當身體受到外來物質感染(如細菌內毒素LPS)或組織受傷，將刺激巨噬細胞活化，並釋出免疫介質，如一氧化氮(NO, nitric oxide)、介白素-6 (IL-6, interleukin-6)、腫瘤壞死因子α (TNF-α, tumor necrosis factor alpha) 與基質金屬蛋白酶(MMPs, matrix metalloproteinases)等，幫助病原體的清除，並藉由免疫細胞的調控，維持免疫功能的平衡。坊間廣泛使用的狼尾草(Pennisetum purpureum S.)生機飲食，被宣稱具健康促進之免疫調節的作用，然而尚無科學之根據，因此本研究探討狼尾草台畜二號酒精或水萃取物對於RAW264.7巨噬細胞免疫調節的影響，以及對於LPS存在下抗發炎的影響。狼尾草台畜二號市售粉末，以99.8 %酒精或二次蒸餾水室溫萃取24小時後的萃取物，於LPS (100 ng/ml)存在或不存在下及狼尾草酒精萃取物(0.625, 1.25, 2.5 mg/ml)或水萃取物(5, 10, 20 mg/ml)處理RAW264.7巨噬細胞24小時後，收集培養基及細胞進行分析。結果顯示，酒精萃取物可增加MMP-9的活性，且可降低LPS所誘導的NO、IL-6、TNF-α生成，且伴隨著iNOS蛋白質、CD14 mRNA表現量的降低。然而對LPS所誘導的NF-κB DNA結合活性無影響。狼尾草水萃物內含nitrite，並可增加巨噬細胞IL-6、TNF-α的產生，然而對iNOS蛋白質表現量以及免疫相關基因iNOS、CD14、TLR4 mRNA無顯著影響，此外水萃物無法對LPS所誘導的NO、IL-6、TNF-α產生無顯著影響。總言之，狼尾草具調節免疫的能力，其中酒精萃取物可增加RAW 264.7巨噬細胞MMP-9活性，水萃物可增加IL-6、TNF-α的產生。同時酒精萃取物可降低LPS誘導RAW 264.7巨噬細胞的NO、IL-6、TNF-α、iNOS蛋白質與CD14 mRNA表現量，而具抗發炎作用。因此認為狼尾草之水萃物具有增加健康者的免疫能力之潛力；酒精萃取物則具有降低發炎者的發炎介質釋出之潛力。

Napiergrass Taishigrass No.2 (Pennisetum purpureum S.) is one of livestock forage species in Taiwan. Recently, napiergrass is considered as one of natural organic diet materials and used to consume for health promotion. However, the effects of napiergrass on health promotion has not been investigated. The aim of this study was to explore the effects of napiergrass Taishigrass No.2 on the modulation of immunity in RAW 264.7 macrophages and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages inflammation. Napiergrass Taishigrass No.2 freeze dry powder was bought from Baoyuan company (Taichung, Taiwan). Napiergrass Taishigrass No.2 was extracted by 99.8% ethanol or water in room temperature for 24 h and condensed for cell culture used. Cells were treated with in the presence or absence of LPS (100 ng/ml) and ethanol extracts of napiergrass (0, 0.625, 1.25, 2.5 mg/ml) or water extracts (0, 5, 10 20 mg/ml) for 24 h. Ethanol extracts of napiergrass increased MMP-9 activities and MMP-9 mRNA expression, but had no effect on MMP-2 activities, implying that napiergrass may have immunomodulatiotory effects. Ethanol extracts of napiergrass suppressed nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS) protein expression and CD14 mRNA expression in LPS-induced RAW 264.7 macrophages, implying that napiergrass may have anti-inflammation activities. However ethanol extracts of napiergrass couldn’t decrease LPS-induced NF-κB DNA binding activity in RAW 264.7 macrophages. In the presence or absence LPS-induced inflammation condition, water extracts of napiergrass increased NO concentration in a dose dependent manner. Under cell-free condition, water extracts of napiergrass showed similar NO results as cell condition. Water extracts had no effect to iNOS protein expression, MMP-9 activities, MMP-2 activities and inflammatory associated mRNA expression. However water of napiergrass extracts increased IL-6 and IL-6 production in RAW 264.7 macrophages. Overall, the results obtained showed that napiergrass have immunomodulatiotory effects by increasing MMP-9 activities, IL-6 and TNF-α production on RAW 264.7 macrophages.

目錄
摘要 I
Abstact III
致謝 IV
目錄 VI
圖目次 VIII
表目次 IX
第一章 緒論 1
第二章 文獻回顧 2
第一節 免疫防禦系統 2
第二節 巨噬細胞與免疫調控 6
第三節 Lipopolysaccharide (LPS)刺激巨噬細胞之發炎反應 10
第四節 免疫介質 14
4-1. 細胞激素 14
4-2. iNOS與NO 15
4-3. Matrix metalloproteinases (MMPs)基質金屬蛋白酶 19
第五節 免疫與疾病 21
第六節 免疫與飲食 23
第七節 狼尾草 25
第三章 研究目的 26
第四章 材料方法 27
第一節 試劑 27
第二節 儀器 30
第三節 狼尾草台畜二號樣本配製 31
第四節 細胞培養 32
第五節 實驗流程 32
第六節 分析項目 34
6-1. 狼尾草酒精萃取物葉綠素a及b含量測定 34
6-2. 狼尾草酒精與水萃取物酚類化合物coumaric acid含量測定 35
6-3. 細胞存活率 36
6-4. 一氧化氮(NO)濃度 37
6-5. 細胞激素IL-6、TNF-α濃度 38
6-6. 基質金屬蛋白酶MMP-9、MMP-2活性 39
6-7. 西方墨點法 40
6-8. Real-time polymerase chain reaction (RT-PCR)分析 42
6-9. NF-κB DNA結合活性 45
第七節 統計方法 46
第五章 結果 47
第一節 狼尾草萃取物之葉綠素a、b及coumaric acid的含量 47
第二節 狼尾草酒精萃取物對細胞存活率的影響 50
第三節 狼尾草酒精萃取物對細胞NO生成的影響 52
第四節 狼尾草酒精萃取物對細胞iNOS蛋白質及mRNA表現量的
影響 54
第五節 狼尾草酒精萃取物對細胞IL-6與TNF-α生成影響 56
第六節 狼尾草酒精萃取物對細胞MMP-9、MMP-2活性與MMP-9
mRNA表現量的影響 58
第七節 狼尾草酒精萃取物對細胞CD14與TLR4 mRNA表現量的
影響 61
第八節 狼尾草酒精萃取物對細胞之NF-κB DNA結合活性的影響 63
第九節 狼尾草水萃取物對細胞存活率的影響 65
第十節 狼尾草水萃取物對細胞NO生成的影響 67
第十一節 狼尾草水萃取物對細胞iNOS蛋白質及mRNA表現量的
影響 69
第十二節 狼尾草水萃取物對細胞IL-6與TNF-α生成影響 71
第十三節 狼尾草水萃取物對細胞MMP-9、MMP-2活性與MMP-9
mRNA表現量的影響 75
第十四節 狼尾草水萃取物對細胞CD14與TLR4 mRNA表現量影響 78
第六章 討論 80
第一節 狼尾草酒精萃取物降低發炎可能之活性成 80
第二節 狼尾草酒精萃取物對MMP的影響可能之原因 84
第三節 狼尾草酒精萃取物對免疫介質影響的可能機制 85
第四節 狼尾草水萃取物對巨噬細胞NO影響的可能之原因 87
第五節 狼尾草水萃取物對巨噬細胞IL-6、TNF-α的影響及機制 89
第六節 狼尾草中之硝酸鹽 90
第七節 狼尾草與生機飲食 91
第七章 結論 92
第八章 參考文獻 93

圖目次
圖一、先天性及後天性免疫系統 4
圖二、巨噬細胞的分化、分布及活化 8
圖三、巨噬細胞對於發炎與傷口癒合的調節 9
圖四、革蘭氏陰性菌之LPS脂多醣 12
圖五、Lipopolysaccharide (LPS)誘導巨噬細胞發炎之訊號傳遞路徑 13
圖六、Nitric oxide (NO)的生成 17
圖七、促發炎細胞激素與一氧化氮產生的關係 18
圖八、實驗流程圖 33
圖九、Coumaric acid之酒精及水溶液的標準曲線 48
圖十、狼尾草酒精萃取物對RAW 264.7細胞存活率的影響 51
圖十一、狼尾草酒精萃取物對RAW 264.7細胞NO生成的影響 53
圖十二、狼尾草酒精萃取物對RAW 264.7細胞iNOS蛋白質(A)及mRNA (B)表現量的影響 55
圖十三、狼尾草酒精萃取物對RAW 264.7細胞IL-6 (A)、TNF-α (B)生成的影響 57
圖十四、狼尾草酒精萃取物對RAW 264.7細胞MMP-9、MMP-2活性影響 59
圖十五、狼尾草酒精萃取物對RAW 264.7細胞MMP-9 mRNA表現量活性的影響 60
圖十六、狼尾草酒精萃取物對RAW 264.7細胞CD14 (A)、TLR4 (B) mRNA表現量的影響 62
圖十七、狼尾草酒精萃取物對RAW 264.7細胞之NF-κB DNA結合活性的影響 64
圖十八、狼尾草水萃取物對RAW 264.7細胞存活率的影響 66
圖十九、狼尾草水萃取物對RAW 264.7細胞(A)或不含細胞狀況下(B) NO的影響 68
圖二十、狼尾草水萃取物對RAW 264.7細胞iNOS蛋白質及iNOS mRNA表現量的影響 70
圖二十一、狼尾草水萃取物對RAW 264.7細胞IL-6、TNF-α生成的影響 73
圖二十二、狼尾草水萃取物與含等量的NaNO2對IL-6 (A)與TNF-α (B)的影響 74
圖二十三、狼尾草水萃取物對RAW 264.7細胞MMP-9、MMP-2活性影響 76
圖二十四、狼尾草水萃取物對RAW 264.7細胞MMP-9 mRNA表現量影響 77
圖二十五、狼尾草水萃取物對RAW 264.7細胞CD14、TLR4 mRNA表現量的影響 79

表目次
表一、免疫細胞上主要辨識的接受器以及其對應的配體 5
表二、引子序列 44
表三、狼尾草酒精及水萃取物葉綠素a、b及coumaric acid的含量 49

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