臺灣博碩士論文加值系統

樟芝 (Taiwanofungus，學名Taiwanofungus camphoratus)為台灣特有的真菌類，具有抗發炎、抗氧化、血管舒張以及抗B型肝炎病毒等生理活性，然而，其詳細抗發炎成分與機制尚未明確。在本研究論文中，我們使用野生型樟芝、液態培養樟芝和固態培養樟芝的冷水、甲醇和熱水三種萃取物，並檢驗它們在微小膠質細胞中的抗發炎作用，並進而探討樟芝萃取物對於預防腦部退化性疾病的產生可能扮演的角色。微小膠質細胞為腦中的免疫細胞，扮演著監控與防禦的角色。然而活化的微小膠質細胞會大量表現誘生型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS)並使iNOS的代謝物一氧化氮 (NO) 大量產生，iNOS和NO的生合成對於發炎反應和腦部退化性疾病的形成扮演重要的角色。首先，以不同濃度及種類的樟芝萃取物處理EOC13.31微小膠質細胞，再以脂多醣 (LPS)和干擾素- (IFN-)誘導EOC13.31微小膠質細胞發炎，並偵測iNOS的表現。西方點墨法及反轉錄-聚合酶連鎖反應顯示，在EOC13.31微小膠質細胞內，野生型樟芝、液態培養樟芝和固態培養樟芝的冷水、甲醇和熱水萃取物皆可抑制LPS和IFN- 所誘導的iNOS蛋白質和mRNA表現；然而，在我們所測試的九種樟芝萃取物中，野生型樟芝甲醇萃取物抑制效果最明顯，且隨著濃度增加其抑制iNOS和TNF-表現的效果越顯著。另外，我們進一步證實由野生型樟芝甲醇萃取物中分離出的CKJ-35-2抑制LPS和IFN- 所誘導的iNOS蛋白質表現的效果最顯著。為了確認其中的抑制機轉，西方點墨法之結果顯示野生型樟芝甲醇萃取物可抑制EOC13.31微小膠質細胞內LPS和IFN- 所誘導p-STAT1 (signal transducer and activator of transcription-1)、p-ERK (extracellular signal- regulated protein kinases)、p-JNK (c-Jun NH2-terminal protein kinases)的活性，除此之外野生型樟芝甲醇萃取物也可抑制 IB(inhibitor B)的降解和p-IB的活性。當我們改以-Amyloid (25 M)刺激細胞發炎時，野生型樟芝甲醇萃取物同樣隨濃度增加而抑制iNOS表現的效果越顯著。綜合以上結果，證實樟芝甲醇萃取物為一種抗發炎物質，可有效抑制腦部微小膠質細胞中iNOS和TNF-的表現，進而抑制微小膠質細胞發炎的情形。此篇研究報告結果顯示樟芝萃取物調節iNOS的特性也許在預防腦部細胞的發炎反應及腦部退化性疾病的形成扮演重要的角色。

Taiwanofungus (Taiwanofungus camphoratus), a medicinal mushroom in Taiwan, is reported to provide several therapeutic benefits including anti-inflammation, antioxidation, vasorelaxation, antihepatitis B surface antigen activities, but the underlying molecular mechanisms are not well understood. In this study, we used three cultured types of wild type, Liquid-state culture, and solid-state culture Taiwanofungus extracts, and to examine their anti-inflammatory effect in microglia cells and the possible role in the protection of neurodegenerative diseases. Since microglia is the immune cell in the brain, it plays a defense and monitoring role. However, activated microglia expresses high level of inducible nitric oxide synthase (iNOS) and its metabolite nitric oxide (NO) that significantly contributes to the pathogenesis of neurodegenerative diseases. First, EOC13.31 microglia was treated with various kinds of Taiwanofungus extracts and lipopolysaccharide (LPS) and interferon- (IFN-), then detected the iNOS expression. Western blot and RT-PCR analysis demonstrated that cold water, methanol, and hot water extracts of wild type, Liquid-state culture, and solid-state culture Taiwanofungus significantly blocked the protein and mRNA expression of iNOS in LPS and IFN- -activated microglia. Among nine extracts of Taiwanofungus, methanol extracts of wild type Taiwanofungus was the most potent inhibitor on the iNOS and TNF- expression, and the inhibition by a dose-dependant manner. The CKJ-35-2 fraction of methanol extracts of wild type Taiwanofungus, effectively inhibited the iNOS expression. To clarify the mechanisms involved, methanol extracts of wild type Taiwanofungus was found to inhibit the LPS and IFN--induced the phosphorylation of extracellular signal-regulated protein kinases (ERK), c-Jun NH2-terminal protein kinases (JNK) and signal transducer and activator of transcription-1 (STAT1). Moreover, methanol extracts of wild type Taiwanofungus also inhibited NF-B activition through the prevention of inhibitor B (IB) degradation and phosphorylation. These results suggests that modulation of iNOS expression by Taiwanofungus extracts may be important in the prevention of inflammation and neurodegenerative diseases.

目錄(Contents) I
中文摘要(Abstract in Chinese) VII
英文摘要(Abstract in English) IX
縮寫表(Abbreviations) XI
緒論( introduction ) 1
壹、腦神經組織與發炎 1
1、腦神經組織介紹 1
2、腦神經組織發炎反應 2

貳、神經退化性疾病與乙型類澱粉質 (-amyloid) 3
1、神經退化性疾病 3
2、乙型類澱粉質 4
3、乙型類澱粉質及相關發炎反應 5
4、iNOS與微小膠質細胞發炎、-Amyloid以及腦部退化性疾病的關係 6

叁、訊號傳遞相關路徑 7
1、有絲分裂-活化蛋白質激酶路徑 （mitogen-activated protein kinase pathways, MAPK pathways） 7
2、Transcription factors (NF-B)： 8
3、訊號傳遞路徑文獻探討： 9

肆、樟芝 (Taiwanofungus) 11
1、簡介 11
2、樟芝的成分 13
3、樟芝生理機能性 13
實驗材料與方法 16
壹、實驗材料 16
1、樟芝 16
2、藥品試劑 16
3、儀器 18
貳、細胞培養實驗: 19
1、EOC13.31: 19
2、EOC細胞培養液的製備: 19

叁、微小膠質細胞的培養: 20

肆、西方墨漬法 (Western-blot) 20
1、細胞蛋白質之收集 20
2、蛋白質含量測定 20
3、1級抗體及2級抗體 21
4、實驗方法 22

伍、反轉錄酶-聚合酶連鎖反應 (Reverse Transcriptase -Polymerase Chain Reaction, RT-PCR) 23
1、萃取核糖核酸 (RNA) 23
2、反轉錄 (Reverse Transcription) 23
3、聚合酶連鎖反應 (Polymerase chain reaction) 24
4、核酸電泳 25

陸、乙型類澱粉質(β-Amyloid)配製 25
1、Aβ25-35 25

柒、動物實驗: 25
1、實驗動物： 25
2、實驗方法： 25

捌、統計分析 26

附錄 27

實驗結果(Results) 29
壹、野生型樟芝的冷水、甲醇以及熱水萃取物均可抑制微小膠質細胞中LPS 和IFN- 所誘導iNOS蛋白質的表現 29

貳、野生型樟芝、液態培養樟芝和固態培養樟芝的甲醇萃取物可抑制微小膠質細胞中LPS 和IFN- 所誘導iNOS的表現 29

叁、野生型樟芝甲醇萃取物隨著濃度增加，抑制微小膠質細胞中LPS 和IFN- 所誘導iNOS蛋白質和mRNA表現的效果越顯著 30

肆、野生型樟芝甲醇萃取物隨著濃度增加而抑制微小膠質細胞中LPS 和IFN- 所誘導TNF- mRNA表現的效果越顯著 30

伍、野生型樟芝甲醇萃取物與微小膠質細胞型態變化的關係 31

陸、野生型樟芝甲醇萃取物內五種成分與微小膠質細胞中LPS 和IFN- 所誘導iNOS的表現的關係 31

柒、野生型樟芝甲醇萃取物與微小膠質細胞中LPS 和IFN- 所誘導的訊號傳遞路徑的關係 32

( 1 )野生型樟芝甲醇萃取物與LPS 和IFN- 所誘導的p-STAT1活性表現的關係 32
( 2 ) 野生型樟芝甲醇萃取物與LPS 和IFN- 所誘導的p-ERK活性表現的關係 33
( 3 ) 野生型樟芝甲醇萃取物與LPS 和IFN- 所誘導的p-JNK活性表現的關係 33
( 4 ) 野生型樟芝甲醇萃取物與LPS 和IFN- 所誘導的p-p38活性表現的關係 34
( 5 ) 野生型樟芝甲醇萃取物與LPS 和IFN- 所誘導的IB蛋白及活性表現的關係 35

捌、野生型樟芝甲醇萃取物隨著濃度增加而抑制微小膠質細胞中-amyloid所誘導iNOS蛋白質表現的效果越顯著 35

玖、野生型樟芝甲醇萃取物和固態培養樟芝甲醇萃取物可抑制老鼠耳朵的發炎水腫現象 36
討論(Discussion) 37
壹、實驗結果摘要與結論 37

貳、實驗結果討論 38
1、樟芝 38
2、微小膠質細胞發炎 39
3、微小膠質細胞發炎訊號傳遞路徑 40

結果圖表 44
Fig. 1 Effects of cold water extracts, methanol extracts and hot water extracts of wild type Taiwanofungus on the LPS & IFN- -mediated iNOS protein expression in EOC13.31 microglia. 44

Fig.2 Effects of cold water extracts, methanol extracts and hot water extracts of wild type Taiwanofungus on the LPS & IFN- -mediated iNOS protein expression in EOC13.31 microglia. 45

Fig.3 Effects of wild type, Liquid-state culture and solid-state culture methanol extracts of Taiwanofungus on the LPS & IFN- -mediated iNOS protein expression in EOC13.31 microglia. 46

Fig.4 Effects of wild type, Liquid-state culture and solid-state culture methanol extracts of Taiwanofungus on the LPS & IFN- -mediated iNOS protein expression in EOC13.31 microglia. 47

Fig.5 Effects of wild type, Liquid-state culture and solid-state culture methanol extracts of Taiwanofungus on the LPS & IFN- -mediated iNOS mRNA expression in EOC13.31 microglia. 48

Fig.6 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated iNOS protein expression in EOC13.31 microglia. 49

Fig.7 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN--mediated iNOS mRNA expression in EOC13.31 microglia. 50

Fig.8 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated TNF- mRNA expression in EOC13.31 microglia. 51

Fig.9 Effect of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated morphology change in EOC13.31 microglia. 52

Fig.10 Effects of 5 fractions of methanol extracts of Taiwanofungus on the LPS & IFN- -mediated iNOS expression in EOC13.31 microglia. 53

Fig.11 Effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated p-STAT1 expression in EOC13.31 microglia. 54

Fig.12 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated p-STAT1 expression in EOC13.31 microglia. 55

Fig.13 Effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated p-ERK expression in EOC13.31 microglia. 56

Fig.14 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated p-ERK expression in EOC13.31 microglia… 57

Fig.15 Effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated p-JNK expression in EOC13.31 microglia. 58

Fig.16 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated p-JNK expression in EOC13.31 microglia. 59

Fig.17 Effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated p-p38 expression in EOC13.31 microglia. 60

Fig.18 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated p-p38 expression in EOC13.31 microglia. 61

Fig.19 Effects of p38 inhibitor and methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated iNOS protein expression in EOC13.31 microglia. 62

Fig.20 Effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN- -mediated IB expression in EOC13.31 microglia. 63

Fig.21 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated p-IB expression in EOC13.31 microglia. 64

Fig.22 Dose-dependent effects of -amyloid -mediated iNOS protein expression in EOC13.31 microglia. 65

Fig.23 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the -amyloid- mediated iNOS protein expression in EOC13.31 microglia. 66

Table.1 Inhibitory effects of methanol extracts of wild type and solid-state culture type Taiwanofungus on TPA-induced ear edema in Mouse.ab 67

Fig.24 Inhibitory effects of methanol extracts of wild type and solid-state culture type Taiwanofungus on TPA-induced ear edema in Mouse. 68

Fig.25 The proposed signaling transduction mechanisms involved in LPS & IFN- -mediated iNOS expression in EOC13.31 microglia…………….67

Fig.S1 Effects of cold water extracts, methanol extracts and hot water extracts of wild type Taiwanofungus on the LPS & IFN- -mediated iNOS and HO-1 protein expression in EOC13.31 microglia. 70

Fig.S2 Dose-dependent effects of methanol extracts of wild type Taiwanofungus on the LPS & IFN-- mediated iNOS ans HO-1 protein expression in EOC13.31 microglia. 71
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