TGF-beta對人類牙髓細胞的影響:p38與TAK1之角色

Abstract

實驗目的：轉型生長因子1 (Transforming growth factor-beta1 ,TGF- β1)對於牙髓組織的修復與牙本質生成扮演重要的角色。本實驗的目 的在於探討TGF-β1對牙髓細胞生長分化所造成影響時TAK1 以及p38所扮演的 角色。 實驗方法：先加入SB203580 (p38抑制劑)，5Z-7-oxozeaenol (TAK1抑制劑)做前處理30分鐘，再使用TGF-β1對人類牙髓細胞做刺激。以MTT 測定，反轉錄鏈聚合反應(RT-PCR)，鹼性磷酸酶染色 (ALP staining)，膠原蛋白定量測定(Sircol Collagen Assay) 來觀察我們的發現。 實驗結果： TGF-β1 (10 ng/ml)並不會誘發牙髓細胞增生，SB203580 (p38抑制劑)亦不會改變TGF-β1 (10 ng/ml)在牙髓細胞的影響。然而5Z-7-oxozeaenol(TAK1抑制劑)卻會降低細胞的存活能力。TGF-β1 (0.1 ng/ml) 些微促進人類牙髓細胞的鹼性磷酸酶的活性會被SB203580 與5Z-7-oxozeaenol所逆轉，而TGF-β1在較高濃度時 (10 ng/ml)會抑制人類牙髓細胞的鹼性磷酸酶的活性而抑制現象無法被SB203580 與5Z-7-oxozeaenol逆轉。透過SB203580 與5Z-7-oxozeaenol，TGF-β1 (5 ng/ml)促進牙髓細胞中膠原蛋白的合成以及TGF-β1 (0.1 ng/ml) 促進鹼性磷酸酶的活性，會逆轉其影響。。然而反轉錄鏈聚合反應實驗亦證實SB203580 與5Z-7-oxozeaenol會抑制牙髓細胞原本因TGF-β1 (10 ng/ml) 促進collagen的基因 表現，但對ALP基因降低表現並無法改變。 結論： TAK1似乎對誘導牙髓細胞的生長能力有所影響。透過TAK1-p38的訊息傳導途徑，TGF-β1 (0.1 ng/ml) 可促進人類牙髓細胞的鹼性磷酸酶的活性以及TGF-β1 (5 ng/ml)可促進牙髓細胞中膠原蛋白的合成。TGF-β1 在高濃度時(10 ng/ml) 無法透過TAK1 - p38路徑抑制人類牙髓細胞的鹼性磷酸酶的活性。

Aim: Transforming growth factor β1 (TGF-β1) plays a role in repair and dentinogenesis in dental pulp cells. The purpose of this study is to investigate whether TGF-β1 stimulates the signaling pathways, TAK1 and p38, thereby influence the morphological changes, cell proliferation, collagen turnover and alkaline phosphatase in human dental pulp cells in vitro. Materials and Methods: Primary-cultured human dental pulp cells were pre-treated with SB203580 (p38 inhibitor) or 5Z-7-oxozeaenol(TAK1 inhibitor) 30 minutes before adding TGF-β1. Morphology of pulp cells was observed under light microscopy (100X). Cell proliferation was evaluated by MTT assay. Cell differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and quantitative assay. Changes in mRNA expression (ALP and collagen) were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Result : In the dental pulp cells, TGF-β1 (10ng/ml) showed little effect on cell number and cell size under light microscopy (100X) and MTT assay. 5Z-7-oxozeaenol(TAK1 inhibitor) showed decrease in the number of dental pulp cells markedly but SB203580 (p38 inhibitor) did not. TGF-β1 (10ng/ml) decreased the ALP activity but SB203580 and 5Z-7-oxozeaenol could not reverse the effect. TGF-β1 (0.1 ng/ml) mildly increased the ALP expression and TGF-β1 (5 ng/ml) increased the collagen formation. These effects could be reversed by SB203580 and 5Z-7-oxozeaenol. According to the RT-PCR, SB203580 and 5Z-7-oxozeaenol could reverse the up-regulatory of collagen expression by TGF-β1 (10ng/ml). However, down-regulatory of ALP gene expression caused by TGF-β1 (10ng/ml) could not be reversed by SB203580 and 5Z-7-oxozeaenol. Conclusion: TAK1 may increase proliferation of human dental pulp cells. TGF-β1 (0.1 ng/ml) can increase ALP activity and TGF-β1 (5 or 10 ng/ml) can induce collagen formation in dental pulp cells via TAK1-p38 pathway. TAK1-p38 pathway may have no influence in down-regulatory of ALP activity in TGF-β1 (10 ng/ml).