| Abstract: | 第一部份:Matrix metalloproteinases (MMPs)在腫瘤的侵襲和轉移時扮演著重要的角色，他們可以分解胞外基質和基底膜，讓癌細胞能夠滲入血管或從血管滲入組織中。在本實驗室先前的研究中，我們探討在老鼠腹腔中接種肉瘤細胞sarcoma 180 cells，於不同接種天數取下老鼠的各種臟器，進行gelatin zymography 分析，觀察肉瘤細胞在生長、侵襲和轉移過程中對老鼠各臟器MMP-2及MMP-9活性變化的影響，結果發現在肝臟組織中MMP-2及MMP-9的活性是同時增加的。因此本研究嘗試深入探討相同實驗設計老鼠肝臟組織中其MMPs的RNA層次變化。我們利用RT-PCR的技術，觀察肝臟組織中其MMP-2及MMP-9的mRNA變化情形。結果顯示，肝臟組織中其MMP-2及MMP-9的mRNA隨著接種天數而增加。綜合上述結果，推論肝臟組織中MMP-2及MMP-9活性的變化是經由mRNA表達改變進而改變protein量所致。另外我們也進行carbonic anhydrase異構變化分析，發現carbonic anhydrase I、II、III的蛋白質變化則是隨著接種天數的增加而逐漸減少。我們也進一步分析其mRNA變化情形，結果發現carbonic anhydrase II的mRNA變化情形和蛋白質變化是不一致的，而carbonic anhydrase I、III則是一致的。我們推想carbonic anhydrase II減少的原因與transcription 無關，可能是translation後分解速率的問題。
第二部份:Matrix metalloproteinases (MMPs)可以分解許多細胞外基質的成份。最近，有許多證據顯示在發炎的牙髓細胞中MMPs 扮演一個重要的角色。可是卻很少人去探討MMPs在人類牙髓細胞中的調控及細菌感染後細胞外基質的分解機制。於是，我們利用了細胞激素Interleukin-l and Transforming growth factor-b (IL-l and TGF-b)、蛋白合成抑制劑cycloheximide (CD)、PKC inhibitors (H7 and Gö6976) 及牙髓致病菌P. endodontalis and P. gingivais 的上清液去探討MMPs在人類牙髓細胞中的調控機制。我們利用gelatin-zymography 去觀察在長時間培養下人類牙髓及牙周韌帶細胞MMP-2及 MMP-9的分泌， 在八天的培養期間，我們發現TGF- b、CD、H7及Gö6976 會抑制MMP-2的分泌，而抑制的強度則是 CD >H7> TGF- b > Gö6976.而 P.endodontalis、P. gingivalis and IL-l則會增加MMP-2 的分泌。而且，在 P. endodontalis 或 P. gingivalis 的刺激下，MMP-2的分泌都會隨著時間或細菌濃度的增加而增加。而不論是處理細胞激素、藥物或是細菌上清液，對MMP-9的分泌都沒有任何影響。以上結果顯示：細胞激素或是藥物可以去調控牙髓細胞中MMP-2的活性。而一些與發炎有關的細胞激素可以增加MMP-2的活性顯示MMP-2在牙髓的發炎可能扮演著重要的角色；而蛋白合成抑制劑及PKC inhibitors可以抑制MMP-2的活性，這也許對牙髓發炎的致病機轉會有幫助，或是在治療牙髓發炎方面提供另一種新的思維。另一方面，在P. endodontalis 或 P. gingivalis刺激之下，使MMP-2活性增加。這表示P. endodontalis 或 P. gingivalis在牙髓及牙尖病變上扮演著重要的角色。 因此，經由細菌所引發的牙髓及牙尖病變可能是經由MMPs這一個路徑。而我們的研究也許可以使經由細菌所引發的牙髓及牙尖病變的治療有一個更明確的方向。
第一部份:Matrix metalloproteinases (MMPs) have been suggested to play an important role in the processes of tumor invasion and metastasis. One of the physiological functions of MMPs is the degradation of extracellular matrix and basement membrane, which is a beneficial factor for the extravasculative and intravasculative penetration of cancer cell during invasion and metastasis. In our previous study, we inoculated sarcoma 180 (S-180) cells into the abdominal cavity of mice to evaluate the changes in the MMPs (MMP-9 and MMP-2) levels and the expression pattern of total protein in all major tissues during the invasion of Sarcoma cells. By using gelatin zymography technique, we found that both MMP-2 and MMP-9 were markedly increased in the liver after the injection of S-180 cells. Therefore, the mRNA levels of MMPs in the liver during the amplification and invasion of S-180 cells were further studied. By using RT-PCR technique, we found that transcription of both MMP-2 and MMP-9 were markedly increased in the liver after the injection of S-180 cells. These results show the change in mRNA level was similar to that of gelatinotic activity of MMP-2 and MMP-9. Furthermore, we found that carbonic anhydrase I, II and III were gradually diminished in the liver after the inoculation of S-180 cells. However, the mRNA level of carbonic anhydrase II was unaffected while those of CAI and CA III were decreased. In conclusion, we suggest that the cause for the decreased protein level of CAII was not on the transcription level.
第二部份:Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes capable of degrading most components of the extracellular matrix. Recently, evidence has shown that MMPs may play a role in tissue degradation in inflamed dental pulp. To date very little is known regarding the regulation of MMPs in human pulp cell cultures and the mechanism of extracellular matrix destruction at the site of bacterial infection. The purpose of this study was to determine the effects to determine the effects of cytokines (interleukin-l and Transforming growth factor-b), protein synthesis inhibitor cycloheximide (CD), protein kinase C inhibitors (H7 and Gö6976) and the supernatants from Porphyromonas endodontalis and Porphyromonas gingivais on the production and secretion of MMPs by primary human pulp and periodontal ligament cell (PDL) cultures in vitro. The results were evaluated by substrate gel zymography from long- term cultures. The main gelatinase secreted by human pulp and PDL cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period, TGF-b, CD, H7, and Gö6976 were found to depress MMP-2 production. The inhibition decreased in an order of CD >H7> TGF-b > Gö6976. P.endodontalis, P. gingivalis and IL-l was found to elevate MMP-2 production. In addition, the stimulation with P. endodontalis or P. gingivalis was in a dose- and time-dependent manner. However, treated with either cytokines or pharmacological agents or P. endodontalis or P. gingivalis had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These observations suggest that the cytokines and pharmacological agents can regulate MMP-2 produced by human pulp cells. Inflammatory cytokines stimulate the production of elevated levels of MMP-2 and MMP-2 might play a role in pulpal innammation. In addition agents that target protein synthesis or the protein kinase C pathway in human pulp cells inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of pulpal inflammation. Such inhibition might contribute to therapeutic efficacy. On the other hand, these results indicate that black-pigmented Bacteroides species play an important role in tissue destruction and disintegration of extracellular matrix in pulpal and periapical diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of microbial-induced pulpal and periapical lesion. An understanding of the actions of these black-pigmented Bacteroides species on pulp and PDL cells may result in new therapies to augment current treatment of pulpal and periapical lesions. |
