BMP-4 對於人類牙根尖細胞分化和間質代謝的影響：Smad1/5/8 和 Smad2/3 訊息傳導路徑的角色

Abstract

實驗目的 探討如何修復受損牙齒組織，並保存和維持受影響牙齒的自然功能，是再生牙髓病學領域中的重要議題。因此，找到合適的生長因子和幹細胞是組織工程學及再生醫學的成功關鍵。來自人類牙根尖的幹細胞（SCAPs）被認為是牙齒組織再生的有效細胞來源，而骨形態蛋白-4（BMP-4）在其中扮演著重要的角色。本研究旨在通過分析相關標誌物的表現，來探討BMP-4對SCAPs的分化潛能和間質代謝的影響，標誌物包括：Osterix（Osx）、N-cadherin、鹼性磷酸（ALP）、第一型膠原蛋白（COL1A1）、尿激型纖溶原激活物（uPA）和纖溶原激活物抑制劑-1（PAI-1）等， 並探討其信號傳導途徑。 實驗方法 使用原代培養的SCAPs作為實驗細胞，並用不同濃度的BMP-4（0, 10, 25, 50, 100, 200 ng/ml）處理24小時後，通過RT-PCR和western blot檢測Osx、N-cadherin、ALP、COL1A1、uPA、PAI-1的表現。另外，也使用p-Smad1/5/8抑制劑（LDN193189）和p-Smad2/3抑制劑 （SB431542）來研究在SCAPs中BMP-4誘發的Smad信號傳導途徑。 實驗結果 加入BMP-4處理24小時後的的SCAPs細胞顯示：Osx、N-cadherin、ALP、COL1A1和PAI-1的mRNA和蛋白質表現量增加，而uPA的表現量降低。加入LDN193189和SB431542後，結果顯示BMP-4在Smad傳導路徑的誘導作用被抑制。LDN193189顯著降低了Smad1/5/8、Smad2/3 的磷酸化，而SB431542則顯著降低了Smad1/5/8的磷酸化。 結論 根據本篇研究結果顯示，SCAPs具有進行多譜系分化的潛力，包括成骨作用、牙齒再生和細胞外間質代謝。根據抑制劑的結果顯示，Smad1/5/8和Smad2/3這兩種途徑可能存在一些交互作用。而TGF-β的信號傳導途徑可能參與了BMP-4的信號傳導途徑。BMP-4藉由Smad傳導路徑有調節SCAPs的分化和細胞外間質代謝的角色。SCAPs和BMP-4的組合使用未來或許可以運用在牙科組織修復工程領域之中。

Aim Regenerative endodontics is a field dedicated to the repair and restoration of damaged dental tissues, in hopes of conserving and maintaining natural function of the affected tooth. Finding the suitable growth factors and stem cells are key factors that contribute to successful tissue engineering. Human stem cells from apical papilla (SCAPs) are known as potent cell sources for dental tissue regeneration, while the bone morphogenic protein-4 (BMP-4) is a growth factor that is vital in the growth and development of human teeth. This study was designed to investigate the impacts BMP-4 may have on the differentiation potential and matrix turnover SCAPs, by analyzing the expression of differentiation related markers, including osterix (Osx), N-cadherin, alkaline phosphatase (ALP), type I collagen (COL1A1), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). The signaling pathways underlying these effects were also explored. Materials and Methods Primary-cultured SCAPs were treated with different concentrations of BMP-4 (0, 10, 25, 50, 100, 200 ng/ml) for 24 hours. The expressions of Osx, N-cadherin, ALP, COL1A1, uPA, PAI-1 were evaluated through RT-PCR and western blot. In addition, pre-treatment of LDN193189 (a p-Smad1/5/8 inhibitor) and SB431542 (a p-Smad2/3 inhibitor) were used to investigate the Smad-dependent pathways induced by BMP-4 in SCAPs. Results SCAPs exposed to BMP-4 for 24 hours showed an increase in the mRNA and protein expressions of Osx, N-cadherin, ALP, COL1A1, and PAI-1, whereas the expression levels of uPA were decreased. Pre-treatment with inhibitors, LDN193189 and SB431542, showed inhibitory effects on the BMP-4-stimulated expressions. Moreover, the addition of LDN193189 significantly reduced the phosphorylation of Smad1/5/8 and Smad2/3. Similarly, when SB431542 was added, the phosphorylation level of Smad1/5/8 was diminished. Conclusion Our results demonstrate that SCAPs have the potential to undergo multi-lineage differentiation, including osteogenesis, odontogenesis, and extracellular matrix turnover. In addition, pre-treatment with LDN193189 significantly decreased the phosphorylation levels of Smad1/5/8 and Smad2/3, while SB431542 also reduced phosphorylation of Smad1/5/8, suggesting these two pathways may have some interaction or crosstalk with each other, and that the TGF-β signaling pathway may possibly play a role in the mechanism of BMP-4 signaling pathways. It is visible through our research that BMP-4 plays an important role in the differentiation and extracellular matrix turnover of SCAPs by regulation through the Smad-dependent signaling pathways. Thus, the knowledge obtained from this study on the combined use of SCAPs and BMP-4 may be a beneficial approach for future clinical applications in dental tissue repair and engineering.