組織型胞漿素原活化劑在人類正常牙髓及發炎牙髓組織的表現

Expression of tissue type plasminogen activator in normal human pulp tissues and inflamed pulp tissues

黃富美(Fu-Mei Huang)

指導教授 ： 周明勇

中山醫學大學/醫學院/醫學研究所/博士(2005年)

https://doi.org/10.6834/CSMU.2005.00001

摘要

牙髓是主宰牙齒最特別的感覺器官，當周圍保護硬組織受到破壞，牙髓組織則容易因外在的刺激（如化學性、物理性、細菌的感染）而產生不同的發炎與修復反應。但是當外來刺激過強或持續的刺激，則會造成牙髓產生不可逆發炎反應，進而阻礙組織的癒合，最後甚至造成牙髓細胞的壞死 。纖維蛋白溶解系統的胞漿素(plasmin)具有造成細胞間質裂解，產生組織破壞的特性，可能是牙髓致病路徑之一。而纖維蛋白溶解系統進行，是藉由細胞分泌胞漿素原活化劑(plasminogen activators; 簡稱PAs)，將胞漿素原(plasminogen)轉化成胞漿素，以執行裂解纖維蛋白的任務。胞漿素原活化劑主要含有組織型(tissue-type plasminogen activator, 簡稱t-PA) 及尿激酶型(urokinase-type)的胞漿素原活化劑。由於先前實驗發現牙髓細胞受發炎介子(inflammatory mediators)刺激後會誘發產生t-PA的表達，推測牙髓發炎可能與t-PA有著密切的關係。但是迄今仍未有任何文獻指出t-PA在牙髓組織的相關位置及可能的調控機轉，本研究收集13個人類正常牙髓組織樣本及17個發炎牙髓組織樣本，製作病理切片，以免疫組織化學染色法，發現t-PA在發炎牙髓組織較正常牙髓組織有強烈的表現(p<0.05)，尤其是在造牙本質細胞層、發炎細胞、造纖維母細胞及血管內皮細胞；並同時抽取正常牙髓組織及發炎牙髓組織RNA以RT-PCR方法比較其間的可能差異，結果發現發炎牙髓組織t-PA mRNA的表現約為正常牙髓組織的3.5倍 (p<0.05)。另外為探討複雜的牙髓發炎的致病機轉，應用體外牙髓細胞培養的研究模型，以加入inflammatory cytokine interleukin-1a (IL-1a) 及Porphyromonas endodontalis (P. endodontalis) 上清液等inflammatory mediators模擬牙髓發炎的情形後，分別再加入SB203580 (p38 inhibitor)、U0126 (MEK inhibitor)、LY294002 (PI3K inhibitor)，在加藥後細胞培養48 小時後以酪蛋白酶譜(casein zymography)和免疫酵素螢光分析(Enzyme-linked immunosorbert assay；簡稱ELISA)方法分析t-PA的活性，以上兩種分析方法所得實驗結果相同，發現interleukin-1a與細菌P. endodontalis上清液可促進牙髓細胞分泌t-PA (p<0.05)，而U0126 、SB203580 和 LY294002可抑制牙髓細胞因interleukin-1a所促進分泌t-PA的活性表現(p<0.05)，而其抑制的效果分別是U0126＞SB203580＞LY294002。而在P. endodontalis上清液刺激下，只有U0126、 SB203580對牙髓細胞所分泌t-PA的活性表現情形有抑制的效果(p<0.05)，而LY294002則不能抑制P. endodontalis 上清液刺激之下，牙髓細胞所分泌t-PA的活性 (p>0.05)。本實驗綜合以上研究所得結論：發現在發炎牙髓組織t-PA表現確實比正常的牙髓組織高很多。因此在發炎的牙髓導致牙髓組織破壞的致病路徑上，t-PA可能扮演重要關鍵角色。另外培養人類牙髓細胞以 IL-1a 與 P. endodontalis的刺激下，對人類牙髓細胞所分泌t-PA的活性都有促進的效果，而對t-PA的調控訊息傳遞路徑可能與MEK 和p38 MAPK路徑較有關。

關鍵字

牙髓炎、組織型胞漿素原活化劑、牙髓細胞、訊息傳遞路徑

並列摘要

The dental pulp is a rather unique sensory organ. Pulpal inflammation and repair were produced in response to the irritations (chemical, mechanical and bacterial infection). If the source of irritation continues or becomes more severe, further inflammation changes may occur. Damage to the pulp tissues continues and the resultant liquefied dead tissue. The plasminogen/plasmin proteolytic system participates in a wide variety of extracellular matrix degradation. Detailed knowledge of plasminogen activators (PA) may be important for understanding the pathogenesis of pulpal disease. The role of the plasminogen-activating system has not been well clarified in human pulp tissue. This study was to investigate the in situ localization of tissue type plasminogen activator in pulpal disease. Thirty pulpal tissue specimens (13 normal and 17 inflamed pulps) were obtained from extracted third molars. Immunohistochemistry was used to identify the in situ localization oft-PA expression in pulp specimens. The levels of t-PA between normal pulp and inflamed pulp tissues were compared using the quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, The results from immunohistochemistry demonstrated that t-PA expression was significantly higher in the inflamed pulp (P＝0.025). t-PA mRNA gene was found more in inflamed pulps when compared with normal pulp tissue (P < 0.05). However, the mechanisms and signal transduction pathways involved in the production of t-PA in human pulp cells are not fully understood. Cultured human pulp cells were treated with various inflammatory mediators interlenkin-1a (IL-1α) and supernatants of Porphyromonas endodontalis (P. endodontalis) in the absence or presence of various kinase inhibitors p38 inhibitor SB203580, MEK inhibitor U0126, and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002 were added to search possible regulatory mechanisms by using casein zymography and enzyme-linked immunosorbent assay (ELISA). The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with IL-1α and P. endodontalis during 48hrs cultured period (p<0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1α-stimulated t-PA production, respectively (p<0.05). The rank orders with respect to decrease the amounts t-PA production stimulated by IL-1α were found to be as follows: U0126 > SB203580 >LY294002. Our findings demonstrated that SB203580 and U0126 significantly reduced the P. endodontalis-stimulated t-PA production, respectively (p<0.05). However, LY294002 was lack of ability to change the P. endodontalis stimulated t-PA production (p>0.05). Conclusions: t-PA expression is significantly higher in inflamed pulp tissue. t-PA may play an important role in the pathogenesis of pulpal inflammation. Our findings demonstrated that IL-1α and P. endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

並列關鍵字

pulpal inflammation、tissue type plasminogen activator、pulp cells、signal transduction pathway

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