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Disruption of kif3a results in defective osteoblastic differentiation in dental mesenchymal stem/precursor cells via the Wnt signaling pathway

机译:通过Wnt信号通路破坏kif3a会导致牙齿间充质干/前体细胞的成骨细胞分化不良

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摘要

The anterograde intraflagellar transport motor protein, kif3a, regulates the integrity of primary cilia and various cellular functions, however, the role of kif3a in dental mesenchymal stem/precursor cell differentiation remains to be fully elucidated. In the present study, the expression of kif3a was knocked down in human dental follicle cells (hDFCs) and human dental pulp cells (hDPCs) using short hairpin RNA. The results of subsequent immunofluorescence revealed that knocking down kif3a resulted in the loss of primary cilia, which led to impairment of substantial mineralization and expression of the differentiation-associated markers, including alkaline phosphatase, Runt-related transcription factor 2, dentin matrix protein 1 and dentin sialophosphoprotein in the hDFCs and hDPCs. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses showed that the expression levels of Wnt3a-mediated active β-catenin and lymphoid enhancer-binding factor 1 were attenuated, whereas the expression of phosphorylated glycogen synthase kinase 3β was enhanced, in the kif3a-knockdown cells. In addition, exogenous Wnt3a partially rescued osteoblastic differentiation in the hDFCs and hDPCs. These results demonstrated that inhibition of kif3a in the hDFCs and hDPCs disrupted primary cilia formation and/or function, and indicated that kif3a is important in the differentiation of hDFCs and hDPCs through the Wnt pathway. These findings not only enhance current understanding of tooth development and diseases of tooth mineralization, but also indicate possible strategies to regulate mineralization during tooth repair and regeneration.
机译:顺行鞭毛内运输运动蛋白,kif3a,调节原发纤毛的完整性和各种细胞功能,但是,kif3a在牙齿间充质干/前体细胞分化中的作用尚待充分阐明。在本研究中,使用短发夹RNA敲低了人牙囊细胞(hDFCs)和人牙髓细胞(hDPCs)中的kif3a表达。随后的免疫荧光结果显示,敲低kif3a会导致原发纤毛损失,从而导致实质性矿化和分化相关标记(包括碱性磷酸酶,Runt相关转录因子2,牙本质基质蛋白1和hDFC和hDPC中的牙本质唾液磷蛋白。逆转录定量聚合酶链反应和Western印迹分析结果表明,Wnt3a介导的活性β-catenin和淋巴样增强剂结合因子1的表达水平降低,而磷酸化糖原合酶激酶3β的表达增强。 kif3a-knockdown细胞。此外,外源性Wnt3a部分挽救了hDFC和hDPC中的成骨细胞分化。这些结果表明,在hDFC和hDPC中对kif3a的抑制破坏了初级纤毛的形成和/或功能,并表明kif3a在通过Wnt途径分化hDFC和hDPC中很重要。这些发现不仅增强了当前对牙齿发育和牙齿矿化疾病的了解,而且还表明了在牙齿修复和再生过程中调节矿化的可能策略。

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