TGF-β1對於人類牙根尖細胞分化的影響:ALK5/Smad2訊息傳遞路徑的角色

Abstract

實驗目的:本實驗目的是透過研究osterix（osx），巢蛋白(nestin)，波形蛋白(vimentin)，結締組織生長因子（CTGF），N-鈣粘蛋白(N-cadherin)，牙骨質蛋白1（CEMP1），牙骨質附著蛋白（CAP）和纖溶酶原激活抑製劑-1（PAI-1）等分化相關標誌物的表現，探索TGF-β1誘導人類牙根尖細胞分化的潛力。並且探索其相關的訊號傳遞途徑。 實驗方法 : 將人類牙根尖細胞暴露於不同濃度（0, 0.5, 1, 5, 10, 25 ng / ml）的TGF-β1下24小時，利用分化相關標誌物如osx，nestin，vimentin，CTGF，N-cadherin，CEMP1，CAP和PAI-1透過反轉錄聚合酶連鎖反應(RT-PCR)，西方點墨法(western blot)以及免疫螢光染色來看其影響基因或蛋白的表現為何。此外，使用SB431542（ALK5 / Smad2抑制劑）的預先處理來研究TGF-β1誘導人類牙根尖細胞的分化作用是否透過Smad依賴性（經典）訊號途徑傳遞。 實驗結果 : 暴露於TGF-β1 24小時的人類牙根尖細胞沒有明顯的形態改變。當加入TGF-β1時，osx，nestin，vimentin，CTGF，N-cadherin，CEMP1，CAP和PAI-1的基因和/或蛋白質表現增加。此外，加入1, 2.5 μM SB431542後減少了TGF-β1誘導的nestin，vimentin，CTGF，N-cadherin和PAI-1的表現。然而，加入抑制劑SB431542並未抑制osx，CEMP1和CAP的表現。 結論 : TGF-β1可能具有誘導人類牙根尖細胞多種分化潛能的能力，例如骨生成，牙本質形成，牙骨質生成，以及影響細胞外基質生成轉換。由TGF-β1誘導的nestin，vimentin，CTGF，N-cadherin和PAI-1的表現是透過ALK5 / Smad2信號傳遞路徑。然而，由TGF-β1誘導的osx，CEMP1和CAP的表現則不是經由此路徑傳遞。TGF-β1及牙根尖細胞應具有應用在臨床再生性牙髓治療的潛力。

Aim : The aim of the study is to investigate the differentiation potentials of human apical papilla cells induced by TGF-β1 by analyzing the expressions of differentiation related markers, including osterix (osx), nestin, vimentin, connective tissue growth factor (CTGF), N-cadherin, cementum protein 1 (CEMP1), cementum attachment protein(CAP), and plasminogen activation inhibitor-1(PAI-1). The signaling pathway conveying the effects was also explored. Materials and methods: Primary-cultured human apical papilla cells were treated with different concentrations (0, 0.5, 1, 5, 10, 25 ng/ml) of TGF-β1 for 24 hours. The expressions of osx, nestin, vimentin, CTGF, N-cadherin, CEMP1, CAP, and PAI-1 were evaluated through RT-PCR, western blot, and immunofluorescence. In addition, pretreatment of SB431542 (as a specific ALK5/Smad2 inhibitor) was used to investigate the Smad-dependent (canonical) pathway in TGF-β1-induced effects on human apical papilla cells. Results: Human apical papilla cells exposed to TGF-β1 for 24 hours showed no obvious morphology change. The mRNA and/or protein expressions of osx, nestin, vimentin, CTGF, N-cadherin, CEMP1, CAP, and PAI-1 showed increased when adding TGF-β1. Besides, pretreatment of 1and 2.5 µM SB431542 reduced TGF-β1 stimulated-expressions of nestin, vimentin, CTGF, N-cadherin, and PAI-1. However, the expressions of osx, CEMP1, and CAP were not inhibited by adding inhibitor SB431542. Conclusions : TGF-β1 may be capable to induce various differentiation potentials of human apical papilla cells such as osteogenesis, odontogenesis and cementogenesis, and to modulate the turnover of extracellular matrix. The expressions of nestin, vimentin, CTGF, N-cadherin, and PAI-1 induced by TGF-β1 were conveyed through ALK5/Smad2 signaling pathway. However, the expressions of Osx, CEMP1, and CAP induced by TGF-β1 were not. The information may benefit the development of new strategies for regenerative endodontics.