实验。CCK-8结果显示实验组对增殖能力没有显著影响。此外,real-time RT-PCR、Western Blot、碱性磷酸酶染色结果表明1×10^-9 mol/L T3诱导促进SCAPs成牙/骨向分化。Western Blot结果表明1×10^-9 mol/L T3激活了ERK通路。U0126抑制牙/骨向分化相关蛋白表达。结论1×10^-9 mol/L T3促进SCAPs成牙/骨向分化,这个过程与ERK通路有密切关联。

Objective To investigate the effect of T3 on the odonto/ostegenic differentiation of SCAPs.Methods SCAPs were isolated and cultured by enzyme digestion method.Different concentrations of T3 media were prepared and cultured for 5 days.The expression and activity of ALP were measured to select the optimum concentration.The CCK-8 was taken to distinguish the proliferation ability between the T3-treated group and the control group.Real-time RT-PCR,Western Blot,and Alkaline Phosphatase Staining(ALP)were performed to evaluate the odonto/ostegenic differentiation potential of the T3-treated SCAPs.The MAPK signal pathway related protein expression was detected via Western Blot.Results The 1×10^-9 mol/L T3-treated group expressed the highest ALP among all groups and it was chosen as the optimal concentration.CCK-8 demonstrated that T3 had no significant effect on the proliferation of SCAPs.Moreover,results of real-time RT-PCR,Western Blot and ALP staining showed that 1×10^-9 mol/L T3 promoted the odonto/ostegenic differentiation of SCAPs.Besides,ERK signal pathway was activated at 15 min after treated with T3.The odonto/ostegenic related protein was suppressed by U0126.Conclusion The 1×10^-9 mol/L T3 promotes the odonto/ostegenic differentiation of SCAPs and this process may be closely related to the ERK signal pathway.