- 學位論文
腫瘤壞死因子(TNFα)對人類牙髓幹細胞的影響
The effect of TNFα on human dental pulp stem cells
摘要
齲齒(dental caries)全世界最盛行的傳染疾病之一，被細菌的產物酸分解的牙本質基質、及細菌的內毒素(LPS)等，都會引發牙髓內的免疫細胞產生一些發炎介質，進而引發一連串的發炎反應。牙髓內發現的發炎前驅的細胞激素包含我們所熟知的間白質(interlukin-1α、interlukin-1β及interlukin-8)還有腫瘤壞死因子(TNFα)。過去的實驗顯示在牙髓細胞礦化過程中MAP4K4的基因表現有顯著的向上調控，在文獻的回顧中也發現MAPK不只與細胞礦化有關更與細胞凋亡與壞死有相關。近年來研究亦發現TNFα可以活化MAP4K4的訊息傳導路徑。因此，我們假設TNFα活化MAP4K4可能可促進牙髓幹細胞的礦化作用而其調控作用可能因作用時間而異。 本研究乃是要探討TNFα對於MAP4K4活化，及其作用時間的長短對人類牙髓幹細胞的影響。其所用的方法是由長時間及短時間給予牙髓幹細胞TNFα的刺激去看對細胞礦化染色的影響。進一步做ALP activity 去看礦化的定量表現，接著藉由間接酵素結合免疫吸附法(indirect ELISA)來看有沒有給予TNFα對於細胞內MAP4K4的濃度及p-MAP4K4濃度的差異及加入MAP4K4的siRNA對ALP activity表現的影響。 結果顯示長時間的給予TNFα對於細胞的礦化都是抑制的現象，短時間內才有促進的表現。短時間的加入TNFα在一個小時之內可以顯著增加ALP activity。此外，加入TNFα在24小時之內皆可以促進MAP4K4的活化。當加入MAPK的siRNA後不管是礦化培養液或是礦化培養液加入TNFα其ALP activity都顯著的下降。因此，由本研究結果可以部份證實TNFα在短期內可以透過活化MAP4K4的訊息傳導路徑促進牙髓幹細胞的礦化表現。
並列摘要
Dental caries is the most prevalent infectious disease in the world. The byproduct of dentin matrix demineralization by bacterial acid, and the bacterial endotoxin, lipopolysaccharide(LPS) would stimulate the immune system cells within the pulp to release proinflammatory cytokines and recruit of more lymphocytes. Moreover, a series of inflammatory response would occur. The proinflammatory cytokines found within the pulp comprise the increased level of interlukin((interlukin-1α、interlukin-1βand interlukin-8) and TNFα. In the past research we found that MAP4K4 (Mitogen-activated protein kinase kinase kinase kinase 4) is up-regulated during the mineralized differentiation process of dental pulp stem cell. Recent study also revealed that MAPK regulate not only the mineralization process, but also apoptosis and necrosis. It was also found that TNFα can activate MAP4K4 signaling pathway .We therefore hypothesized that TNFα may affect the mineralization process through activation of MAP4K4. The purpose of the study is to investigate the effect of TNFα on human dental pulp stem cells. The materials and methods used in this study was treating dental pulp stem cell with TNFα for different time periods to see the effects on the expression of ALP by staining and the activation of ALP was also measured. Then, we treat dental pulp stem cell with TNFα and see the effect on the cytoplasm level of MAP4K4 and p-MAP4K4 thr`ough indirect ELISA. At last, MAPK inhibitor is added in to make sure the expression of ALP activity. Our result indicated that by treating with TNFα for a long period, the mineralization of dental pulp stem stem cells were inhibited, but the mineralization expression is enhanced after short time treatment with TNFα. With ELISA analysis, TNFα promotes the activation of MAP4K4 was shown. When MAPK siRNA is inserted, the expression of ALP activity is downregulated, with or without adding TNFα to the culture medium . It is concluded that TNFα will promote the mineralized differentiation via MAP4K4 signaling pathway in short time period, but long treated time inhibit cell mineralization and may eventually lead to cell necrosis and apoptosis.