前列腺素E2對人類牙髓細胞分化及基因表現的影響

實驗目的: 前列腺素E2 (Prostaglandin E2，PGE2)對於牙髓組織的發炎與修復性牙本質的形成扮演了重要的角色。其中PGE2具有多變的接受器( EP1、EP2、EP3、EP4)，分別調控了不同的下游訊息傳導，而在牙髓細胞中，主要是有EP1、EP2、EP3 接受器。本實驗目的在於探討PGE2所誘導的訊息傳導途徑及其對牙髓細胞分化及基因表現的影響。並且觀察EP4 接收器是否調控人類牙髓細胞的分化。實驗方法: 使用不同濃度的PGE2及其接受器(receptor)的(agonist): 19ROH-PGE2(EP2 agonist)、Sulprostone(EP1/EP3 agonist)、CAY10598(EP4 agonist) 對人類牙髓細胞做刺激，之後在顯微鏡下觀察細胞形態的改變，並且在96小時及五天後做MTT測定和鹼性磷酸酶染色(ALP staining)。在某些實驗中，加入PGE2 和CAY10598 培養 30分鐘或 5天，PGE2更觀察0~120分鐘的變化，之後做反轉錄鍊聚合反應(RT-PCR) 來看鈣化相關基因 (RUNX-2,ALP,Osterix,osteocalcin)及AP-1 轉錄因子(AP-1 transcription factors(c-fos,FosB,c-jun,JunB))的表現。另外在PGE2的訊息傳導的實驗中，加入了H89(PKA的抑制劑) 和dorsormorpin (AMPK的抑制劑)，以RT-PCR來觀察對PGE2誘導產生的AP-1轉錄因子及ALP表現的影響。實驗結果: 人類牙髓細胞的形態不受到PGE2、19ROH-PGE2及Sulprostone的影響，但CAY10598 10uM會使細胞核變大及深染，細胞變圓。根據MTT測定，可發現CAY10598 5uM和10uM會抑制細胞的生長。在ALP染色的實驗中可發現PGE2、19ROH-PGE2、Sulprostone、CAY10598都可使ALP的活性在低濃度上升在高濃度下降。根據反轉錄鏈聚合反應的實驗可發現加入PGE2(5uM~10uM) 5天，可使鈣化相關因子(ALP、Runx2、Osterix、osteocalcin)的表現量上升，且其表現的趨勢與AP-1轉錄因子中的c-fos、FosB及JunB的表現一致。而在120分鐘內可使AP-1 轉錄因子(c-fos、FosB、JunB)表現上升。加入PGE2及CAY10598不同濃度30分鐘後都可發現和5天有不同的結果: c-fos、fosB在低濃度時表現上升在高濃度時下降，然而JunB在每個組別中都是在高濃度及給予較久的刺激時間時表現上升，c-jun則相反之。加入了H89(PKA抑制劑)和dorsomorpine(AMPK的抑制劑)可看到PGE2的作用被抑制的現象。結論: PGE2 藉由PKA/AMPK的路徑來調控人類牙髓細胞的分化。 EP1/2/3/4的agonists都能使牙髓細胞的ALP activity上升。而牙髓細胞對PGE2的反應會因刺激的時間有所不同。 EP4接受器雖在人類牙髓細胞表現量低，但在牙本質生成上具有重要的角色。

關鍵字

牙髓細胞；前列腺素E2 ；鹼性磷酸酶AP-1轉錄因子

並列摘要

Aim: During pulpal inflammation, PGE2 level was elevated and known to be an important mediator to regulate the function of dental pulp cells. This can be due to binding of PGE2 to cell surface receptors such as EP1, EP2 and EP3 prostaglandin receptor to stimulate downstream signaling. The aim of the study is to investigate the effect of PGE2 and EP4 receptor activation on the differentiation and gene expression in human dental pulp and its signal transduction pathway. Materials and Methods: Primary-cultured human dental pulp cells were treated with PGE2 and it agonists (19ROH-PGE2, EP2 agonist; sulprostone, EP1/EP3 agonist; CAY10598, EP4 agonist) with different concentration and then observed the morphological change of pulp cell under contrast microscope. Cell proliferation was evaluated by MTT assay. Effects of PGE2 and CAY10598 on cell differentiation and mineralization during different stimulation periods or with different concentrations were evaluated by alkaline phosphatase (ALP) staining and reverse-transcriptase polymerase chain reaction(RT-PCR). We observed both the AP-1 transcription factors (c-fos, FosB, c-jun, JunB) and the mineralization markers (ALP, Runx2, Osterix, osteocalcin) mRNA change in human dental pulp after stimulation. Cell were pretreated with H89 (PKA inhibitor), dorsomorphin (AMPK inhibitor) 30 minutes before adding PGE2. Signaling of PGE2 was investigated by RT-PCR. Results: The cell morphology didn’t affect by PGE2、19ROH-PGE2 and Sulprostone. However, the nuclei turned into lager and hyperchromatic after stimulation by CAY10598 10 uM. MTT assay also indicated CAY10598 inhibited the growth of pulp cells at high concentration (5μM-10μM). Cells under the treatment of PGE2 and its agonists all showed an increase in ALP activity at lower concentration and decreased at higher concentration. In 5 days cultured group, PGE2 (5μM-10μM) can induce mineralization markers (ALP, Runx2, Osterix, Osteocalcin) expression, which was consistent with the expression of AP-1 transcription factors (c-fos, FosB, JunB). PGE2 also can induce expression of c-fos, FosB and JunB during 120 mins, while c-jun decreased time dependently. In 30mins culture group, PGE2 and CAY10598 induce c-fos、FosB at lower concentration (0.1μM-0.5μM) compared with 5 days cultured group. Interestingly, the expression of junB were always enhanced at higher concentration or longer stimulation period, but c-jun decreased dose-dependently and time-dependently. H89 (PKA inhibitor) and dorsomorphin (AMPK inhibitor) attenuated the effects of PGE2 . Conclusion: PGE2 may be involved in dental pulp inflammationand repair process via induction of AP-1 transduction of AP-1 transcription factors and mineralization markers via EP2 and EP4 receptors through PKA/AMPK singaling pathway. EP1/3 agonist (sulprostone)、EP2 agonist (19ROH-PGE2) and EP4 agonist(CAY10598) can increase the ALP activity of dental pulp cells. The responsiveness of human dental pulp cells to PGE2 may change during the culture period. Although EP4 is the least receptor in human dental pulp, but its activation by CAY10598 still has significant effects on dentinogenesis.

並列關鍵字

Dental pulp cells ； PGE2 ； ALP ； Runx2 ； Osterix ； Osteocalcin ； AP-1 transcription factors

參考文獻

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前列腺素E2對人類牙髓細胞分化及基因表現的影響

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