- 學位論文
IL-1β對人類牙髓細胞表現及分泌細胞激素 IL-6和IL-8的影響：TAK1和IRAKs的角色
Effect of IL-1β on IL6 and IL-8 Expression/production in Dental Pulp Cells: Role of TAK1 and IRAKs
摘要
在牙髓的急性及慢性發炎中，Interleukin 1 β (IL-1β)為一常見的促炎性細胞因子，而IL-6和IL-8則是其下游的發炎因子。近年來在IL-1β的細胞訊息路徑中有兩個重要的訊息分子transforming growth factor β-activated kinase-1 (TAK1)和interleukin-1 receptor-associated kinases (IRAKs)被發現，後續亦有研究探討其應用於治療發炎性疾病的可能性，然而這兩個訊息因子對於牙髓細胞中Interleukin 1 β (IL-1β)促進IL-6及IL-8表現及分泌的影響目前並不清楚 實驗設計：將培養皿中的人類牙髓細胞分成不同的組別，分別經過5z-7-oxozeaenol (TAK1抑制劑)或IRAK1/4抑制劑處理後，暴露於含有Interleukin 1 β (IL-1β)的細胞培養液中。細胞會進行3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT)實驗已確定所加入的藥劑不會造成細胞死亡，之後藉由螢光染色來觀察TAK1和IRAKs磷酸化的情形，以西方墨點法來檢測IL-6和IL-8分泌的情形，以RT-PCR來檢測IL-6和IL-8的mRNA表現，並且以ELISA定量IL-8的分泌狀況。 實驗結果：牙髓細胞在經過IL-1β (0.1-10 ng/ml)刺激後可以觀察到IL-6及IL-8的表現明顯增加，此外也可以觀察到IRAK1和TAK1有磷酸化的情形。細胞在經過低於毒性濃度的5z-7-oxozeaenol (1 and 2.5 μM)和IRAK1/4抑制劑(10, 20 and 40 μM)的處理後，可以觀察到IL-6和IL-8的表現受到了抑制。 結論：實驗的結果證實IL-1β可以藉由促進IL-6和IL-8的分泌來調控牙髓細胞的發炎反應和修復，而其中的調控與TAK1和IRAKs有關，抑制TAK1或是IRAKs或許可以成為將來調控牙髓發炎反應及修復的方法。
並列摘要
Objective: Interleukin 1 β (IL-1β) is a pro-inflammatory cytokine involved in the acute and chronic inflammatory processes of dental pulp. IL-6 and IL-8 are two inflammatory mediators. However, the role of transforming growth factor β-activated kinase-1 (TAK1) and interleukin-1 receptor-associated kinases (IRAKs) in responsible for the effects of IL-1β on the IL-6 and IL-8 expression/secretion of dental pulp cells are not clear. Design: Human dental pulp cells were exposed to IL-1β with/without pretreatment with 5z-7-oxozeaneaeol (a TAK1 inhibitor) or IRAK1/4 inhibitor. The 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay was performed to estimate the viable cell number. The expression of p-TAK1 and p-IRAK1 were observed by immunofluorescence. The protein expression of IL-6 and IL-8 was tested by western blot and the expression of IL-6 and IL-8 mRNA was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). The secretion of IL-8 was measured by enzyme-linked immunosorbant assay. Results: Exposure of dental pulp cells to IL-1β (0.1-10 ng/ml) stimulated the IL-6 and IL-8 expression and secretion by dental pulp cells. The phosphorylation of TAK1 and IRAK1 can also be observed after exposed dental pulp cells to IL-1β. Pretreatment and co-incubation of pulp cells by 5z-7oxozeaenol (1 and 2.5 μM) and IRAK1/4 inhibitor (10, 20 and 40 μM) at non-toxic concentrations could prevent the IL-1β-induced IL-6 and IL-8 expression. 5z-7oxozeaenol and IRAK1/4 also attenuated the IL-1β-induced IL-6 and IL-8 secretion of dental pulp cells. Conclusions: These results indicate that IL-1β can be important in the pathogenesis of pulpal inflammatory diseases and repair via stimulation of IL-6 and IL-8 expression and secretion. These events are associated with TAK1 and IRAK1/4 signaling pathways of dental pulp cells. Blocking of TAK1 and IRAK1/4 may have potential use to control inflammation of dental pulp in the future.