臺灣博碩士論文加值系統

近年來，中草藥因成分天然與低副作用，故廣被研究以為治療疾病之用。和厚朴酚，帶有雙酚結構，存在於玉蘭科植物的毬果，葉以及樹皮內，已被廣泛應用於中醫藥。文獻指出，其能抗氧化、抗發炎、抗癌與抗菌，故可做為健康食品的補充。但其真正作用機轉，目前仍不清楚。在此，我們將針對和厚朴酚的抗癌與抗發炎進行探討，我們以IV5細胞做為和厚朴酚抗癌的研究對象，另以RAW264.7細胞進行抗發炎研究。IV5部分，Honokiol有效的抑制細胞生長與移行能力，western分析也發現Honokiol對Src的活性 (Src pY416) 以及FAK的活性 (FAK pY397、FAK pY861) 的抑制程度與其劑量及作用時間皆有關。特別的是，Honokiol亦可抑制由LPS所引起的巨噬細胞的位移。相對於Src與FAK在LPS刺激巨噬細胞的角色，Honokiol有效的抑制Src與FAK的活性。有趣的是，在myeloid中專一表達的Src family kinases (Lyn、Fgr、Hck)，其表現量在LPS刺激下並未受到任何影響，Honokiol也對其無任何作用。這些結果顯示，Honokiol能有效的減弱LPS所引起的巨噬細胞活動，主要是透過調控Src表現量與活性。

Mounting evidence indicates that honokiol possesses anti-tumorigenic and anti-inflammatory property. However, the underlying mechanisms are still vague. In this study, we demonstrated that honokiol inhibited proliferation and migration of v-Src transformed fibroblasts (IV5) in a dose- and time-dependent manner. Western analysis revealed that honokiol treatment suppressed the activity of Src (as reflected by Src Pi-Y416) and FAK (as reflected by FAK Pi-Y397) in IV5 cells. Interestingly, honokiol also exerted inhibitory effect on LPS-mediated macrophage migration. Consistent with the involvement of Src and FAK in macrophage mobility in response to LPS stimulation, honokiol suppressed LPS-elicited upregulation of Src and the activation of both Src and FAK in a dose- and time-dependent fashion. In contrast, the expression of myeloid Src family kinases (i.e. Lyn, Fgr and Hck) was not affected by honokiol in LPS-exposed macrophages. These results implicate that through downregulation of the expression and activity of Src, honokiol could effectively impair LPS-initiated macrophage locomotion.

目錄
英文摘要 (Abstract in English) I
中文摘要 (Abstract in Chinese) II
第一章 序論 (Introduction) 1
第一節 免疫系統 (immune system) 2
第二節 巨噬細胞 (macrophages) 4
第三節 Toll-like receptors (TLRs) 及Lipopolysaccharides (LPS) 5
第四節 iNOS (inducible nitric oxide synthase) 7
第五節 Src kinase 9
第六節 Honokiol 11
第二章 研究動機 (Motive) 12
第三章 實驗材料與方法 (Materials and Methods) 14
第一節 實驗材料 15
一、 細胞: 15
二、 試劑: 15
三、 抗體 16
四、 實驗儀器與耗材 18
五、 實驗藥品 19
第二節 實驗方法 21
一、 細胞培養 (Cell culture): 21
二、 細胞收集 (Lysates): 23
三、 蛋白質濃度測定 24
四、 蛋白質電泳 (SDS-PAGE): 24
五、 西方點墨法 (western blot analysis): 26
六、 細胞移行分析 (cell migration assay): 27
七、 iGEMDOCK 28
第四章 結果 (Results) 30
一、 Honokiol 可抑制 IV5細胞生長能力 31
二、 Honokiol 可抑制 IV5整體蛋白tyrosine phosphorylation 32
三、 Honokiol 可抑制 IV5 細胞的v-Src Tyr-416 phosphorylation 33
四、 Honokiol 可抑制 IV5 FAK Tyr-397與Tyr-861 phosphorylation 34
五、 Honokiol 可抑制IV5細胞移行能力 35
六、 Honokiol 可抑制LPS所誘導巨噬細胞之移行能力 36
七、 Honokiol 可抑制LPS所誘導巨噬細胞之整體蛋白tyrosine 37
八、 Honokiol 可抑制巨噬細胞內LPS誘導的Src expression與 38
九、 Honokiol 可抑制不同TLRs所誘導巨噬細胞移行能力 39
十、 分子模擬Honokiol與ANP ligand-binding pocket鍵結 40
十一、 分子模式預測Honokiol與 Src之間的交互作用 41
十二、 分子模擬Honokiol與Src間鍵結1 42
十三、 分子模擬Honokiol與Src間鍵結2 43
第五章 討論 (Discussion) 44
第六章 參考文獻 (Reference) 49
第七章 圖表 (Figure) 60
Fig. 1. Growth inhibition of IV5 cells by honokiol. 61
Fig. 2. Honokiol treatment leads to the reduction of total protein tyrosyl phosphorylated in IV5. 62
Fig. 3. Honokiol treatment reduces the level of Tyr-416 phosphorylation Src. 63
Fig. 4. Honokiol treatment reduces the level of Tyr-397 and Tyr-861 phosphorylation FAK. 64
Fig. 5. Honokiol reduces IV5 cells migration motility. 65
Fig. 6. Honokiol inhibits LPS induce macrophage migration. 66
Fig. 7. Honokiol treatment reduces the total proteins tyrosyl phosphorylation in LPS-stimulated macrophages. 67
Fig. 8. Honokiol treatment reduces the level of Src in LPS-stimulate macrophages. 68
Fig. 9. Honokiol reduces poly I:C-, PGN-, and CpG-induce Src expression and migration in macrophages. 69
Fig. 10. Analysis docking conformations of the Honokiol in the ANP ligand-binding pocket of src protein (PDB ID: 2SRC) using iGEMDOCK. 70
Fig. 11. Prediction of potential Honokiol-binding pockets in src protein using iGEMDOCK. 71
Fig. 12. Analysis computational conformation of Honokiol with whole Src protein using iGEMDOCK. 73
Fig. 13. Analysis computational conformation of Honokiol in Src protein with best docking position using iGEMDOCK. 74

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