- 學位論文
以Real-time RT-PCR的方式定量端粒酶反轉錄酶(hTERT)在胃癌的表現及其與幽門螺旋桿菌的關係
Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in gastric adenocarcinoma and its relationship with helicobacter pylori infection by using LightCycler real-time quantitative RT-PCR
摘要
目前癌症已位居十大死因之第一位，其中胃癌位居消化道癌症的第三位。對於其致癌的原因目前仍尚未有非常確定的機轉，不過基本上是多種因素造成的，包括環境因素及宿主本身的因素等。自從1982年澳洲馬歇爾 (Marshall) 發現幽門螺旋桿菌的存在後，陸續的研究都証實幽門螺旋桿菌和許多消化性疾病如胃炎、胃潰瘍、十二指腸潰瘍、胃淋巴瘤甚至於胃癌有關。1994年 世界衞生組織 ( WHO)更是正式地將幽門螺旋桿菌列為是胃癌的致癌因子之一。而最近的研究也指出端粒酶和細胞的長生不死有關。 一般體細胞會藉由端粒的縮短來控制分裂的次數及細胞壽命，而癌細胞能活化端粒酶作用，使得端粒不再縮短，所以細胞可以繼續分裂而形成永生不死。在80%以上的腫瘤組織及一些不死化細胞株可以發現端粒酶是呈現活化狀態，因此端粒脢這種酵素的激活化被認為可能是細胞癌化及不死化的重要因子。本研究主要是想瞭解幽門螺旋桿菌及端粒酶在胃癌癌化過程中可能扮演的角色。我們總共收集了39位經由胃內視鏡及切片証實為胃腺癌之病患，以內視鏡取其胃竇及胃體的組織進行幽門螺旋桿菌的檢測包括病理組織、尿素酶及細菌培養、同時病人也接受碳13尿素呼氣試驗。幽門螺旋桿菌的判定為若細菌培養為陽性或其它三種檢查有兩種以上為陽性者則判定為陽性；否則則判定為陰性。另外以內視鏡取癌組織及正常組織以Real time RT-PCR來定量測定端粒酶活性(hTERT) 的表現，另外進一步評估其與幽門螺旋桿菌感染及病人臨床分期之間的關係。 在結果方面，我們將分成三部份來討論。 第一部份為幽門螺旋桿菌感染與端粒酶活性之相關。我們的研究中發現胃癌組織的端粒酶表現 (NhTERT為69.13 + 82.56) 較周邊正常組織的端粒酶表現(NhTERT為48.69 + 57.39)來得高，不過沒有統計上的差異 (p=0.208)。而在幽門螺旋桿菌感染陽性的26個胃癌組織中其端粒酶活性表現(NhTERT)為78.44 + 82.61較周邊正常組織的NhTERT為34.29 + 22.13為高 (p = 0.07)。 第二部份為telomerase index及over-expression與幽門螺旋桿菌感染之關係。Telomerase index 是以胃癌組織端粒酶活性的表現(NhTERT)和周邊正常組織端粒酶活性的表現(NhTERT)的比值；而over-expression則是telomerase index大於1。在有幽門螺旋桿菌感染的26個樣本中其over-expression較沒有幽門螺旋桿菌感染者為高 (p=0.02) 而在telomerase index也發現有幽門螺旋桿菌感染者較沒有幽門螺旋桿菌感染者來得高(p=0.09)。 第三部份則是探討臨床分期與端粒酶活性的關係。胃癌的臨床分期是以國際聯合癌症協會的TNM 系統來分期，不論是以胃癌組織中端粒酶活性的表現(NhTERT)來比較或是以telomerase index來比較都沒有差別。 結果發現在有幽門螺旋桿菌感染的樣本中端粒酶活性的表現 (包括NhTERT、telomerase index及是否有over-expression)在胃癌組織中的表現都比周邊正常組織來得高，其中是否有over-expression更是有統計上的差異。這個結果指出幽門螺旋桿菌感染會增加胃癌組織中端粒酶活性的表現，表示幽門螺旋桿菌的感染在癌化過程中可能經由增加端粒酶活性而扮演著重要的角色。
並列摘要
Malignancy has been the top of the ten leading cause of death in Taiwan and gastric cancer is the third one of the gastrointestinal tract malignancy. Although the mechanism of the carcinogenesis is not very clear yet, it was thought to be multiple factors, including environment and host factors. Since Marshall discovered the H. pylori, it is found to be involved in many upper digestive diseases such as gastritis, peptic ulcer diseases, mucosa-associated lymphoid tissue lymphoma and even gastric adenocarcinoma. In 1994, WHO recognized H. pylori as a definite carcinogen for gastric cancer. Nowadays, more and more interesting in telomerase and cell immortality had been reported. Telomere DNA shortens at chromosome ends with division of human somatic cells and leads to cell death, but cancer cells express telomerase activity so that they can be indefinite replication and immortality. Over 80% of tumors and some germ cell lines express telomerase activity. Therefore, telomerase activity was thought to be the important factor of carcinogenesis and immortality. In our study, we would like to understand the role that H. pylori infection and telomerase in the carcinogenesis of gastric cancer. We collect 39 patients all of them are gastric adenocarcinoma diagnosed by gastroendoscopic examination and pathology. The H. pylori infection status was evaluated by histology, culture, CLO test and 13C-UBT. H. pylori infection was defined as positive if the culture was positive or if two of the other three tests (histology, CLO test and 13C-UBT ) were positive. Otherwise, it was defined as negative for H. pylori infection. Besides, we also collect cancer tissue and peripheral cancer tissue by endoscopic biopsy to quantitative detection of human telomerase catalytic subunit (hTERT) by using LightCycler Telo TAGGG real time RT-PCR. In the first part of our study, we compared the correlation between helicobacter pylori infection and telomerase activity. We found that telomerase activity (NhTERT =hTERT mRNA copies/PBGD mRNA copies/1000) in the gastric cancer tissue was higher than that in the peripheral normal tissue (69.13 + 82.56 and 48.69 + 57.39; p=0.208). Among the 26 samples with helicobacter pylori infection, the telomerase activity is higher than those without helicobacter pylori infection (78.44 + 82.61 and 34.29 + 22.13; p = 0.07). The second part is the relationship between telomerase index and over-expression and helicobacter pylori infection. Telomerase index is the ratio of telomerase activity (NhTERT) in gastric cancer tissue to that in peripheral normal tissue; over-expression is defined as telomerase index is higher than 1. Telomerase index is higher in the group with helicobacter pylori infection that that without infection and also is the over expression (p=0.09 and p=0.02). The third part of this study is to evaluate the relationship between clinical stage and telomerase activity. We found that there is no significant between stages and telomerase activity either NhTERT or telomerase index. Our result showed that among the group of helicobacter pylori infection, the expression of telomerase activity (including NhTERT、telomerase index and over-expression) is higher in the gastric cancer tissue than those in the peripheral normal tissue, while the over expression showed significantly higher. This may point out that helicobacter pylori infection can augment the telomerase activity in the gastric cancer and maybe explained by that helicobacter pylori infection plays an important role in the carcinogenesis by increasing telomerase activity.