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標題: | TGF-β1對牙髓細胞之訊息傳導機制與其對牙髓分化的影響 Signaling transduction pathway of TGF-β1 and its effect on differention of human dental pulp cell |
作者: | Po-Shuen Lin 林柏萱 |
指導教授: | 鄭景暉(Jiiang-Huei Jeng) |
共同指導教授: | 林俊彬(Chun-Pin Lin) |
關鍵字: | 牙髓細胞,轉型生長因子,鹼性磷酸酶,Runx-2, Dental pulp cells,TGF-β1,Alkaline phosphatase,Runx-2, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 實驗目的:轉型生長因子(Transforming growth factor-beta, TGF-β) 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所誘導的訊息傳導途徑以及TGF-β1對牙髓細胞生長分化所造成影響的機制。
實驗方法:使用TGF-β1對人類牙髓細胞做刺激,在某些實驗中則先加入U0126,Noggin,SB431542 等抑制劑做前處理。另外以西方點墨法(Western Blot Analysis),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:在西方點墨法的分析中,TGF-β1會誘導下游的ERK, Smad 2/3, Smad 1/5/8 等訊息的活化。藉由Smad 2的訊息傳導途徑,TGF-β1 (5 ng/ml & 10 ng/ml) 會抑制人類牙髓細胞的細胞存活率與鹼性磷酸酶的活性。透過反轉錄鏈聚合反應的實驗可以發現TGF-β1 (5 ng/ml & 10 ng/ml) 會造成ALP 與Runx-2的基因表現都有下降的趨勢。TGF-β1 (5 ng/ml)也會促進牙髓細胞中膠原蛋白的合成。這些影響在使用U0126 (MEK/ERK抑制劑),Noggin (Smad 1/5/8抑制劑)做前處理的組別中沒有看到顯著的改變。但在使用SB431542作前處理的組別中 2 則可看到TGF-β1的作用有被抑制的現象。 結論:TGF-β1在人類牙髓細胞中的訊息傳導是複雜的,除了傳統的Smad 2/3之外,還包括了Smad1/5/8,ERK等途徑。透過Smad 2/3的途徑TGF-β1可能抑制細胞內的ALP,Runx-2的表現並提高膠原蛋白的合成,此作用與牙髓的修復與牙本質的再生有某種程度的影響。 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 two signaling pathways, MEK/ERK and Smad2 to mediate Runx2 expression, collagen matrix deposition and alkaline phosphatase in human dental pulp cells. Materials and Methods: Primary-cultured human dental pulp cells were treated with TGF-β1. in some experiments, cells were pretreated with U0126 (a MEK/ERK inhibitor), Noggin (a BMP antagonist), or SB431542 (an ALK5/ Smad 2/3 inhibitor) 30 minutes before adding TGF-β1. Signaling of TGF-β1 was investigated by western-blot assay. Cell proliferation was evaluated by MTT assay. Cell differentiation and mineralization was evaluated by alkaline phosphatase (ALP) staining. Changes in mRNA expression were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Results: In human dental pulp cell, TGF-β1 induce the expression of ERK, Smad 2, Smad 1/5/8 signaling. Cell under the treatment of TGF-β1 (5 ng/ml & 10 ng/ml) showed a decrease in ALP activity and gene expression of ALP and Runx-2. TGF-β1 (5 ng/ml) induced collagen formation in dental pulp cell. Pretreatment of U0126 (a MEK/ERK inhibitor) and Noggin (a Smad 1/5/8 inhibitor) was not effective to prevent these events. SB431542 (an ALK5/ Smad 2/3 inhibitor) attenuate the TGF-β 1-induced decline of ALP activity and Runx-2 expression. In conclusion, TGF-β1 down-regulates Runx-2 and ALP activity of human dental pulp cells via ALK5/Smad2/3 signaling. Conclusion: Signal transduction of TGF-β1 in human dental pulp cell is complex. In addition to the well-known Smad2/3 pathway, Smad 1/5/8 and MAPK/ERK also take part in this complex system. These events are crucial in the mechanism of pulpal repair and regeneration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25896 |
全文授權: | 未授權 |
顯示於系所單位: | 臨床牙醫學研究所 |
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