探討Lactobacillus kefiri K影響第一型糖尿病之可能機制

Investigation of possible mechanism of Lactobacillus kefiri K on its affecting type 1 diabetes

黃舜瑜(Shun-Yu Huang)

指導教授 ： 陳明汝

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703147

摘要

本研究於體外試驗中，將模擬人類腸道上皮細胞之Caco 2單層膜細胞與活菌Lb. kefiri K進行共培養後，發現細胞緊密結合蛋白基因OCLN表現量顯著上升(p < 0.05)。此外，不管是熱致死處理或是活菌Lb. kefiri K皆可以藉由提高小鼠骨髓分離之樹突細胞表面分子MHC classⅡ、CD40、CD86之表現量進而使其成熟。從這些結果推測Lb. kefiri K可能透過提高腸道黏膜屏障及調節免疫反應進而影響第一型糖尿病(T1D)。
在本研究第一次動物試驗方面，在連續注射5天STZ誘導8週齡C57BL/6 小鼠T1D前，連續給予7天菌株K(此為第0週)，其空腹血糖值於第1週開始顯著高於STZ組，而在第8週K組小鼠口服糖耐量曲線下面積會顯著高於STZ組(p < 0.05)，這些糖尿病相關症狀的加劇與先前文獻並不一致。有趣的是，第一次試驗小鼠血清中GLP-1及胰島素之濃度結果卻與先前文獻相似，而迴腸緊密結合蛋白之表現量在各組間皆無顯著差異，在培氏斑及腸繫膜淋巴結中，發現給予了活菌之組別CD4+ T細胞之比率顯著降低(p < 0.05)。造成此次試驗結果與先前文獻不一致之原因，我們推測可能包含了菌株的變異、實驗鼠之週齡及個體差異等因素。
為了進一步探討這些因素是否會影響試驗結果，本研究重新活化菌株K並同時使用5及8週齡小鼠進行第二次試驗。試驗結果顯示給予5及8週齡誘發T1D小鼠菌株K皆可以減緩T1D相關症狀包含空腹血糖值、口服糖耐量及其曲線下面積，暗示週齡之不同並非第一次試驗與先前文獻不一致之因素。此外，第二次動物試驗中發現給予菌株K之組別，在三個淋巴相關組織中，CD4+ T細胞之比例相對於控制組並無顯著差異。
綜上述，我們推測兩次體內試驗結果不同可能與菌株重新活化相關，而是否兩次使用之菌株有所差異進而造成T1D病程發展不同，則需要更進一步之探討。

關鍵字

乳酸菌 ； 免疫調節 ； 腸道黏膜屏障 ； 糖尿病 ； 升糖素類似胜肽

並列摘要

In the present study, we investigated the possible mechanisms of Lb. kefiri K involving in its ameliorating hyperglycemia in vitro and in vivo. In vitro, the human epithelial Caco 2 cell monolayer was co-cultured with live or heat-killed Lb. kefiri K. The gene expression of occludin, a tight junction protein, was upregulated after treatment with live Lb. kefiri K. Additionally, Lb. kefiri K also accelerated dendritic cells (DC) maturation by modulating the expression of MHC classⅡ, CD40 and CD86, the surface markers on murine bone marrow dendritic cells (BMDC). The in-vitro results suggested that the possible mechanisms of Lb. kefiri K to ameliorate hyperglycemia might involve in enhancing intestinal integrity and modulating immune response.
We further investigated the mechanisms in vivo. After 1-week administration of 108 CFU Lb. kefiri K per mouse daily, eight-week-old male C57BL/6 mice were induced type 1 diabetes (T1D) by multiple low dosage of STZ. The Lb. kefiri K treatment was continued for nine weeks. Results indicated that no significant difference was found on gene expression of tight junction protein among the groups. The Lb. kefiri K treated group showed the significantly higher fasting blood glucose level and lower percentage of CD4+ T cells in Peyer’s patches and mesenteric lymph node (p < 0.05) than the STZ group. Lb. kefiri K treatment also indicated to increase GLP-1 level in serum (p < 0.1). Interestingly, the in-vivo findings were inconsistence with our previous study. The differences might be due to the age of mice, strain and individual differences.
To further investigate possible factors involved, five and eight-week-old male C57BL/6 mice were used in the 2nd experiment. In addition, the strain K also be re-activated from stock. Results indicated that administration of live Lb. kefiri K ameliorated T1D associated symptoms including fasting blood glucose, oral glucose tolerance test and the area under curve on both five and eight-week-old mice (p < 0.05). It demonstrated that age might not be the factor influencing the 1st in vivo result. Besides, there was no significant difference between CD4+ T cell percentages in Peyer’s patches and mesenteric lymph node of each group.
Above all, the different results in vivo might be due to the re-activated strain K. There might be difference between cultures of strain K used in two experiments. However, further study is necessary to investigate possible reasons.

並列關鍵字

lactic acid bacteria ； immune-regulatory effect ； gut mucosal barrier function ； diabetes ； glucagon-like peptide-1

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