以動物模式評估益生菌之免疫調節作用及腸道保護作用

Evaluate immune regulatory effects and intestinal protection of probiotics in animals model.

王凱文(Kai-Wen Wang)

指導教授 ： 詹明修 ； 盧敏吉

中山醫學大學/醫學院/醫學研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00162

摘要

氣喘屬於一種慢性發炎疾病，病灶可見於肺臟及氣管組織。發病時的症狀包括有喘鳴聲、胸痛以及最危險的呼吸道阻塞。若發生呼吸道阻塞未即時給予治療可能導致氣喘病人死亡。氣喘的這些症狀通常是終生的且容易復發，現今許多研究證實了氣喘發生的機制是由於Th1及Th2免疫反應失調所導致，而普遍認為CD4+ Th2這類細胞是驅使並影響氣喘病程的主因。這些細胞會在肺部產生許多發炎性細胞激素，包括Interleukin-4、Interleukin-5、Tumor necrosis factor-α等。目前越來越多研究皆傾向從天然物中找尋有療效的物質，而益生菌也是其中之一;所謂的益生菌是一群活的微生物，並且對健康有幫助的。先前的研究指出樹狀突細胞 (dendritic cell) 可藉由Toll like受體與益生菌或腸道常在菌接觸，因此我們認為益生菌或許能藉由此機制平衡免疫反應。首先，我們將U937單核球細胞與食品工業研究所提供之106株潛力益生菌株進行共同培養，並偵測MCP-1、IL-10、IL-6及TNF-α分泌量。初步篩選出29株益生菌，並將其與人類週邊單核球細胞 (Peripheral blood mononuclear cells, PBMC) 共同培養並偵測IFN-γ。除去市面已有的菌種後，最後選定了七株做進一步的研究。接著先以無菌鼠評估挑選的益生菌是否有能力調節免疫反應，結果發現肺臟沖洗液中IL-4、IL-5分泌量皆是減少的，而脾臟細胞培養中IL-10及IFN-γ分泌量則是增加的。在OVA誘導氣喘動物模式中，我們以C57BL/6品系小鼠進行實驗，此種小鼠與無菌鼠相同是天生傾向Th1免疫反應的。然而從細胞激素分泌看來，結果顯示在C57BL/6品系小鼠身上益生菌無法影響過敏性氣喘的發展。接著以BALB/c小鼠進行分析以確認結果，氣喘小鼠經餵食混合益生菌 (三菌組或七菌組) 後皆無法有效降低氣喘造成的發炎反應。比起單純氣喘的組別，氣喘組餵食益生菌似乎是有害無益的，而組織切片也發現肺部發炎程度增加，包括：嗜伊紅性球浸潤、氣管平滑肌增生。另外，我們把克雷白氏菌 (K. pneumoniae) 當作病原菌感染小鼠，接著餵食數天的益生菌。結果發現餵食益生菌的組別比起控制組有較高的死亡率，而且比起三菌組Mix3，餵食的菌種越多 (Mix7) 存活率就越低。有趣地是，若先每天餵食小鼠益生菌再感染病原菌則能避免小鼠死亡，除此之外糞便中病原菌的菌落數也會顯著的減少。因此我們所使用的益生菌對OVA誘導氣喘動物模式的影響並不顯著，但是能保護小鼠不受克雷白氏菌的感染。

關鍵字

益生菌 ； 氣喘 ； 免疫調節 ； 腸道保護

並列摘要

Asthma has been known as a kind of chronic inflammation occurred in lung and airways. It has characterized by several symptoms, such as wheezing、chest pain and the most dangerous one, airway obstruction. It causes death of asthma patient when happens without emergency treatment. All of these symptoms usually appear recurrent episodes during the life. To date, many research approved that mechanism of asthma was Th1/Th2 imbalance. Most researchers agree that CD4+ Th2 cells initiate and promote asthma, they express many pro-inflammatory cytokines including IL-4, IL-5 and TNF-α in lung. Recently, growing research have exclusively focused on the therapeutic effects of natural substances, the probiotics is one of those candidates. Probiotics has defined as a group of living microorganisms which produce beneficial effects on health. Previously studies demonstrated that dendritic cells interacted with bacteria whether probiotics or normal flora via Toll like receptors. We proposed that probiotics might able to accomplish balanced immune response. Firstly, U937 cells were cultured with 106 potential probiotic strains obtained from FIRDI for screening the secretion levels of MCP-1, IL-10, IL-6 and TNF-α. Twenty nine strains had been selected, and cultured with human PBMC to evaluate the ability of IFN-γ secretion in advance. After excluding commercialize strains, 7 strains has been chosen for further studies. In addition, healthy germ-free mice were be used to test whether the mixture of seleted probiotics could play a role in regulation of immunity. Results showed the expressions of IL-4, IL-5 were decreased in bronchoalveolar lavage, and IL-10, IFN-γ increased in splenocyte culture. Furthermore, in OVA-sensitized asthmatic animal model, we use specific pathogen free (SPF) C57BL/6 mouse, which had inherently Th1-biased immune response same with germ free mouse. As the cytokine secreting profile shown, probiotics mixture is poor to affect asthmatic development on C57BL/6 mouse. BALB/c mice were used to confirm the affect on C57BL/6 mice, whereas mice fed by probiotics mixture (Mix3 or Mix7) did not benefit to reduce inflammation caused by OVA-triggering. Administration of probiotics seems even more farmful than asthma induced only. The same results also found in lung sections, the increases of inflammation were demonstrated by histochemistry stains of eosinophils and airway smooth muscle. On the other hand, we use high dose of K. pneumoniae as a pathogen to infect mice, after fed with probiotics mixture for indicated days. Probiotics groups have higher mortality comparing with control group. Also, rather than Mix3, more strains (Mix7) caused decline of survival rates. Interesting, mice were prevente to death and colony of pathogen has significantly decreased in feces by K.P. infection when administrated daily probiotics supplementation ahead. In conclusion, our selected probiotics were poor to affect OVA-sensitized animal model, but it protected mice from K.P. infection.

並列關鍵字

probiotics ； asthma

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