凝血酶誘導牙周韌帶細胞結締組織生長因子表現之研究

Abstract

牙周病的產生主要是破壞牙齒的支持結構,而牙周病的治療主要是以發炎控制進而希望利用再生的技術，希望恢復牙齒周遭健康的支持組織。近年來，有需多的研究紛紛探討再生因子應用在重建牙周組織的部分。結締組織生長因子(CTGF)在某些研究當中已被證實具有誘導骨細胞分化的能力，然而在不同的組織中仍是有相當的差異性。 我們利用不朽化牙周韌帶細胞，以反轉錄聚合酶連鎖反應以及西方墨點法觀察凝血酶(thrombin)是否能誘發牙周韌帶細胞結締組織生長因子的表現。 並且探討凝血酶誘導結締組織生長因子訊息傳遞路徑。結果發現，凝血酶能以劑量與時間相關性來刺激牙周韌帶細胞CTGF的表現。使用凝血酶受器N端合成胜肽PAR1促效劑SFLLRN，和凝血酶有類似的效果；絲胺酸蛋白酶抑制劑PPACK可以完全抑制凝血酶誘發的CTGF表現，顯示凝血酶是透過其蛋白酶活性，酶切活化PAR1，來引發後續的訊息傳導。當使用不同的訊息傳導路徑抑制物來前處理牙周韌帶細胞時，其結果顯示： ASK1抑制劑thioredoxin、ROS 抑制劑NAC、JNK抑制劑 SP600125會顯著降低凝血酶誘導的CTGF表現。其次，使用抗氧化劑N-acetyl-L-cysteine (NAC)、Rac-GTPase抑制劑NSC-23766、NADPH oxidase (NOX)抑制劑Plumbagin和DPI亦能顯著降低凝血酶誘導的CTGF表現。凝血酶可能是透過 PAR1/ROS/ASK1/JNK此一路徑來誘導CTGF表現，且NOX可能是此路徑中ROS的來源。 我們另外發現，凝血酶(thrombin)能誘導成骨细胞分化的重要轉錄因子RUNX2的基因表現。凝血酶(thrombin)是否能誘導牙周韌帶細胞骨分化則有待進一步研究。

The periodontal disease means destruction of the tooth supporting tissue, and the treatment of periodontitis mainly control the inflammation and further regenerate the health tooth supporting tissue through regenerative technique. Recently, there are more and more researches focus on the application of growth factor on periodontal reconstruction. In some researches, connective tissue growth factor (CTGF) has been proved its osteogenetic potential, however, it still showed large variety among different cells and tissues. We used immortalized periodontal ligament cells to investigate if thrombin could induce CTGF expression through real-time PCR and western blot, and we also research the signal transduction pathway of thrombin-induced CTGF expression. In this study, we showed that thrombin caused a concentration- and time-dependent expression of CTGF in periodontal ligament cells. The effect of thrombin could be mimicked with the protease-activated receptor 1 (PAR1) agonist peptide, SFLLRN, and could be completely inhibited by a serine protease inhibitor, PPACK, indicating that thrombin mediated this effect via the proteolytic cleavage and activation of PAR1. We further used several signaling inhibitors to pretreat periodontal ligament cells and the results were: ASK1 inhibitor (thioredoxin), ROS inhibitor (NAC), JNK inhibitor (SP600125) could significantly reduce the level of thrombin-induced CTGF. In addition, antioxidant N-acetyl-L-cysteine (NAC), Rac-GTPase inhibitor (NSC-23766), NADPH oxidase inhibitors (Plumbagin and DPI), also had the inhibitory effects on the thrombin-induced CTGF. Thrombin probably induced CTGF expression through PAR1/ROS/ASK1/JNK pathway, and NOS could be the source of ROS. The study also reported that thrombin could induce the gene expression of Runx2, the main transcriptional factor of osteogenesis. However, whether thrombin could induce osteogenesis in periodontal ligament cells is still uncertain and further researches are needed.