人類胃上皮幹細胞在幽門螺旋桿菌致癌機制之研究

幽門螺旋桿菌與胃癌的關係最早是在1998 年Takeshi 的蒙古沙鼠動物試驗中被證實，而癌變(Carcinogenesis)是由許多不同基因突變所累積成的結果，從幽門螺旋桿菌致癌分子模式中可以發現，幽門螺旋桿菌所造成的癌症是經由許多不同基因改變累積而成的，從病理學上的研究胃癌的形態可以分為瀰漫型以及腸型胃癌，在瀰漫型胃癌中，幽門螺旋桿菌感染會經由刺激端粒酶的再活化，p53 的突變，Cadherin 的突變或遺失以及c-met 的表現而直接造成癌症的產生。但是在腸型胃癌中，幽門螺旋桿菌感染所造成的端粒酶再活化是首先被偵測於腸化生階段而p53 和APC 基因的去活化是被發現在於化生不良階段, 另外c-met, k-sam, c-erB-2, cyclin E 基因的過度表現則是被認為和早期胃癌較有相關性。目前關於幽門螺旋桿菌致癌的研究主要是被應用於動物模式，但有越來越多的研究顯示癌症的產生可能是來自於幹細胞的變異，於是本實驗的目的是希望可以建立一個具有胃上皮幹細胞特性的細胞株並將此細胞株應用於幽門螺旋桿菌致癌的分子模式中。本實驗的設計首先經由胃鏡取下人類正常胃黏膜組織再利用 collagenase 及dispase 進行分解，接著利用K-NAC 培養基持續培養與觀察及篩選其生命期較長且具有分裂與分化能力之細胞，並以無菌 cloning cylinders 加以選殖分離。最後，將選殖出來的細胞株與幽門螺旋桿菌共同培養後，以同步定量聚合酶連鎖反應（real-time PCR）偵測其端粒酶的活性以及利用反轉錄聚合酶連鎖反應（RT-PCR）偵測其p53， c-met 以及OPN 的mRNA 表現情況。在我們的實驗結果當中發現ST2-8 細胞株除了具有對稱分裂的能力以外還同時可以以不對稱分裂分化成為其他型態的細胞，例如類上皮細胞、類分泌細胞以及類神經細胞。另外經過計算後發現其累積細胞群落複製等級（cumulative population doubling level）為37，顯示其具有比正常細胞更長之生命期。而於細胞非依賴附著生長（AIG）之實驗中ST2-8 細胞株可形成小的細胞群落，並其頻率為1.7％。在免疫染色方面ST2-8 其vimentin、CK-7、CK-8、CK-18、CK-19、 Mucin-5AC 及Epithelial membrane antigen 均呈陽性反應。並且利用 RT-PCR 發現此細胞株具有Oct-4 基因的表現。而在幽門螺旋桿菌致癌機制研究方面，發現當我們將ST2-8、幽門螺旋桿菌（C1283）及 MNNG 共同培養5 天後，其端粒酶的活性表現比ST2-8 分別與幽門螺旋桿菌或MNNG 共同培養時有顯著性升高的趨勢。但其p53， c-met 以及OPN 的mRNA 表現情況卻沒有顯著性之差異。所以，綜合以上的研究結果發現ST2-8 細胞株是一株具有幹細胞特性的胃上皮細胞株並且此細胞株是可以被應用於幽門螺旋桿菌致癌機制的探討上。

關鍵字

人類胃上皮幹細胞；幽門螺旋桿菌；胃癌

並列摘要

The association between Helicobacter pylori and gastric adenocarcinoma is intriguing. Although much speculation has accumulated, prospective study in human can not be available. Fortunately, it has been proved by Takeshi et al. in 1998 that Helicobacter pylori infection can lead to gastric carcinogenesis. The experiment consisted in direct inoculation of Mongolian gerbils with Helicobacter pylori. Serial pathological change was demonstrated from gastritis to adenocarcinoma. Since gastric epithelium of human adult replaces in about 1 to 3 days, pathological change of gastric epithelium resulted from chronic infection with Helicobacter pylori has been proposed to originate from gastric stem cells. However, there are no exact molecular markers for identifying human gastric stem cells but some properties that belong to progenitor cells. This preliminary study is aimed at evaluating some possible markers for stem cells and early cells and utilizing these properties to characterize cells similar to progenitor cells and stem cells. Later, we plan to investigate gastric carcinogenesis effects of Helicobacter pylori while co-culture with these early cells. Endoscopic biopsy specimens were utilized for cultivation of stem cells and progenitor cells. Human adult gastric epithelium sampled from biopsy were digested with collagenase and dispase and then cultivated with K-NAC media. Colonies with more proliferation and differentiation were selected and then isolated with sterile cloning cylinders. There isolated cell colonies were then co-cultivated with Helicobacter pylori. Real-time PCR for activity of telomerase and RT-PCR for m-RNA of p53, c-met and OPN were later evaluated. In this study, we found a cell colony, called ST2-8, was much longer lived than other cell colonies. The cumulative population doubling level was 37. Both symmetric and asymmetric cell differentiation were observed by both the same shape and many different shapes of cells. The frequency of AIG (anchorage of independent growth) of ST2-8 was 1.7%. There was positive immunostaining of ST2-8 with the markers of vimentin、CK-7、 CK-8、CK-18、CK-19、Mucin-5AC and epithelial membrane antigen. And the cell line expression Oct-4 gene with RT-PCR. Besides, we also co-cultivated ST2-8 colony with Helicobacter pylori (C1283) and MNNG. The activity of telomerase was elevated after 5 days culture as compared with the activity of initial co-culture. The expression of mRNA of p53, c-met, and OPN was not significantly different. So, we concluded that the gastric epithelium cell colony ST2-8 has the characteristics of stem cells and we can apply the molecular studies concerning the carcinogenesis on stem cell-like colonies.

並列關鍵字

Human Gastric Stem cells ； Helicobacter pylori ； Stomach cancer

參考文獻

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人類胃上皮幹細胞在幽門螺旋桿菌致癌機制之研究

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