TGF-β1對人類牙髓細胞生成膠原蛋白，前列腺素E2，第二型環氧酶的影響與其訊息傳導機制的探討

Abstract

實驗目的：轉型生長因子(Transforming growth factor-beta, TGF-β) 在發生蛀牙時，產生牙髓組織的修復與牙本質母細胞的分化與基質生成扮演重要的角色。TGF-β1經由許多不同的訊息傳導路徑來發揮它的效果，像是SMAD 路徑與 MAPK路徑。 本篇論文分兩部分來探討TGF-β1對牙髓組織修復與再生的影響。第一部分討論不同訊息傳導路徑對於牙髓細胞中TGF-β1在膠原蛋白(collagen)，第三型基質金屬蛋白酶 (metalloproteinase-3, MMP-3) 與第一型組織抑制金屬蛋白酶 (tissue inhibitor metalloproteinase-1, TIMP-1)生成的影響。第二部分探討TGF-β1與第二型環氧酶(cyclooxygenase-2, COX-2)、前列腺素E2 (prostaglandin E2, PGE2)這兩種重要的發炎相關酵素的關係。並且進一步討論TGF-β1的不同訊息傳導路徑對於第二型環氧酶的影響。 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 (MEK/ERK抑制劑) ，SB431542 (ALK5/Smad2抑制劑) 等訊息傳導路徑的抑制劑做前處理。第一部分使用膠原蛋白定量測定(Sircol Collagen Assay) 測量牙髓細胞中膠原半白的量。培養液中的pro-collagen I, TIMP-1 and MMP-3的量以酵素免疫分析法（Enzyme-Linked ImmunoSorbent Assay, ELISA）來測定。第二部分以酵素免疫分析法（Enzyme-Linked ImmunoSorbent Assay, ELISA） 來測定PGE2的量。以反轉錄鏈聚合反應(RT-PCR)、西方點墨法(Western Blot Analysis)來測定COX-2 mRNA 與蛋白質的量。 實驗結果：TGF-β1 會促進牙髓細胞生成膠原蛋白，也會增加pro-collagen I 與 TIMP-1 的產生，但是對於MMP-3會有輕微的抑制傾向。 SB431542與 U0126這兩種訊息傳導路徑的抑制劑會抑制 TGF-β1所誘導的膠原蛋白與TIMP-1的產生。 TGF-β1 (1-10 ng/ml)會增加牙髓細胞內 COX-2與 PGE2的產生。 SB431542會抑制牙髓細胞內TGF-β1對於 COX-2的作用。而 U0126對於TGF-β1也有稍微抑制的現象。 結論：TGF-β1 會參與牙髓細胞受傷後產生的修復與再生過程，這個現象是經由ALK5/Smad2/3與MEK/ERK的訊息傳導路徑去刺激膠原蛋白與TIMP-1的生成。TGF-β1也透過這個訊息傳導路徑去增加COX-2 與PGE2的生成，這與牙髓細胞受傷後早期的發炎現象與之後的修復有關。

Aim: TGF-β1 is an important growth factor which shown to influence odontoblast differentiation and matrix deposition in reactionary dentinogenesis during dental caries. TGF-β1 exerts its effects through many signaling pathways, such as SMADs and MAPKs. In this study, there are two parts investigating effects of TGF-β1on pulpal repair and regeneration. In the first part, we investigated the differential signaling pathways responsible for effects of TGF-β1 on collagen turnover, metalloproteinase-3 (MMP-3) and tissue inhibitor metalloproteinase-1 (TIMP-1) production in human dental pulp cells. In the second part of this study, we investigated the relationships between TGF-β1, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2), two important proinflammatory cytokines of pulpal inflammation in in human dental pulp cell and further clarify the differential signaling transduction pathways of TGF-β1 that influence COX-2 production. Materials and Methods: Pulp cells were exposed to TGF-β1 with/without pretreatment of SB431542 (an ALK5/Smad2/3 inhibitor) and U0126 (a MEK/ERK inhibitor). In the first part of this study, sircol collagen assay was used to measure cellular collagen content. Culture medium pro-collagen I, TIMP-1 and MMP-3 levels were determined by enzyme-linked immunosorbant assay (ELISA). In the second part of this study, ELISA was used for measurement of PGE2 levels. RT-PCR and western blot were used to determined COX-2 mRNA and protein, respectively. Results: TGF-β1 increased the collagen content, pro-collagen I and TIMP-1 production, but slightly decreased MMP-3 production of pulp cells. SB431542 and U0126 prevented the TGF-β1-induced increase of collagen content and TIMP-1 production of dental pulp cells. Exposure to TGF-β1 (1-10 ng/ml) increased the COX-2 level of cultured pulp cells. Exposure to TGF-β1 (0.1-10 ng/mL) significantly increased PGE2 level in dental pulp cells. Under the pretreatment of SB431542, the stimulatory effect of TGF-β1 on COX-2 level of pulp cells was inhibited. Similarly, U0126 also partly inhibited the TGF-β1-induced COX-2 expression. Conclusion: TGF-β1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with ALK5/Smad2/3 and MEK/ERK signaling. TGF-β1 increased the COX-2 and PGE2 level of cultured pulp cells. This effect was associated with ALK5/Smad2/3 and MEK/ERK pathways. These events are important in the early inflammation, repair and regeneration of dental pulp in response to injury.