TGF-β1 對於牙根尖細胞生長與分化的影響

Effect of TGF-β1 on the growth and differentiation of human apical papilla cells

吳怡樺(I-Hua Wu)

指導教授 ： 鄭景暉

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02739

摘要

實驗目的: 轉型生長因子（Transforming growth factor-beta, TGF-β）對於細胞生長、細胞分化以及牙本質生成扮演著重要角色，但其訊息傳導路徑還未被完全了解，並且對於牙根尖細胞之特性也尚未了解。本實驗之目的在於探討TGF-β1 對牙根尖細胞生長與分化所造成的影響與機制。
實驗方法：使用TGF-β1 對於人類牙根尖細胞做刺激與培養，在一些
實驗另有先加入抑制劑：SB431542 或U0126 做前處理之後，以MTT、
直接計算活細胞數量、鹼性磷酸酶染色及活性測定（ALP staining &
activity quantitative assay ）、膠原蛋白定量測定（Sircol collagen assay）、Alizarin Red 染色、鈣離子定量測定以及反轉錄聚合酶反應（RT-PCR）等實驗，來觀察我們的發現。
實驗結果：在細胞生長或是細胞外基質（extracellular matrix）方
面，TGF-β1 主要是藉由Smad 2/3 的路徑，刺激人類牙根尖細胞的細胞存活率以及膠原蛋白的生成；並且，TGF-β1 在較低的濃度下（0.5-1ng/ml）會促進鹼性磷酸酶的活性，在較高濃度（5-10 ng/ml）則是有抑制的效果，並且ERK 的抑制劑U0126 可逆轉低濃度時的促進效果，卻無法逆轉其高濃度的抑制效果，這代表了TGF-β1 在不同的濃度下會引發不同的訊息傳導途徑。而在鈣化誘導方面，TGF-β1 則是有抑制鈣化小節（nodule）以及鈣離子的形成。
結論：TGF-β1 在人類牙根尖細胞中的訊息傳遞是相當複雜的，除了常見的Smad 2/3 途徑之外，還包含了ERK 等其他Non-Smad 途徑。經
由ALP 活性測定實驗我們可得知，TGF-β1 會隨著濃度的不同而對細
胞有著相異的效果，並且濃度不同所引發訊息傳導途徑也會有所不
同。種種的觀察對於人類牙根尖細胞未來是否有機會運用在牙根再
生，有某種程度上的進ㄧ步理解。

關鍵字

牙根尖細胞 ； 轉型生長因子 ； 鹼性磷酸酶鈣化

並列摘要

Aim：Transforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and osteogenesis / odontogenesis. The purpose of this
study is to investigate the effects of TGF-β1 on human apical papilla cells. We hypothesize that TGF-β1 can stimulate the two signaling pathways, MEK/ERK and
Smad 2/3 to mediate alkaline phosphatase (ALP) expression, collagen matrix deposition and calcium deposition in human apical papilla cells.
Materials and Methods：Primary-cultured human apical papilla cells were treated with TGF-β1. In some experiments, apical papilla cells were pretreated with SB431542 ( an ALK5 / Smad 2/3 inhibitor) or U0126 ( a MEK/ERK inhibitor) 30 minutes before adding TGF-β1. Cell viability and cell growth were examined by MTT assay or direct counting of viable cell numbers. Collagen content was determined by Sircol Collagen assay. Cell differentiation and mineralization were evaluated by alkaline phosphatase（ALP）staining, ALP activity quantitative assay, Alizarin Red staining, and calcium
quantitative analysis. Changes in mRNA expression were determined by reverse-transcription Polymerase Chain Reaction（RT-PCR）.
Results：In human apical papilla cells, TGF-β1 increased cell numbers and cell viability. Cells under the treatment of TGF-β1 (>0.1 ng/ml) would induce collagen formation.
Pretreatment of U0126（a MEK/ERK inhibitior）was not effective to reverse the effects of TGF-β1 on cell growth and matrix formation; but SB431542（an ALK5/ Smad 2/3
inhibitor）could prevent those effects. In the differentiation, TGF-β1 down-regulates ALP activity in the lower concentration （0.5-1 ng/ml) and up-regulates it in the higher concentration（5-10 ng/ml）. SB431542 can reverse the effect of TGF-β1 at lower or higer concentration; but U0126 only can reverse the effect of TGF-β1 at lower concentration. Besides, TGF-β1 inhibits the mineralization on apical papilla cells.
Conclusion：Signal transduction of TGF-β1 in human apical papilla cells is complex. Different cell sources, different concentrations of growth factor, presence of absence of
serum or culture conditions can affect the effects of TGF-β1. Besides, the well-known Smad 2/3 pathway, non Smad pathway（ex. ERK）may also take part in this complex
system. These results highlight our future use of growth factors in pulpal repair and dentinogenesis.

並列關鍵字

apical papilla cells ； TGF-β1 ； alkaline phosphate ； mineralization

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