Zhang Zhichun, Guo Qingru, Tian Hua, Lv Ping, Zhou Chunyan, Gao Xuejun
Department of Cariology and Endodontology, School and Hospital of Stomatology, Peking University, Beijing, People's Republic of China.
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, Beijing, People's Republic of China.
J Endod. 2014 Oct;40(10):1593-9. doi: 10.1016/j.joen.2014.07.009. Epub 2014 Aug 16.
Wingless-type MMTV integration site family, member 10A (WNT10A) plays crucial roles in odontogenesis. The aim of this study was to investigate the effects of WNT10A on human dental pulp cells (DPCs), which contain a mixed population of cells, including stem and progenitor cells, and participate in dentin repair or dentin-pulp regeneration.
Healthy human premolars extracted for orthodontic reasons were used as a study model. The expression of WNT10A protein in dental pulp was determined by immunohistochemistry. The messenger RNA expression of WNT10A and Wnt-related genes was analyzed by semiquantitative reverse-transcription polymerase chain reaction. DPCs were enzymatically separated from pulp tissues, cultured, and passaged. The biological effects of WNT10A on DPCs were investigated using recombinant lentivirus encoding WNT10A complementary DNA. WNT10A-induced changes in DPC proliferation were assessed by methyltetrazolium assay and flow cytometry. In order to determine the effects of WNT10A on DPC differentiation, the activity of alkaline phosphatase (ALP), an early marker of odontoblastic differentiation, was assessed using an ALP activity assay kit, and the expression levels of odontoblast-specific genes, including DSPP, DMP1, ALP, and COL1A1, were detected by quantitative polymerase chain reaction and Western blot.
WNT10A protein was clearly identified in the cytoplasm of DPCs. Semiquantitative reverse-transcription polymerase chain reaction indicated the expression of WNT10A and Wnt-related genes in pulp tissues as well as in passaging DPCs. Lentiviral overexpression of WNT10A enhanced proliferation of DPCs and down-regulated ALP activity and the expression of odontoblast-specific genes.
WNT10A promotes the proliferation of DPCs and negatively regulates their odontoblastic differentiation.
无翼型MMTV整合位点家族成员10A（WNT10A）在牙齿发育过程中发挥着关键作用。本研究旨在探讨WNT10A对人牙髓细胞（DPCs）的影响，人牙髓细胞包含多种细胞，包括干细胞和祖细胞，并参与牙本质修复或牙髓-牙本质再生。
以因正畸原因拔除的健康人前磨牙作为研究模型。采用免疫组织化学法测定牙髓中WNT10A蛋白的表达。通过半定量逆转录聚合酶链反应分析WNT10A及Wnt相关基因的信使核糖核酸表达。将DPCs从牙髓组织中酶解分离、培养并传代。使用编码WNT10A互补DNA的重组慢病毒研究WNT10A对DPCs的生物学作用。通过甲基四氮唑法和流式细胞术评估WNT10A诱导的DPCs增殖变化。为了确定WNT10A对DPCs分化的影响，使用碱性磷酸酶（ALP）活性检测试剂盒评估成牙本质细胞分化早期标志物ALP的活性，并通过定量聚合酶链反应和蛋白质免疫印迹法检测包括牙本质涎磷蛋白（DSPP）、牙本质基质蛋白1（DMP1）、ALP和I型胶原α1链（COL1A1）在内的成牙本质细胞特异性基因的表达水平。
在DPCs的细胞质中可清晰鉴定出WNT10A蛋白。半定量逆转录聚合酶链反应表明WNT10A及Wnt相关基因在牙髓组织以及传代的DPCs中均有表达。WNT10A的慢病毒过表达增强了DPCs的增殖，并下调了ALP活性和成牙本质细胞特异性基因的表达。
WNT10A促进DPCs的增殖，并对其成牙本质细胞分化起负向调节作用。
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