數株乳酸桿菌胞外多醣抑制 4NQO 誘導人類腸道上皮細胞 Int-407 細胞毒性及人類結腸癌細胞 HT-29 增生之研究

Effects of Lactobacillus spp. Exopolysaccharides on the Anticytotoxicity of Human Intestine Cell Line Int-407 and Inhibition of Colon Cancer Cell Line HT-29 Proliferation

劉素霞(Sue-Xia Low)

指導教授 ： 游若篍

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01854

摘要

結腸直腸癌 (colorectal cancer, CRC) 是目前常見的癌症，約 70% 結腸直腸 癌與飲食習慣相關。益生菌及益生質具有維持人體腸道內正常微生物菌相、減緩乳糖不耐症、降低膽固醇、調節免疫系統、抗腫瘤及抗致突變等諸多生理功能，有研究指出部分原因可能與其所產之胞外多醣 (exopolysaccharides, EPS) 有關。 本研究以五株乳酸桿菌胞外多醣為為材料進行試驗，首先探討其對雙叉桿菌的助生性效果，接著利用致突變劑 4-nitroquinoline-1-oxide (4NQO) 誘導人類腸道上 皮細胞 Int-407 產生細胞及基因毒性，評估降低細胞及基因毒性的能力，並探討抗細胞毒性的機制。最後以 MTT assay 及流式細胞儀分析其抑制人類結腸癌細 胞 HT-29 增生及細胞週期阻滯的能力。結果顯示，乳酸桿菌胞外多醣對雙叉桿菌皆有助生性效果，以 L. acidophilus BCRC 14079 胞外多醣效果最佳。在抗細胞毒性方面，五株乳酸桿菌胞外多醣皆可顯著提高經誘導損傷後 Int-407 的細胞存活率，但在不同濃度下有最佳的效果。利用分段式預反應探討其抗細胞毒性機制，結果顯示主要為阻斷效應 (blocking effects) 及生物抗細胞毒性效應 (bioanticytotoxic effects)。由彗星電泳結果顯示，乳酸桿菌胞外多醣處理組 DNA 拖尾程度顯著小於 4NQO 組。在抑制人類結腸癌細胞 HT-29 增生方面，隨作用時間增加，細胞存活率越低，且在 48 小時作用下呈現濃度效應，以 200 ug/mL 之 L. casei 01 胞外多醣效果最佳，抑制率為 31.2%。流式細胞儀分析結果顯示，乳酸桿菌胞外多醣能使 HT-29 細胞週期停滯於 G0/G1 phase，並顯著增加 sub-G1 phase，誘導細胞走向凋亡。本研究結果顯示，乳酸桿菌胞外多醣具有助生效果，也能有效的保護腸道細胞降低損傷，抑制癌細胞的增生。

關鍵字

乳酸桿菌胞外多醣 ； 結腸直腸癌 ； 助生性 ； 抗細胞基因毒性 ； 抑制癌細胞增生

並列摘要

Colorectal cancer commonly known as bowel cancer, is one of the leading causes of cancer. About 70% of colorectal cancer is related to dietary habits. Probiotics and prebiotics have received an increasing amount of attention in recent years mainly because of their positive health effects such as maintenance of human intestinal microflora, reduction of lactose intolerance, cholesterol-lowering activities, immunomodulating, antitumor and antimutagenic activity. Studies had shown that the beneficial effects could be attributable to positive physiological effect of exopolysaccharides (EPS). In this study, we employed EPS produced by five Lactobacillus spp. as materials. At first, we investigated the prebiotic effects of EPS on the growth of bifidobacteria. Then EPS were studied for their anticytotoxicity and antigenotoxicity activities against 4-nitroquinoline-1-oxide (4NQO) induced human intestine cell line Int 407. In addition, MTT assay and flow cytometry were used to explore the antiproliferation and cell cycle arrest of human colon cancer cell, HT-29. The results showed that EPS had the prebiotic effects on the growth of bifidobacteria, especially EPS produced by L. acidophilus BCRC 14079. Besides that, EPS could reduce cytotoxicity of Int-407 induced by 4NQO. The possible anticytotoxic mechanisms of EPS were blocking effects and bioanticytotoxic effects. From the results of comet assay, we observed that the DNA tails significantly reduced in the EPS groups, showed the antigenotoxicity effects of EPS. Furthermore, MTT assay showed that the proliferation of HT-29 cells was inhibited after treatment with EPS, and in a time- and dose-dependent manner. At 200 ug/mL, EPS produced by L. casei 01 strongly inhibited the proliferation of HT-29 cells, with a sustained diminution of the number of viable cells throughout the 48 h treatment period. The results of flow cytometry analysis showed that EPS treatment enabled the HT-29 cell cycle arrest at G0/G1 phase, and a significant increase in the sub-G1 phase which induced cell apoptosis. Conclusively, all of EPS produced by Lactobacillus spp. in this study had the prebiotic effects, and protect the intestinal cells from damage effect, antiproliferation of cancer cells and reducing the possibility of suffering from colorectal cancer.

並列關鍵字

exopolysaccharides (EPS) ； colorectal cancer ； prebiotic ； anticytotoxicity ； antigenotoxicity ； antiproliferation

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