2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649304摘要：牙髓炎是一種多因性的疾病，常由於微生物如P. gingivalis等的感染引起牙髓組織的發炎反應。由於包覆在硬組織內的牙髓組織於血液供應上受到限制，持續存在的發炎反應最終常會導致牙髓組織壞死。然而到目前為止，對於這個發炎與破壞的機制並不完全清楚。 許多關於牙周組織初期免疫反應的研究已經顯示，牙周組織的上皮細胞能夠藉由細胞激素的釋放，調控下游免疫反應。細胞表面的類鐸受器(Toll-like receptor，簡稱TLR)，能夠辨認病原相關的分子特徵(pathogen-associated molecular pattern，簡稱PAMP)，啟動組織的初期免疫反應，進而調節更進一步的後繼免疫反應 (adaptive immunity)。P. gingivalis為常見的牙周及牙髓致病菌，其LPS為TLR ligand之一。目前已經知道牙髓組織會表現TLR2和TLR4受體，而TLR4與NOD2之活化可能是啟動牙髓炎的主要機制。而本研究團隊目前已初步得知，P. gingivalis的LPS雖然對牙髓細胞之生長影響不明顯，但是對間質蛋白代謝具有影響。牙髓組織細胞和致病菌的交互作用影響了牙髓發炎反應的進行。本研究將針對牙髓細胞早期免疫反應的功能角色加以探討，尤其是TLR在牙髓細胞的表現，以及TLR在接受牙髓病菌刺激後的訊息傳導及對細胞激素表現的影響，以期對牙髓炎致病機轉有更進一步的了解。 本研究將分為三個部分進行。第一部分首先以牙科患者的健康牙髓組織檢體進行培養，以第三代至第十代的細胞進行體外研究；以TLR ligand---P. gingivalis LPS及其DNA與TLR2/TLR4 agonist刺激培養牙髓細胞，以西方墨點法偵測下游轉錄因子是否磷酸化，同時用聚合酶連鎖反應及電泳等方式測定刺激前後TLR與細胞激素的變化。第二部分是以免疫組織化學染色法(IHC)觀察健康與疾病之牙髓組織中TLR2、TLR4及其下游相關轉錄因子的表現。第三部分利用聚合酶連鎖反應針對健康與疾病牙髓細胞中TLR2、TLR4，以及與發炎反應相關之細胞激素如：MIP、MCP-1、IL-1、IL-8、TNF-α等的表現加以定量比較，再與染色結果交叉分析各因子間表現的相關性，從中找出牙髓細胞TLR可能的訊息傳遞路徑。 本研究利用目前已發展成熟之牙髓組織培養技術進行研究，並藉由臨床牙髓組織的觀察及功能分析，希望了解 TLR 在牙髓細胞的表現、對於牙髓早期發炎反應的影響、及相關之訊息傳導路徑。有助於對牙髓炎的致病機轉有更進一步的認識，進而能突破目前臨床治療的瓶頸，提供臨床醫師另一種治療的思考方向。<br> Abstract: Pulpitis is a multi-factor disease, which is frequently induced by microorganisms such as Porphymonas gingivalis (P. gingivalis). Persisted inflammatory reaction of pulp tissue usually results in pulp necrosis, because the blood supply of pulp tissue was limited and confined within the hard tooth structure. However, the mechanism of pulp tissue inflammation and consequently tissue destruction is not clear. Studies have proved that gingival epithelial cells could regulate adaptive immune response through cytokines releasing. Gingival epithelial cells express Toll-like receptors (TLR) which can recognize pathogen-associated molecular patterns (PAMPs), initiate the earliest response to danger signals, and regulate innate or adaptive immune response. It is also well known that lipopolysaccharide (LPS) of P. gingivalis, which is one of TLR ligand, relates to the severity of periodontitis and pulpitis. According to the literature review, pulp tissue expresses TLR2 and TLR4, and the activity of TLR4 and NOD2 may initiate pathogen-related pulpitis. Our previous studies showed that P. gingivalis LPS may not have prominent effect on the viability of pulp cells, but it induced some inflammatory cytokines production in human dental pulp cells. In this proposal, we want to investigate the expression pattern of TLR and proinflammatory cytokines of human dental pulp cells from subjects with or without pulpitis to elucidate the possible role of TLR in the pathogenesis of pulpitis. Pulp tissue from pulpitis-free subjects are collected and cultured in vitro. The third to tenth generations of cultured pulp cells will be used for this study. To evaluate the effects of TLRs activation on pulp cells, primary human dental pulp cell cultures will be stimulated with different TLR-ligands, including LPS and DNA of P. gingivalis at different time intervals. For understanding the possible signal pathway, cellular RNA and proteins will be extracted for RT-PCR and Western blot analysis. Another pulp tissue from puplitis-affected and pulpitis-free subjects are collected. Expression of TLR2, TLR4 and associated transcription factors, such as NF- κ B, MAPK and IRF3 etc., will be examined in with immunohistochemical staining (IHC). RT-PCR will be used to quantify proinflammatory cytokines: MIP, MCP-1, IL-1,IL-8, TNF-α etc., expressed in health and diseased human dental pulp tissue. Results of IHC staining and RT-PCR will be analyzed and interpreted to elucidate the possible correlation of TLR and the inflammatory status of pulp tissue. The in vivo and in vitro results can delineate the molecular interaction involved in the early immune response of pulp cells against pulpal pathogens. This will be informative and fundamental in understanding the function of pulp cells and their interaction with pathogens in the pathogenesis of pulpitis. The understanding of innate immune responses of pulp tissues can offer us a new perspective to develop novel prevention and therapeutic strategies against pulpitis.牙髓細胞牙髓炎類鐸受器細胞激素pulp cellspulpitistoll-like receptor (TLR)cytokine