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  • 學位論文

探討Lactobacillus mali改善腸炎疾病之功效

Investigation of the effects of Lactobacillus mali on ameliorating colitis disease

指導教授 : 陳明汝

摘要


益生菌近年已被做為改善不同腸胃性功能障礙的替代性療法。Lactobacillus mali 為本實驗室先前自糖液克弗爾中分離而出之潛力益生菌菌株,於體外和體內試驗中皆表現明顯的抗腸炎功效。本試驗之目的為探討Lb. mali 改善腸道腸炎疾病之功效,包含預防出血性大腸桿菌O157:H7 感染以及減緩腸炎相關癌症之發展。第一階段試驗探討Lb. mali 對出血性大腸桿菌O157: H7 所誘發腸炎小鼠模型的預防功效。試驗選用八週齡之Balb/c 小鼠依據組別每日分別給予磷酸鹽緩衝生理食鹽水、不同劑量之乳酸桿菌 (108 CFU/隻 和 5 × 108 CFU/隻) 並持續兩周,試驗後七天進行誘導出血性大腸桿菌O157: H7,分別於第0、4、7 天給予出血性大腸桿菌 (2 ×109 CFU/200μL)以誘發小鼠感染腸炎。結果顯示,正對照組有75%小鼠糞便溶血反應檢測為陽性;對比之下,餵予低劑量和高劑量乳酸桿菌之小鼠,僅有14%結果為陽性。除此之外,小鼠給予Lb. mali 能有效避免出血性大腸桿菌O157: H7 入侵肝臟和脾臟。迴腸組織切片也顯示Lb. mali 能藉由維持腸道上皮屏障完整而降低出血性大腸桿菌之感染。然而血清中免疫球蛋白(immunoglobulin, Ig) 包含IgG, IgM 及IgA 的含量並無顯著差異。 試驗進一步研究Lb. mali 抗腸炎功效是否具有減緩腸炎相關癌症發展之機能性。體外試驗使用人類結腸癌細胞株Caco-2 進行細胞聚集試驗。結果指出,與益生菌株共培養之細胞有較高細胞黏著性表現; 在動物試驗, 使用八週齡C57BL/6JNarl 小鼠,依據組別每日給予不同劑量Lb. mali (2 ×108 CFU/隻和109 CFU/ 隻) 。持續2 週後, 小鼠以單次腹腔注射前致癌物質氧化偶氮甲烷 (azoxymethane, AOM ) (200μg/20g),7 天後替換飲用水至2%硫酸葡聚糖納鹽 (dextran sodium sulfate, DSS)誘導潰瘍性結腸炎7 天,而後置換滅菌飲用水14 日,DSS 循環共進行3 次以誘導腸炎相關癌症。結果顯示,相較於正對照組,小鼠給予Lb. mali 可減緩潰瘍性結腸炎之臨床症狀,包含減緩體重下降、結腸長度以及較低的直腸出血評分。結腸組織切片亦指出餵食益生菌之小鼠,其良性腫瘤比例與負對照組相比有提高之趨勢。除此之外,藉由結腸組織切片之免疫組織化學染色發現給予Lb. mali 後,結腸癌之細胞增生因子 (proliferating cell nuclear antigen,PCNA) 表現量有下降之趨勢但各組組織切片皆缺乏腫瘤抑制蛋白p53 之表現。 綜觀上述,本試驗顯示Lb. mali 藉由其減緩腸道發炎之功效,而具有改善出血性大腸桿菌感染和減緩腸炎相關癌症發展之潛力。然而,有關其詳細之抗腸炎之機制仍須進一步探討。

並列摘要


Probiotics has been used as alternative food to ameliorate various gastrointestinal disorders. Lactobacillus mali (Lb. mali), one of the potential probiotics isolated from sugary kefir of our laboratory previously, has been shown clearly anti-colitis effects on both in vitro and in vivo assays. The aim of this study was to investigate the effects of Lactobacillus mali on ameliorating colitis disease, including enterohemorrhagic Escherichia coli (EHEC) O157:H7-induced colitis, a group of Shiga toxin-producing foodborne pathogens, and on lowering the risk of the chronic colitis-associated cancer (CAC). For EHEC infection animal model, eight-week-old Balb/c mice were oral administrated daily with either phosphate buffered saline or different dosages of Lb. mali (108 and 5 × 108 CFU) for 7 days. This was followed by intragastric challenges with EHEC O157:H7 (2 × 109 CFU), which were conducted at day 0, 4, and 7 after the end of Lb. mali treatment. The results showed that the mice daily administrated with Lb. mali could reduce the symptoms of EHEC infection. The fecal occult blood tests showed that 6 out of 8 of the mice (75%) in the EHEC infection group had fecal occult blood present, whereas only 2 out of 14 (14%) were found to have fecal occult blood present in the Lb. mali pretreatment group. Also, pre-treatment of Lb. mali significantly protected the liver and spleen from EHEC invasion in mice. Further, histological analysis showed that Lb. mali could protect intestinal epithelial monolayer against EHEC infected colitis. However, the immunoglobulin (Ig) in serum, including IgG, IgM, and IgA, did not show significant differences among the groups. To further investigate the anti-colitis effects of Lb. mali on decreasing the onset of CAC, human epithelial colorectal adenocarcinoma Caco-2 cell were used for cell aggregation assay in vitro. The results indicated that cell treated with probiotics showed better cell adhesion abilities. In animal model, eight-week-old C57BL/6JNarl mice were oral administrated daily with either phosphate buffered saline as a control, and or different dosages of Lb. mali (2 × 108 and 109 CFU) for 14 days. At the beginning of experiment, mice were given a single intraperitoneal injection with azoxymethane (10 mg/kg). After 7 days, they were treated with 2% dextran sodium sulfate (DSS) solution in drinking water for consecutive 7 days followed by autoclaved water for 14 days. DSS cycle was repeated for 3 times. During the DSS-induced period, fecal occult blood was measured by a scoring system for evaluating colitis severity. After scarifying, colonic and other organ tissues were collected for analysis. The results showed that mice treated with Lb. mali maintained longer colon length than non-treated groups. Similarly, hematoxylin and eosin staining colonic tissue showed less infiltration in Lb. mali groups compared with non-treated groups. In addition, the expression level of proliferating cell nuclear antigen is lower in Lb. mali-treated group. In conclusion, our studies suggested that Lb. mali have the potential to ameliorate the EHEC infection and attenuate the development of CAC due to their abilities to reduce the level of intestinal inflammation. However, further investigations of the possible mechanisms on probiotics in modulating colitis diseases are necessary.

參考文獻


黃懿儂。2010。台灣黏質發酵乳之機能性研究。國立臺灣大學動物科學技術學研
魏士弘。2014。篩選可刺激類升糖素胜肽-1 分泌之益生菌及探討其減緩高血糖症
陳彥伯。2013。克弗爾分離菌株Lactobacillus kefiranofaciens M1 對腸道功能調節
陳希嘉。2008。藉由變性梯度膠體電泳及寡核苷酸微陣列技術鑑定乳酸菌之研究。
沈澤臻。2014。藉由小鼠與犬隻模式探討Lactobacillus kefiranofaciens M1 於異位

被引用紀錄


湯芯瑜(2016)。Lactobacillus kefiranofaciens M1對不同壓力之反應及其於噴霧乾燥和冷凍乾燥後存活之探討〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU201600980

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