miR-146a-5p在Notch2调控牙周膜干细胞成骨分化机制的作用研究

miR-146a-5p modulates the osteogenic differentiation of periodontal ligament stem cells by targeting Notch2

目的:探索miR-146a-5p在Notch2分子调控牙周膜干细胞向成骨细胞分化中的关键作用,为口腔疾病的治疗及组织工程修复提供基础的理论依据。方法:培养hPDLSCs,采用RT-PCR检测对其成骨诱导前后成骨分化相关基因ALP、OSX及Notch2基因水平、miR-146a-5p的表达变化;通过Western Blot检测hPDLSCs成骨诱导分化后14d成骨相关蛋白ALP、OSX以及Notch2改变;将miR-146a-5p转染hPDLSCs,通过RT-PCR及Western Blot分别检测成骨相关基因ALP、OSX以及Notch2 mRNA蛋白水平。结果:RT-PCR检测结果表明,hPDLSCs成骨诱导7d、14d,ALP、OSX及Notch2 mRNA水平表达增高(P<0.05),同时在蛋白水平上ALP、OSX、Notch2分子表达增高(P<0.05);RT-PCR显示成骨诱导14d,miR-146a-5p表达下降;miR-146a-5p转染hPDLSCs后,成骨相关蛋白ALP、OSX以及Notch2表达降低(P<0.05)。结论:miR-146a-5p在hPDLSCs向成骨细胞方向分化过程中能够抑制Notch信号通路分子以及成骨相关基因ALP、OSX表达,参与hPDLSCs向成骨细胞方向分化。

Objective:This study aims to demonstrate the crucial role of miR-146a-5p in modulating the osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)by targeting Notch2,providing a theoretical basis for treatment and tissue engineered repair of oral diseases.Methods:Stable hPDLSCs were cultured and induced to differentiate into osteoblasts by changing the micro-environment of the media.RT-PCR was applied to observe the alterations of osteogenesisrelated genes.The Notch2 gene and miR-146a-5p were also detected;the changes of osteogenic proteins ALP,OSX and Notch2 were analyzed byWestern Blot.hPDLSCs were transfected with miR-146a-5p overexpression plasmid,and RT-PCR and Western Blot were respectively used to test the gene and protein levels of osteogenesis-related genes ALP,OSX,and Notch2 mRNA.Results:RT-PCR showed that expression of osteogenesis-related genes(P<0.05),Notch2 mRNA(P<0.05),and related proteins(P<0.05)were increased after osteogenic differentiation of hPDLSCs.While 14 days later,RT-PCR found that miR-146a-5p expression was inhibited;when the miR-146a-5p was overexpressed,compared with the control group,the expression of osteogenesis-related proteins and Notch2 was decreased(P<0.05).Conclusion:miR-146a-5p can inhibit the expression of molecules in the Notch signaling pathway and osteogenesis-related genes ALP and OSX,which express and participate in the osteogenic differentiation of hPDLSCs.