- 學位論文
探討Akkermansia muciniphila與Amuc_1100*蛋白對肥胖小鼠體重控制及腸道屏障功能之影響
Effect of Akkermansia muciniphila and Amuc_1100* on weight control and intestinal barrier function of obese mice
摘要
本試驗旨在探討A. muciniphila與Amuc_1100*蛋白對肥胖動物體重控制及腸道屏障功能的影響，試驗分別以細胞試驗及動物試驗進行之。動物試驗使用50隻8週齡小鼠，隨機分配至5個處理組，分別為一般飼糧對照組、高脂飼糧對照組、高脂飼糧菌液組、高脂飼糧蛋白組、高脂飼糧變性蛋白組。後四組先餵飼高脂飼糧3週誘發肥胖，挑選體增重達20%以上之小鼠，管餵菌液或蛋白持續5週，於試驗結束時進行採樣及犧牲。結果顯示A. muciniphila與Amuc_1100*蛋白可減少部分小鼠肝臟中的油滴堆積，但A. muciniphila、Amuc_1100*與Amuc_1100*變性蛋白對小鼠體重與其他代謝指標均無顯著影響，腸道緊密連接蛋白之表現量亦無顯著差異。此外，三種處理均不會產生肝腎毒性或進一步引起發炎反應。細胞試驗部分，以Caco-2細胞株測試Amuc_1100*蛋白對腸道細胞屏障功能之影響，其結果顯示對腸道細胞之緊密連接並無顯著促進效果；另以HEK-Blue細胞株表現人類類鐸受體二型（toll-like receptor 2, TLR2）之活性測試系統，進行Amuc_1100*蛋白對TLR2活化能力的分析，結果顯示1000 ng/ml之Amuc_1100*蛋白可活化之TLR2反應數值與0.3 ng/ml的Pam2CSK4（TLR2活化劑）相當。綜而言之，A. muciniphila與Amuc_1100*系列之蛋白質可能僅有預防動物肥胖之功能，對肥胖動物之減重效果有限；而細胞試驗結果顯示Amuc_1100*無法明顯促進腸道細胞的屏障功能，這些結果或許跟Amuc_1100*需Pam2CSK4的3300倍濃度才能產生相同的TLR2刺激活性有關。
並列摘要
The objective of this study was to investigate the impact of A. muciniphila and Amuc_1100* on weight control and gut barrier function in obese animals. The following experiment was divided into two parts, including in vitro and in vivo studies. For in vivo study, 50 mice were randomly divided into 5 groups. 4 groups of mice were fed with high fat diet to induce obesity for 3 weeks. Mice with ≥ 20% weight gain were selected to continue the following experiment. Mice were fed by high fat diet with oral gavage of A. muciniphila, Amuc_1100* and denatured Amuc_1100*. PBS with 10% glycerol were used as placebo. The experiment lasted for 5 weeks. Results showed that A. muciniphila and Amuc_1100* decreased lipid accumulation in liver of obese mice. No significant differences in mice weight gain, metabolic profile and expression of intestinal tight junction with A. muciniphila, Amuc_1100* and denatured Amuc_1100* treatment. None of the treatments contributed to hepatotoxicity or nephrotoxic. In vitro studies part, Caco-2 cells were treated with Amuc_1100* for transepithelial electrical resistance (TEER) assay and the results showed no significant difference in TEER value. In addition, HEK-Blue cells expressed human toll-like receptor 2 (TLR2) and reporter gene were utilized to investigate whether Amuc_1100* can stimulate TLR2 responses. The results showed that the activation value of 1000 ng/ml of Amuc_1100* presented the similar value of 0.3 ng/ml of Pam2CSK4, which is an activator of TLR2. The studies showed both A. muciniphila and Amuc_1100* only decreased lipid accumulation in liver and had no effect on weight control or intestinal barrier function of obese mice. It was suggested that A. muciniphila and Amuc_1100* should be utilized in prevention of obesity instead of weight loss of obese animals. Results of in vitro studies also showed that the protein had no effect to enhance the tight junction between Caco-2 cells. These results may due to 3300 times of Pam2CSK4 concentration was required for Amuc_1100* to activate the same level of TLR2 signaling.