Notch信号通路在牙髓和牙周细胞矿化及组织损伤修复中的调控作用研究

Modulatory effect of Notch signaling on dental pulp cells and periodontal ligament cells undergoing odontogenic/osteogenic differentiation and tissue regeneration

目的探讨Notch信号通路在人牙髓细胞(dental pulp cells,DPCs)和牙周韧带细胞(periodontal ligament cells,PDLCs)向成牙本质/成骨样细胞分化及组织损伤修复中的调控作用。方法酶消化法分离培养DPCs和PDLCs,实时荧光定量反转录聚合酶链反应(RT-PCR)检测Notch信号通路相关基因Notch1、Notch2、DLL1和DLL3mRNA在DPCs和PDLCs矿化诱导过程中表达水平变化;建立大鼠牙髓和牙周联合损伤动物模型,观察4周后牙髓牙本质复合体及牙周附着装置修复情况,免疫组织荧光染色检测Notch1在牙髓和牙周联合损伤修复动物体内模型的表达和分布。结果 DPCs和PDLCs经矿化诱导,Notch1、Notch2、DLL1mRNA表达均上调,与对照组间的差异有统计学意义(P<0.05);DLL3mRNA表达上调,与对照组间差异无统计学意义(P>0.05)。免疫荧光示Notch1蛋白在损伤处的新生牙髓及牙周韧带组织细胞浆强表达,而在正常牙髓及牙周韧带组织基本无表达。结论在DPCs和PDLCs诱导成牙本质/成骨分化的过程中,Notch1、Notch2、DLL1呈现相似的表达变化趋势上调,Notch1信号在牙髓及牙周损伤修复的新生组织明显激活,提示Notch信号通路参与DPCs和PDLCs成牙本质/成骨分化,且在牙髓牙本质复合体和牙周附着装置的损伤修复中起重要的调控作用。

Objective The purpose of this study was to investigate the expression of Notch signaling including Notch1,Notch2,DLL1 and DLL3 in human dental pulp cells （DPCs） and periodontal ligament cells （PDLCs） undergoing odontogenic/osteogenic differentiation and the distribution of Notch1 in rat dental pulp and periodontal ligament with injury. Methods DPCs and PDLCs were isolated by enzymatic digestion and preceded to odontogenic/osteogenic induction for 14 days,and the cells without induction served as controls. The expressions of Notch1,Notch2,DLL1 and DLL3 were investigated by real-time fluorescence quantitative RT-PCR. Lewis rat model with dental pulp and periodontal ligament injury was created and the distribution of Notch1 was determined by immunofluorescent staining. Results The mRNA levels of Notch1,Notch2 and DLL1 were increased as DPCs and PDLCs were induced along odontogenic/osteogenic lineage. Statistical differences showed the mRNA levels in the induced groups were significantly higher than control group （P〈0.05）,albeit DLL3 didn＇t show any significant difference between control group and induction group （P〈0.05）. Immunofluorescent staining also revealed the Notch1 signal was activated in the newly formed dental pulp and periodontal ligament tissue after injury compared with the normal ones. Conclusions The Notch signaling pathway including Notch1,Notch2 and DLL1 showed similar up-regulated expression pattern in DPCs and PDLCs undergoing odontogenic/osteogenic induction. Moreover,Notch1 was activated expressed in dental pulp and periodontal ligament tissues with injury. In conclusion,Notch signaling pathway may play an important modulatory role in the odontogenic/osteogenic differentiation of DPCs and PDLCs,as well as the dental tissue regeneration.