Tang Jia, Saito Takashi
Division of Biochemistry, Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
Division of Clinical Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
Stem Cells Int. 2017;2017:2546261. doi: 10.1155/2017/2546261. Epub 2017 Nov 19.
The purpose of the current study was to investigate the effects of nephronectin (Npnt) in human dental pulp stem cells (hDPSCs).
Npnt was coated to nontissue culture-treated polystyrene (non-PS) plates. The presence of immobilized protein on the surface was detected by polyclonal rabbit primary anti-Npnt antibody. Then the cell number was counted and compared with PBS-, bovine serum albumin- (BSA-), fish scale type I collagen- (FCOL1-), and human fibronectin- (Fn-) coated wells. Cell proliferation was assessed using CCK-8 assay. Cell morphology was observed under light microscopy and fluorescence microscopy. Lastly, the mRNA expression profiles of integrins, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and mineralization capacity of hDPSCs were investigated by real time RT-PCR and alizarin red staining, respectively.
Npnt mediates hDPSC adhesion and spreading partially via the Arg-Gly-Asp (RGD) motif. Npnt enhanced the mRNA expression of ITGA1, ITGA4, ITGA7, and ITGB1 on day five. Npnt downregulated DSPP but significantly upregulated BSP mRNA expression at day 28. Further, Npnt and FCOL1 accelerated the matrix mineralization in hDPSCs.
The current findings implicate that Npnt would be favorable to recruit hDPSCs and conducive to mineralization in hDPSCs. The combination of Npnt with hDPSCs may offer a promising approach for hard tissue regeneration.
本研究旨在探讨肾连蛋白（Npnt）对人牙髓干细胞（hDPSCs）的影响。
将Npnt包被在未进行组织培养处理的聚苯乙烯（non-PS）平板上。用兔抗Npnt多克隆一抗检测表面固定蛋白的存在。然后计数细胞数量，并与用磷酸盐缓冲液（PBS）、牛血清白蛋白（BSA）、鱼鳞I型胶原（FCOL1）和人纤连蛋白（Fn）包被的孔进行比较。使用CCK-8法评估细胞增殖。在光学显微镜和荧光显微镜下观察细胞形态。最后，分别通过实时逆转录聚合酶链反应（RT-PCR）和茜素红染色研究hDPSCs整合素、牙本质涎磷蛋白（DSPP）、骨涎蛋白（BSP）的mRNA表达谱及矿化能力。
Npnt部分通过精氨酸-甘氨酸-天冬氨酸（RGD）基序介导hDPSC的黏附和铺展。在第5天，Npnt增强了ITGA1、ITGA4、ITGA7和ITGB1的mRNA表达。在第28天，Npnt下调DSPP但显著上调BSP的mRNA表达。此外，Npnt和FCOL1加速了hDPSCs中的基质矿化。
目前的研究结果表明，Npnt有利于募集hDPSCs并有助于hDPSCs矿化。Npnt与hDPSCs的组合可能为硬组织再生提供一种有前景的方法。
