內化細菌毒性和噬菌體對腸道上皮細胞增生與癌化之探討

Abstract

背景：結腸直腸癌 (colorectal cancer, CRC) 的發生率在台灣已經連續9年佔據十大癌症之首。而發炎性腸道疾病 (inflammatory bowel disease, IBD) 的患者有很高的風險會進展成CRC。先前許多證據指出腸道菌叢與IBD及CRC的病理發展是有關的，但詳細的機制目前尚未清楚。目的：探討含有毒性因子之細菌入侵腸道上皮細胞後是否會直接影響細胞的增生週期，並利用噬菌體抑制細菌之促癌作用。方法：將腸癌模式小鼠大腸上皮細胞分離之大腸桿菌 (Escherichia coli) 與人類大腸癌細胞株Caco-2及T84細胞共培養4小時以測量內化菌落數，並於24小時後利用流式細胞技術觀察細胞週期之改變。利用PCR技術分析內化細菌之毒性因子，並將細菌之fimA、fimH及htrA基因作單獨或共同剔除，隨後與Caco-2細胞共培養後測量內化菌落數及觀察細胞增生情形。噬菌體實驗則是將噬菌體、細菌、Caco-2細胞三者共培養後測量內化細菌菌落數及觀察細胞增生情形。結果：小鼠腸道上皮細胞分離的E. coli感染Caco-2或T84細胞後，都會侵襲至細胞中並促使細胞加速增生。若將細菌毒性因子包括fimA、fimH或htrA基因單獨或共同剔除，則細胞增生情形減少。將針對E. coli有專一性的噬菌體與細菌和Caco-2細胞共培養後，可降低內化菌落數並且減少細胞增生的情形。結論：小鼠腸道上皮細胞分離之細菌透過毒性因子fimA/fimH/htrA內化至細胞中並促進細胞加速增生，推論是細菌促癌的機制。而利用噬菌體可有效抑制細菌促進上皮細胞增生的能力。

Background: The incidence of colorectal cancer (CRC) had been the highest among other types of cancers for 9 years in Taiwan, and patients with inflammatory bowel disease (IBD) had a higher risk. Previous studies showed that gut microbiota were related to IBD and CRC development, but the mechanism is still unclear. Aim: To investigate whether the invasion of intestinal bacteria with virulence factors and endogenous plasmids may promote epithelial cell proliferation, and the inhibitory effect of bacteriophage on bacteria-induced epithelial cell hyperproliferation. Methods: Escherichia coli were isolated from mouse colonocytes of colitis-associated cancer model. Human Caco-2 cells and T84 cells were apically exposed to E. coli for 4 hours to measure the internalized bacterial colony forming units. After 24 hours, the cell cycle progression was measured by flow cytometry. Bacterial virulence genes were determined by PCR; bacteria were deleted of fimA, fimH, and htrA genes individually or simultaneously and then exposed to Caco-2 cells for measuring bacterial internalization and cell proliferation. In addition, Caco-2 cells were exposed to bacteriophage and bacteria for measuring bacterial internalization and cell proliferation. Results: The mouse colonocytic isolated E. coli were internalized into Caco-2 and T84 cells, and caused cell proliferation. The internalized E. coli expressed type 1 fimbriae, fimA, fimH gene or stress protein, htrA gene. Single or multiple gene deletion of fimA, fimH, and htrA in E. coli reduced Caco-2 cell proliferation. Addition of phage in the co-culture of E. coli and Caco-2 cells decreased bacterial internalization and cell proliferation. Conclusions: The mouse colonocytic isolated bacteria invaded epithelial cells and promoted cell proliferation via a fimA/fimH/htrA-dependent mechanism, which may contribute to protumorigenic activity. Bacteriophages inhibited the epithelial hyperproliferation induced by bacterial internalization.