p38β在矿化诱导液诱导的人牙髓细胞成牙本质向分化中的作用

The role of p38β in odontoblastic differentiation of human dental pulp cells

目的研究p38β丝裂原激活的蛋白激酶(MAPK)对矿化诱导液诱导的人牙髓细胞(h DPC)成牙本质向分化的影响。方法体外培养hDPC,Western blot检测hDPC矿化诱导0、3、7、14 d后p38β蛋白表达情况;构建3条慢病毒载体p38β鄄shRNA并分别转染hDPC,Western blot及实时荧光定量聚合酶链反应(PCR)检测p38β蛋白及基因表达;设立空白对照组、矿化诱导(OM)组、OM+空载体(NC)组、OM+sh鄄p38β组共4组。成牙本质向矿化诱导1、7、14 d,实时荧光定量PCR检测成牙本质向分化指标牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)的基因表达;Western blot检测成牙本质向分化指标DSPP蛋白的表达;成牙本质向矿化诱导14 d,茜素红染色检测矿化结节的形成情况。结果 Western blot结果表明正常牙髓组织中存在p38β蛋白;成功构建p38β稳定低表达的hDPCs(实时荧光定量PCR显示三条序列干扰效率分别为55.4%、68.3%、54.2%);实时荧光定量PCR显示沉默p38β的hDPCs经矿化诱导后,成牙本质向分化指标DSPP、ALP基因表达水平显著降低,Western blot结果显示成牙本质向分化指标DSPP蛋白表达水平显著降低;茜素红染色结果显示,矿化诱导+sh鄄p38β组与空白对照组较另外两组的矿化结节数量明显减少。结论 p38β在OM诱导的h DPC成牙本质向分化中发挥作用。

Objective To investigate the role of p38β MAPK in odontoblastic differentiation of human dental pulp cells（hDPCs）. Methods HDPCs obtained from the healthy third molars or premolars were cultured. Western blot were used to detect the expression of p38β in hDPCs after OM induced for0, 3, 7 and 14 days. Lentivirus shRNA were constructed and transfected into hDPCs, The protein and mRNA levels of p38β were detected by western blot and quantitative real-time PCR. Then hDPCs were treated with odontogenic medium for 1, 7 and 14 days. The DSPP protein and mRNA levels were analyzed by western blot and quantitative real-time PCR, and the mRNA levels of ALP were also analyzed by quantitative real-time PCR. The formation of mineralized nodules were examined by Alizarin red staining. Results Western blot analysis showed that the expression of p38β can be detected in hDPCs. Quantitative real-time PCR showed that DSPP and ALP mRNA levels were obviously decreased after stable low expression of p38β, the levels of DSPP protein also decreased. Mineralization nodules were less observed in p38β-shRNA group after the induction of odontogenic differentiation of HDPCs for14 days. Conclusion P38β MAPK is involved in OM-induced odontoblastic differentiation of HDPCs.