鄭景暉Jeng, Jiiang-Huei臺灣大學:臨床牙醫學研究所林柏萱Lin, Po-ShuenPo-ShuenLin2010-05-262018-07-092010-05-262018-07-092009U0001-2207200900022000http://ntur.lib.ntu.edu.tw//handle/246246/184184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。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。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作前處理的組別中可看到TGF-β1的作用有被抑制的現象。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.aterials 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.esults: 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.onclusion: 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.中文摘要.......1bstract.......3hapter I Literature Review..5.1 Reparative mechanism of pulpal-dentin complex.......5.2 Transforming Growth Factor –beta superfamily.8.2.1 TGF-β superfamily....8.2.2 TGF-β isoforms and their peptide structure.....9.2.3 TGF-β in dental pulp.9.3 Receptors of TGF-β...11.4 Signal transduction pathways of TGF-β1........12.4.1 Ligand binding & Receptor recruitment.12.4.2 Smad Phosphorylation & Co-mad binding.....13.4.3 Translocation into nucleus & DNA transcription.......13.4.4 I-Smads..14.4.5 TGF-β & MAPK pathway...14.5 Regulation of Alkaline phosphatase (ALP) and its role in tissue mineralization..15.5.1 Alkaline phosphatase.15.5.2 Regulation of ALPase..16.6 Runx2 and its role in cell differention.....17hapter II The purposes of the study.19hapter III Materials & Methods.....20.1 Materials..20.2 Culture of human dental pulp cells.20.3 Effect of TGF-β on human dental pulp cells...21.4 Alkaline phosphatase (ALP) staining......21.5 Reverse Transcription Polymerse Chain Reaction (RT-PCR)........22.5.1 Isolation of total RNA.23.5.2 RNA Quantitation....24.5.3 Reverse transcription (RT)..24.5.4 Polymerase Chain Reaction (PCR).......25.6 Western Blot Analysis.27.6.1 Sample preparation..27.6.2 Protein Concentration Determination...27.6.3 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)...28.6.4 Western Blot..28.7 MTT assay........30.8 Collagen Content Assay.....30.9 Statistical analysis.....32hapter IV Results..33.1 Morphological observation on human dental pulp cell.33.2 Signaling transduction pathways on human dental pulp cell by TGF-β1 - western blot analysis.......33.3 Effect of TGF-β1 on cell viability of pulp cells- MTT assay.......34.4 Effect of TGF-β1 on ALP activity of pulp cells......35.5 Effect of TGF-β1 on ALP activity of pulp cells and its modulation by SB431542, Noggin and U0126.36.6 Effect of TGF-β1 on ALP and Runx-2 mRNA expression of pulp cells..37.7 Effect of SB431542 on the TGF-β1-induced alterations of ALP and Runx-2 expression in dental pulp cells..37.8 Effect of TGF-β1 on type I collagen formation of dental pulp cell and its modulation by SB431542, Noggin and U0126.....38hpater V Discussion.......39hapter VI Conclusion......44eferences...45application/pdf2174600 bytesapplication/pdfen-US牙髓細胞轉型生長因子鹼性磷酸酶Runx-2Dental pulp cellsTGF-β1Alkaline phosphataseRunx-2http://ntur.lib.ntu.edu.tw/bitstream/246246/184184/1/ntu-98-R95422016-1.pdf