Biomedical Science Team, Chang Gung University of Science and Technology, Kwei-Shan, Taoyun City, Taiwan; Department of Dentistry, Chang Gung Memorial Hospital, Taipei, Taiwan.
Graduate Institute of Clinical Dentistry, National Taiwan University Medical College, Taiwan; Department of Dentistry, National Taiwan University Hospital, Taiwan.
J Adv Res. 2022 Sep;40:95-107. doi: 10.1016/j.jare.2021.12.006. Epub 2021 Dec 28.
Basic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration.
The purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling.
SCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining.
SCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05).
bFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.
碱性成纤维细胞生长因子（bFGF）在成牙本质细胞分化和牙本质基质沉积中发挥关键作用，从而有助于牙髓-牙本质的修复和再生。
本研究旨在阐明 bFGF 对根尖乳头干细胞（SCAP）中纤溶酶原激活因子、TIMP-1、ALP；和 SPARC（骨粘连蛋白）表达/产生的影响；以及 MEK/ERK、p38、Akt 和 TAK1 信号通路的参与情况。
用 bFGF 处理 SCAP，并用/不用预处理和共孵育各种信号转导抑制剂（U0126、SB203580、LY294002 和 5Z-7-oxozeaenol）。通过免疫荧光染色分析 FGF 受体（FGFRs）、PAI-1、uPA、p-ERK、p-TAK1 和 p-p38 的表达。通过实时 PCR 和 ELISA 测定 SCAP 的基因表达和蛋白分泌。通过 ALP 染色评估 ALP 活性。
SCAP 表达 FGFR1、2、3 和 4。bFGF 刺激 PAI-1、uPA、uPAR 和 TIMP-1 的 mRNA 表达（p<0.05）。bFGF 诱导 PAI-1、uPA 和可溶性 uPAR 的产生（p<0.05），但抑制 SCAP 的 ALP 活性和 SPARC 产生（p<0.05）。bFGF 刺激 SCAP 的 ERK、TAK1 和 p38 磷酸化。U0126（MEK/ERK 抑制剂）和 5Z-7-oxozeaenol（TAK1 抑制剂）减弱了 bFGF 诱导的 SCAP 的 PAI-1、uPA、uPAR 和 TIMP-1 的表达和产生，但 SB203580（p38 抑制剂）没有。LY294002、SB203580 和 5Z-7oxozeaenol 不能逆转 bFGF 引起的 ALP 活性抑制。有趣的是，U0126 和 5Z-7-oxozeaenol 阻止了 bFGF 诱导的 SPARC 产生的下降（p<0.05）。
bFGF 可能通过调节 PAI-1、uPA、uPAR 和 TIMP-1 来调节纤溶和基质转化，但 bFGF 抑制了 SCAP 的分化（ALP、SPARC）。这些事件主要受 MEK/ERK、p38 和 TAK1 调节。bFGF 和 SCAP 的联合使用可能通过调节纤溶酶原激活系统、迁移、基质转化和 SCAP 的分化来促进牙髓/根修复和再生。
