臺灣博碩士論文加值系統

肥胖現今已成為全球公共衛生中一大議題，且與代謝症候群及台灣十大死因呈高度相關。瘦體素（leptin）由脂肪細胞分泌，能夠刺激腦部下視丘（hypothalamus）產生飽足感，在腦部發揮抑制食慾的功能。雖然肥胖者瘦體素濃度普遍較高，但因肥胖者的瘦體素受器敏感度降低，產生瘦體素抗性（resistance），故無法依靠瘦體素減低熱量攝取而降低體重。Suppressor of cytokine signaling（SOCS）蛋白為瘦體素的負調控劑，它透過抑制signal transducer and activator of transcription-3（STAT3）的磷酸化來避免下視丘中瘦體素濃度過高。鼠李糖乳桿菌（Lactobacillus rhamnosus GG, LGG）會參與調控SOCS3表現。本實驗目的為探討LGG與宿主之間能量代謝的關係。本研究使用八周齡C57BL/6J品系公鼠，隨機分成四組後，餵飼高脂飼料誘導肥胖與瘦體素抗性，其中兩組每日管餵鼠李糖乳桿菌108與1010 CFU，連續餵食10周後，體重與攝食量並無顯著差異，肝臟切片顯示給予LGG能夠預防高脂飲食所造成的脂肪肝。餵食10周後以腹腔注射人工瘦體素，並發現LGG能夠提高瘦體素敏感度，在測量下視丘中SOCS3之後，給予高劑量LGG小鼠，注射食鹽水之後，SOCS3表現低；在注射人工瘦體素之後，SOCS3表現量立即升高，表示LGG會使下視丘仍對於瘦體素保持高度敏感。迴腸組織切片顯示L. rhamnosus GG的給予，能夠使小腸腔內絨毛保持長度，絨毛長度與腺窩比例保持於健康狀態，不被高脂飲食所影響。經由變性梯度膠體電泳與次世代定序（next-generation sequencing）分析之結果顯示，飲食的不同會立即改變腸道微生物的組成，食用高脂飼糧會使分解多醣類微生物增加，使得宿主吸收額外的熱量，造成肥胖。而L. rhamnosus GG能夠改變腸道菌相，使得腸內益菌增加，維持腸道健康。經由此研究，能夠更加闡明益生菌抗肥胖功能的調控機制，並改善人類與動物因肥胖所引起相關疾病的發生，也讓微生物與能量代謝的關係有更進一步的發現與突破。

Obesity, which has reached epidemic proportions globally, may induce metabolic syndromes in relation to cause of death. Leptin can suppress food intake and thereby lead to weight loss through binding leptin receptor on the hypothalamus. Suppressor of cytokine signaling (SOCS) proteins is one of the mediator reducing leptin sensitivity by inhibition of the phosphorylation of signal transducer and activator of transcription-3 (STAT3), an intracellular leptin-signaling mediator. Lactobacillus rhamnosus GG (LGG) was proved to be highly correlated with SOCS3. The purpose of this study is to expand the comprehension of the relation between L. rhamnosus GG and host energy metabolism. 8-week-old of C57BL/6J male mice were devided into treatment groups, ND (normal diet / PBS treatment) , HFD (high-fat diet / PBS treatment), LL (high-fat diet / 108 CFU L. rhamnosus GG) and LH (high-fat diet / 1010 CFU/mL L. rhamnosus GG). The grow performance, leptin sensitivity, and gut microbiota composition of the mice were assayed. The results show that, L. rhamnosus GG may decrease lipid deposition, high-dosed L. rhamnosus GG effectively maintained leptin sensitivity. In LH group, their body weight dropped off more clearly after leptin i.p. injection than saline i.p. injection. Also, the levels of serum leptin were significantly higher in L. rhamnosus GG -treated mice than those in the PBS-treated mice (p<0.05). In addition, leptin-injected mice presented significantly higher expression of SOCS-3 in hypothalamus than saline-injected mice (p<0.001) in LH group. Finally, L. rhamnosus GG could establish a healthy intestinal environment to prevent the damage cause by pathogens. Ileum sections showed that L. rhamnosus GG -treated mice had significant longer villi length and villi length-crypt depth ratio than HFD group. In conclusion, L. rhamnosus GG plays an important role in modulating serum leptin levels and giving rise to the enhancement of leptin sensitivity and intestinal health in diet-induced obesity.

口試委員審定書..............i
誌謝.......................ii
中文摘要...................iii
英文摘要...................iv

壹、文獻探討...............................1
第一節 肥胖................................1

一、肥胖的現況.............................1
二、肥胖的成因.............................3
(一)遺傳與生理因素.........................3
(二)環境因素..............................4
(三)生活型態及內分泌.......................4
(四)其他..................................4

三、肥胖與發炎反應.........................5

第二節、肥胖與食慾調控.....................6

一、食慾調控途徑與物質.....................6
二、瘦體素................................9
(一)瘦體素................................9
(二)瘦體素作用途徑........................9
(三)瘦體素抗性...........................10
(四)瘦體素抗性之機制......................10

第三節、腸道微生物........................13

一、腸道微生物與肥胖......................14
二、影響腸道微生物組成之因素...............15
(一)遺傳.................................15
(二)飲食.................................15
(三)免疫系統..............................16
(四)其他.................................16

三、益生菌與益生質........................17

四、益生菌對於能量衡定之機能性.............18
(一)調節腸道菌相.........................18
(二)影響宿主生理代謝機制..................18

五、Lactobacillus rhamnosus GG..........19

貳、材料與方法.......................... 20

第一節、實驗架構.........................20

第二節 L. rhamnosus GG 對於能量代謝與腸道微生物的影響.....21

一、實驗材料.............................21
(一)益生菌株.............................21
(二)試驗動物.............................21

二、實驗方法.............................21
(一)菌株製備.............................21
(二)試驗設計.............................22
(三)試樣收集與分析........................26
(四)統計分析.............................33

參、結果與討論............................36

第一節、Lactobacillus rhamnosus GG 對於能量代謝之影響...36
一、L. rhamnosus GG 對於食源性肥胖小鼠生長性狀影響.......36
二、L. rhamnosus GG 降低肝臟脂肪堆積...................40

第二節、L. rhamnosus GG 對於瘦體素敏感度之影響..........43
一、L. rhamnosus GG 對於瘦體素敏感度測試影響............43
二、L. rhamnosus GG 影響血液中瘦體素濃度...............44
三、L. rhamnosus GG 對於下視丘中 SOCS3 表現量影響......48

第三節、L. rhamnosus GG 對於腸道微生物之調節...........51
一、L. rhamnosus GG 保護腸道上皮型態完整性.............51
二、腸道微生物組成分析................................54

肆、結論......................................66

伍、參考文獻..................................67

圖目錄

圖1-1肥胖所引起的發炎反應與巨噬細胞浸潤.............5
圖1-2瘦體素於中樞神經系統之訊號傳導途徑與其阻斷物....12
圖1-3腸道微生物與宿主彼此間的影響..................16
圖2-1高脂飼糧誘導小鼠肥胖之試驗設計................24
圖 2-2 瘦體素敏感度試驗設計.......................25
圖 2-3 Illumina 雙邊定序技術流程.................34
圖 3-1 餵食 Lactobacillus rhamnosus GG 十週後對於食源性肥胖小鼠之體重影響........37
圖 3-2 餵食 Lactobacillus rhamnosus GG 十週後對於食源性肥胖小鼠之能量攝取影響.........38
圖 3-3 餵食 Lactobacillus rhamnosus GG 十週後對於食源性肥胖小鼠體重與副睪脂肪重量之影響.....39
圖 3-4 餵食 Lactobacillus rhamnosus GG 十週後小鼠肝臟組織切片圖............................41
圖 3-5 餵食 Lactobacillus rhamnosus GG 十週後小鼠肝臟組織以蘇丹黑染色之切片....42
圖 3-6 餵食 Lactobacillus rhamnosus GG 十週後之瘦體素敏感度測試之小鼠體重變化....45
圖3-7餵食Lactobacillus rhamnosus GG十週後對於小鼠血清中瘦體素濃度之影響.....46
圖3-8餵食Lactobacillus rhamnosus GG十週後對於小鼠血清中TNFα濃度之影響......47
圖 3-9 餵食 Lactobacillus rhamnosus GG 十週後對於小鼠下視丘中 SOCS3 表現量之影響.......50
圖 3-10 餵食 Lactobacillus rhamnosus GG 十週後對於小鼠腸道上皮型態之影響..........52
圖 3-11 餵食 Lactobacillus rhamnosus GG 十週後對於小鼠迴腸之組織型態學之影響..53
圖 3-12 餵食 Lactobacillus rhamnosus GG 小鼠糞便之變性梯度電泳膠片圖..................57
圖 3-13 餵食 Lactobacillus rhamnosus GG 之小鼠糞便經次世代定序後依據編碼所得組別之讀數.......59
圖 3-14 餵食 Lactobacillus rhamnosus GG 之小鼠腸道微生物經次世代定序分析之菌門比例.........60
圖 3-15 餵食 Lactobacillus rhamnosus GG 之小鼠腸道微生物經次世代定序分析之菌綱比例............61
圖 3-16 餵食 Lactobacillus rhamnosus GG 之小鼠腸道微生物中桿菌綱含量.................62
圖 3-17 餵食 Lactobacillus rhamnosus GG 之小鼠腸道微生物中菌門等級之主成分分析.......63
圖 3-18 餵食 Lactobacillus rhamnosus GG 之小鼠腸道微生物中菌綱等級之主成分分析........64
圖 3-19 餵食 Lactobacillus rhamnosus GG 之小鼠腸道微生物中菌目等級之主成分分析........65

表目錄

表 1-1 肥胖的定義..................2
表 1-2 肥胖相關之疾病..............................2
表 1-3 影響中樞神經調控食慾之物質..................8
表 2-1 一般飼糧之能量來源比例......................23
表 2-2 高脂飼糧之能量來源比例......................23
表 2-3 小鼠誘導肥胖模型之試驗組別..........24
表 2-4 引子列表....................................28
表 2-5 序列條碼清單................................35
表 3-1 餵食 Lactobacillus rhamnosus GG 小鼠糞便經品質檢查及過濾後所得次世代定序之序列統計.......................58

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