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Intestinal Epithelial Response to Bacterial Endocytosis in Colitis-associated Colon Cancers
colorectal cancer,Escherichia coli,bacterial endocytosis,cell proliferation,
|Publication Year :||2017|
|Abstract:||背景：結腸直腸癌 (colorectal cancer, CRC) 的發生率在台灣已經連續八年蟬聯冠軍。而發炎性腸道疾病 (inflammatory bowel disease, IBD) 的病患罹患CRC的風險較正常人高出兩倍以上。過去對於CRC的研究主要著重在基因以及生活飲食習慣的探討，但最近逐漸發現，CRC的形成也與腸道菌叢有密切關連性。例如在IBD患者腸黏膜中所發現的黏附侵入性大腸桿菌 (Adherent-invasive Escherichia coli, AIEC) 可以透過活化巨噬細胞發炎反應而間接地影響癌症進展；具有pks基因可產生毒素colibactin的大腸桿菌 (E. coli) 則會導致上皮細胞之去氧核醣核酸 (deoxyribonucleic acid, DNA) 受損而促進腫瘤生成。然而時至今日，對於腸道菌是否可透過內化 (internalization) 至腸道上皮細胞，而影響細胞增生速率和促進腫瘤的形成則尚未明瞭。目的：探討細菌及毒性因子之入侵是否影響腸道上皮細胞的增生或死亡。方法: 給予BALB/c小鼠三個循環的氧化偶氮甲烷 (azoxymethane, AOM) 及葡聚醣硫酸鈉 (dextran sodium sulphate, DSS) 以誘導CRC產生，抗生素處理組則會經口給予含有抗生素的飲用水一週。爾後觀察小鼠腫瘤形成量及細菌內吞至大腸上皮細胞的數量，並將內化細菌分離利用16S rDNA定序辨認菌株種類。在細胞實驗的部分，將人類大腸癌細胞株Caco-2與小鼠大腸上皮細胞分離出的菌株進行共培養4小時測量細菌內吞之情形，並利用細胞計數和流式細胞術觀察24及48小時後Caco-2細胞增生情形。此外，測量細胞DNA的片段化程度代表細胞凋亡 (apoptosis)，而培養液中乳酸脫氫酶之活性則代表細胞壞死 (necrosis) 的程度。結果：給予AOM/DSS會導致小鼠結直腸腫瘤的形成，以及增加大腸上皮細胞的內吞細菌量；而口服抗生素處理後可有效降低腫瘤量及減少大腸上皮細胞的細菌量。這些內化至腸道上皮細胞的細菌包括有Escherichia coli、Enterobacter cloacae、Enterococcus faecalis、Staphylococcus。將這些細菌與Caco-2細胞共培養進行實驗則可以發現，小鼠腸道上皮內化的E. coli和人類AIEC LF82具有最強的入侵能力，且可以在細胞中存活至少五天。小鼠腸道上皮分離的E. coli含毒性因子fimA、fimH、htrA，但不含pksR和dsbA；其入侵能力和毒性因子皆會隨著重複內化至細胞中而增加。此外，小鼠腸道上皮細胞分離的E.coli感染Caco-2細胞後會導致Caco-2細胞數目增加和細胞週期加速；但若將細菌的毒性因子htrA基因剔除，Caco-2細胞增生加速現象則隨之消失。最後，E.coli亦會導致細胞凋亡但無細胞壞死情形。結論：抗生素處理可抑制小鼠腸道腫瘤的形成和降低腸道上皮細胞的細菌內吞量，顯示細菌與致癌有關。腸道上皮細胞內吞的E.coli表現毒性因子htrA，且會促進上皮細胞增生，因此在腫瘤形成的過程中可能扮演著重要的角色。|
Background: The incidence of colorectal cancer (CRC) had been the highest among other types of cancers for 8 years in Taiwan; patients with inflammatory bowel disease (IBD) had a higher risk. Beyond the notion of heredity and living habits, colonic bacteria also play critical roles in tumor formation. The IBD-associated adherent-invasive Escherichia coli (AIEC) activate macrophages to promote tumorigenesis indirectly through inflammatory response. The pks+ colibactin-producing E. coli caused epithelial cell DNA damage to promote tumor growth. However, it remains unclear whether bacterial internalization may increase epithelial cell proliferation and tumorigenesis. Aim: To investigate whether the invasion of intestinal bacteria and virulence factors may promote epithelial proliferation and/or cell death. Methods: BALB/c mice were subjected to chemical induction of CRC by three cycles of azoxymethane (AOM)/dextran sodium sulfate (DSS). In some groups, antibiotics were administered to AOM/DSS (A/D) mice. Bacteria isolated from mouse colonocytes were sequenced for 16S rDNA. Human Caco-2BBe cells were apically exposed to the mouse internalized bacteria for 4 hours for measurement of bacterial endocytosis. Cell proliferation was measured by cell number counting and flow cytometry. Cell death was determined by DNA fragmentation and lactodehydrogenase assay. Results: Mice subjected to AOM/DSS developed colorectal tumors and exhibited increased bacterial internalization in colonocytes. Treatment with antibiotics reduced the tumor burden, as well as the endocytosed bacterial counts. The mouse endocytosed bacterial strains included Escherichia coli、Enterobacter cloacae、Enterococcus faecalis、Staphylococcus. By in vitro co-culture experiments with Caco-2 cells, higher levels of bacterial internalization were found after challenge with mouse isolated E. coli and human AIEC LF82 strains, compared to Enterobacter, Enterococcus and Staphylococcus. Moreover, the mouse internalized E. coli survived in Caco-2 cells for at least 5 days. The mouse internalized E. coli expressed virulence factors such as fimA, fimH and htrA, but not pksR or dsbA; the bacterial invasive ability and virulent levels were both increased following sequential passage into Caco-2 cells. Lastly, mouse isolated E. coli incresaed cell proliferation and cycle progression of Caco-2 cells, of which deletion of bacterial htrA gene abolished the cellular hyperproliferative effects. In addition, mouse isolated E.coli also induced apoptosis, but not necrosis, of Caco-2 cells. Conclusions: Antibiotic treatment inhibited colon tumorigenesis in mouse models, which may be partly through reducing bacterial endocytosis. Internalized E. coli expressed virulence factors and induced epithelial hyperproliferation, suggesting that the bacteria may play an important role in tumorigenesis.
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