乳酸桿菌較雙歧桿菌容易誘導小鼠脾臟細胞分泌IFN‐gamma及Th1細胞分化

Lactobacillus is more potent than Bifidobacterium in inducing IFN‐gamma secretion and Th1 lineage differentiation in murine splenocytes

顏湘庭(Xiang-Ting Yan)

指導教授 ： 陳彥旭

共同指導教授 ： 吳登強(Deng-Chyang Wu)

高雄醫學大學/醫學院/醫學研究所/碩士(2011年)

摘要

背景：在目前的臨床研究上，益生菌可以改善人體的免疫反應，其中乳酸桿菌和雙歧桿菌是微生物中最常作為益生菌的菌屬。雖然益生菌對腸道黏膜免疫有良好的效果，但是對於調控免疫的相關機制目前還不清楚。
目的: 本研究探討乳酸桿菌與雙歧桿菌對腸道黏膜細胞性免疫(cellular immunity)的影響，尤其針對如何影響Naive CD4+ T細胞的分化做討論。
材料和方法：在我們的研究中使用三株乳酸桿菌，L. paracasei 33、L. casei 01 和 L. paracasei D11a；三株雙歧桿菌，B. lactis Bb12、B. lactis HN019, 和 B. longum。將C57BL/ 6小鼠犧牲之後取出脾臟細胞，再以Multiplicity of infection (MOI) 30與益生菌共同培養12、24和48小時之後，收集上清液測ELISA，細胞抽RNA轉cDNA檢測調控CD4+ T細胞分化的轉錄因子情形。
結果：我們主要分析8種細胞激素的表現量，即IFN-gamma、TNF-alpha、IL- 4、5、6、10、12和17，分別將脾臟細胞與六株益生菌共同培養12、24和48小時。IL-4、IL-5和TGF-beta的分泌量小於標準品測不到，而乳酸桿菌和雙歧桿菌刺激脾臟細胞後細胞激素IFN gamma、IL-10、TNF alpha、IL-6都有相同趨勢的上升。 乳酸桿菌和雙歧桿菌刺激脾臟細胞後都可以讓CD4+ T細胞走向Th1 lineage分化的T-bet轉錄因子顯著上升，其他CD4+ T細胞分化的轉錄因子，如Th2的GATA-3、Th17的ROR-γt、iTreg的Foxp3，在48小時後都沒有上升。並且，A菌刺激脾臟細胞產生IFN gamma與IL-12的能力比B菌還強，刺激屬於 第一型輔助性T細胞(Th1)的轉錄因子(T-bet)表現量比B菌還高。
結論：乳酸桿菌與雙歧桿菌雖分屬不同的細菌門，但對腸道免疫系統的影響是一致的。結果顯示，乳酸桿菌促使脾臟細胞增加T-bet的反應和增加細胞激素IL-12以及IFN?ngamma的分泌的能力比雙歧桿菌強；更可以進一步抑制其他的轉錄因子ROR-gt、GATA-3及Foxp-3的表現，以及抑制細胞激素IL-4、IL-5、IL-17的分泌，走向Th1的分化路徑。

關鍵字

益生菌 ； 乳酸桿菌 ； 雙歧桿菌 ； 腸道免疫 ； Th1 ； IFN gamma ； T-bet

並列摘要

Background: Lactobacillus and Bifidobacterium are the most common microbes used as probiotics. Although probiotics are good for mucosal immunity in the gut, it remains unclear how Lactobacillus and Bifidobacterium affect the naive CD4+ T cells differentiation, which is critical in intestinal mucosal immunity.

Materials and methods: Three strains of Lactobacillus, L. paracasei 33, L. casei 01 and L. paracasei D11a, and three strains of Bifidobacterium, B. lactis Bb12, B. lactis HN019, and B. longum, were used in this study. Multiplicity of infection (MOI) 30 of each strain was applied to the experiments. The splenocytes were isolated from C57BL/ 6 mice. Culture supernatant was collected for ELISA. Cell pellets were collected from real-time PCR analysis of the critical transcription factors for naive CD4+ T cells differentiation.

Results: We examined the expression of the 8 cytokines, namely IFNγ, TNFα, IL-4, 5, 6, 10, 12 and 17, after the six strains of probiotics were co-cultured with splenocytes for 12, 24 and 48 hrs. No expression of IL4, IL5 and TGFβ was detected in both groups, whereas the levels of IL-6 and 17 were similar in both groups. The level of IL12 was higher in Lactobacillus group than Bifidobacterium group at each time point (p<0.05). The expression of IL-10, IFNγ, and TNFα reached significantly high level at 48 hrs in both Lactobacillus and Bifidobacterium groups. This was compatible with the 8-fold higher expression of T-bet, which is a Th1 cell specific transcription factor, at 48 hrs.

Conclusion: Both Lactobacillus and Bifidobacterium induce murine splenocytes toward Th1 expression, especially after 48 hrs’ co-culture. Transcription factors, including GATA3, RORγt and FOXP3, were inhibited after 48 hrs’ co-culture. However, whether these results collectively exert pro-inflammatory or immunosuppressive effects in vivo awaits further validation.

並列關鍵字

probiotics ； Lactobacillus ； Bifidobacterium ； intestinal immunity ； Th1 ； interferon-gamma ； T-bet

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