- 學位論文
以基因小鼠模式研究牙周病致病菌牙齦卟啉單孢菌之促進胰臟癌之致病機制
Studies on the pathogenesis of periodontits pathogen Porphyromonas gingivalis-envoked pancreatic cancer progression in transgenic disease mouse model
摘要
胰腺癌 (Pancreatic cancer) 是全球惡性腫瘤死亡的主要原因之一，由於胰臟位置的特性，產生腫瘤時不容易被發現，當確診時胰臟癌通常為末期，存活期短，但目前對胰腺癌在早期階段成長速度的發展仍不清楚。因此我們想利用可100％發展成PanIN病變及在較長的等待時間可進展至PDAC 的3、6、12及24週PDX-1-Cre;LSL-Kras G12D (KC) 鼠來了解胰腺癌早期的依序進展。另外研究也指出牙齦卟口林單孢菌 (Porphyromonas gingivalis, P.gingivalis) 會增加兩倍以上罹患胰腺癌的風險，因此我們以傳統慣用雨蛙素 (caerulein) 誘導急性胰臟發炎加速胰腺癌惡化之動物模式為對照，評估實驗小鼠有無牙齦卟口林單孢菌感染是否同樣加速引發胰腺癌惡化。發現無經雨蛙素或牙齦卟口林單孢菌誘導的小鼠體重、脾臟及胰臟重均未出現顯著差異；但24週時Kras基因轉殖小鼠 (KC鼠) 會出現性別差異，母鼠胰臟重量會高於公鼠胰臟重量。在胰臟組織切片蘇木紫與伊紅染色方面得知無經雨蛙素或牙齦卟口林單孢菌誘導的KC母鼠較公鼠的胰管病變更後期，而經雨蛙素誘導的KC鼠胰管病變也比牙齦卟口林單孢菌誘導的更後期。胰臟病變會有胰腺非典型導管增生 (atypical ductal hyperplasia)，可作為細胞增殖指標的PCNA表現量在KC鼠中隨著週齡及誘導增多。癌相關成纖維細胞會促進腫瘤發生；而上皮細胞轉型成間質細胞是造成腫瘤轉移的機轉之一，因此隨著時間的增加再加上誘導，都會增加α-SMA、N-cadherin、SNAIL及twist的表現。在文獻中已確定胰腺發炎會增快胰腺癌的進展，而在我們的in vitro實驗中發現，加入牙齦卟口林單孢菌共同培養時也會刺激脾臟細胞及Raw264.7細胞分泌IL-1β、IL-6、TNF-α等發炎細胞激素。且主要是由牙齦卟口林單孢菌本體刺激免疫系統產生發炎反應，並推測出巨噬細胞也許不是IL-6的主要分泌細胞及可能為TNF-α的主要產生細胞。因此我們認為雖然牙齦卟口林單孢菌誘導造成的慢性低度感染所促進胰腺癌發展的惡化不像雨蛙素誘導造成的急性發炎顯著，但也明顯比未誘導時導致胰管病變進程提前。綜合以上結果，我們認為牙齦卟口林單孢菌造成的發炎反應會加速胰腺癌的惡化程度。
並列摘要
Pancreatic cancer is one of the worldwide major causes of malignant tumor deaths. The location of the pancreas is privacy, and it's not easy to be found at the beginning. It always causes a definite diagnosis with low survival rate at the last stage. However, the development of pancreatic cancer at the early stage of development is still unclear. Therefor we used 3,6,12 and 24 week-old PDX-1-Cre;LSL-Kras G12D (KC) mice that can develop to murine PanIN lesions with 100% penetrance, and progress to PDAC with a long latency, to understand the progress of early pancreatic cancer in sequence. In addition, the researcher also pointed out that Porphyromonas gingivalis (P.gingivalis) was associated with a twofold increased risk of pancreatic cancer. However, compared to the traditional caerulein induced acute pancreas inflammation animal model, which can promote the pancreatic cancer, while the assessment of mice infected with P.gingivalis, whether the acceleration caused by pancreatic cancer gotten worse. We found mice without using caerulein or P.gingivalis, the mice body weight, spleen and pancreas weight were not obvious in difference, but at 24 week-old Kras transgenic mice (KC mice) were using P.gingivalis that induced the spleen and pancreas were obviously lower with caerulein inducing by weight. On the other hand, the spontaneous mice development of pancreatic cancer, the pancreas weight in KC mice in 24 week-old will emerge the gender differences, female mice will be higher than the male mice. In pancreas tissue sections, we found that KC female mice without caerulein or P.gingivalis induction were more severe than male mice. Besides, we also found that KC mice with caerulein induction were more severe than those by P.gingivalis. The pancreas lesion occur together with a typical ductal hyperplasia, and the cell proliferation index indicated the PCNA expression without induction in KC female mice at 12 week-old and male mice at 24 week-old began to increase. Caerulein-induced acute inflammatory may increase the progression of pancreatic cancer and it has been identified from the literature. In our experiment we also found that the co-culture with P.gingivalis will stimulate the secretion of IL-1β,IL-6 and TNF-α inflammatory cytokines of spleen and Raw264.7 cells. Although chronic low infection caused by P.gingivalis-induced is not more obvious than by caerulein-induced which lead to the development of pancreatic cancer. It makes the time of occurrence of the lesion more forward than the mice that was not induced. Cancer-associated fibroblasts (CAFs) can significantly promote tumorigenesis, and epithelial-mesenchymal-transition is one of the mechanisms of tumor metastasis; so as time increases and induced by P.gingivalis or caerulein will increase α-SMA, N-cadherin, SNAIL and twist in pancreatic tissue performance. In in vitro experiments also found that P.gingivalis itself mainly stimulating the immune system to produce inflammation. By speculating the macrophage cells may not secreted IL-6 and the TNF-α may producing cells. As the results above, we believe that the inflammation caused by P.gingivalis would accelerate the deterioration degree of pancreatic cancer.
參考文獻
延伸閱讀
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