- 學位論文
TGF-β1對於脫落乳牙牙髓幹細胞之訊息傳導機制 及其生長與分化的影響
The signal transduction pathway of TGF-β1 and its effect on the growth and differentiation of dental pulp stem cells from human exfoliated deciduous teeth
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摘要
實驗目的：轉型生長因子（Transforming growth factor-beta1, TGF-β1）調控許多生理機制，且被認為在牙齒組織受傷的防禦系統中扮演重要的角色，近十年才被發現的脫落乳牙牙髓幹細胞（Dental pulp stem cells from human exfoliated deciduous teeth, SHED）被認為是良好的幹細胞來源而受到矚目，然而關於TGF-β1和SHED之間的作用目前仍尚未了解，而本實驗之目的在於研究TGF-β1對於SHED在細胞生長與分化的影響，還有其可能調控的訊息傳導路徑。 實驗方法：加入不同濃度的TGF-β1至細胞培養液中，我們利用細胞存活率分析（MTT assay）、 膠原蛋白定量測定（Sircol collagen assay）、 鹼性磷酸酶染色及活性測定（ALP staining & ALP activity quantitative assay ）去檢測TGF-β1對於SHED在細胞生長、細胞外基質的生成、細胞分化的影響，此外，我們也加入不 同的抑制劑，包含SB431542、U0126、5Z-7-Oxozeaenol、SB203580四種，探測可能參與調控細胞反應的訊息傳導路徑。利用酵素連結免疫吸附法（ELISA）去評估不同訊息傳導路徑之間的交互作用，而基因表現的改變則以反轉錄聚合酶反應（RT-PCR）的結果來呈現。 實驗結果：TGF-β1刺激促進SHED的細胞生長和細胞外基質的生成，而這個作用可以被SB431542、5Z-7-Oxozeaenol、SB203580所抑制，而U0126的加入則沒有明顯的影響。在細胞分化的部份，低濃度的 TGF-β1 (0.5-1 ng/ml)會促進鹼性磷酸酶的活性，然而高濃度的 TGF-β1 (5-10 ng/ml)則會有抑制的效果，SB431542能阻斷TGF-β1在低濃度和高濃度的作用，U0126和5Z-7-Oxozeaenol則只能阻斷低濃度 TGF-β1的影響，而SB203580本身就對鹼性磷酸酶的活性就有強烈的抑制作用。此外，磷酸化Smad2的表現可以很快的被TGF-β1誘發，且能被SB431542所抑制，然而5Z-7-Oxozeaenol的加入則是可以完全阻斷TGF-β1對於Smad2的磷酸化。 結論：TGF-β1對於SHED細胞反應的訊息調控是非常複雜的，除了Smad和non-Smad訊息傳導路徑之間可能存在的交互作用，不同濃度的TGF-β1還會經由不同的訊息傳導路徑來調控產生不同的細胞反應，本實驗的結果提供我們對於乳牙牙髓修復的機制有進一步的了解，對於未來在牙髓組織再生方面的研究也有所幫助。
並列摘要
Aim : Transforming growth factor-β1 (TGF-β1) regulates many biological process and is thought to be important in response to dental tissue injury. Dental pulp stem cells from human exfoliated deciduous teeth (SHED) were discovered in the recent ten years and considered a desirable stem cell source. The purpose of this study is to investigate the effects of TGF-β1 on SHED. We hypothesize that TGF-β1 can affect cell viability, collagen production and alkaline phosphatase (ALP) expression in SHED via both Smad and non-Smad MAPK pathway, which might have cross-talk with each other. Materials and Methods : Primary-cultured SHED were treated with different concentration of TGF-β1. We used MTT assay, Sircol Collagen assay, ALP staining and ALP activity quantitative assay to detect the effect of TGF-β1 on cell viability, matrix formation, and cell differentiation. Besides, SHED were pretreated with SB431542 (an ALK5-Smad2/3 inhibitor), U0126 (a MEK-ERK1/2 inhibitor), 5Z-7-Oxozeaenol (a TAK1 inhibitor), or SB203580 (a p38 MAPK inhibitor) for examining the related signaling pathways. Phosphorylation of Smad2 was examined by Enzyme-linked immunosorbent assay (ELISA) for evaluating the cross-talk within the Smad and non-Smad MAPK pathway. Changes in mRNA expression were determined by reversetranscription Polymerase Chain Reaction (RT-PCR). Results : TGF-β1 stimulated cell proliferation and matrix production of SHED in a dose-dependent manner, which could be reversed by pretreatment of SB431542, 5Z-7-Oxozeaenol and SB203580; nevertheless, it was not suppressed by U0126. In cell differentiation, low concentration of TGF-β1 (0.5-1 ng/ml) up-regulated the ALP activity of SHED, whereas high concentration (5-10 ng/ml) down-regulated its expression. SB431542 could reverse the effect of TGF-β1 at both low and high concentrations. SB203580 itself down-regulated ALP activity significantly; however, 5Z-7-Oxozeaenol only reversed the effect of TGF-β1 in low concentration. Concerning the signal transduction, the expression of phospho-Smad2, which was quickly induced by TGF-β1 and repressed by SB431542, was found to be completely abolished by 5Z-7-Oxozeaenol. Conclusion : The regulation about signal transduction of TGF-β1 in SHED is complicated. In addition to the cross-talk of Smad and non-Smad pathway, different concentration of TGF-β1 can mediate different cell response via different signaling pathways. These results are crucial to the mechanism of pulpal repair, that can be useful for further investigation of pulpal regeneration.
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延伸閱讀
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- 王慧茹（2013）。探討類胰島素生長因子-1受體之訊息傳遞於無血清培養之牙髓幹細胞特性影響〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://doi.org/10.6831/TMU.2013.00012
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