儘管目前已有許多藥物可用於治療停經後骨質疏鬆症，但是對於低副作用的替代藥物還是有其需求，本研究主要在評估新日本靈芝水萃物(GNES)對RANKL誘發蝕骨細胞生成的效應並探討其作用機制，結果顯示GNES依劑量效應不僅抑制RAW 264.7小鼠吞噬細胞分化成蝕骨細胞，且會抑制骨質吸收坑洞(resorption pit)的生成，在作用機制的結果亦發現，GNES會增加細胞HO-1蛋白表現，降低細胞內活性氧生成及減少NFATc1蛋白進入細胞核內，以HO-1 siRNA抑制細胞HO-1蛋白表現，發現可逆轉GNES抑制蝕骨細胞分化，活性氧生成及核內NFATc1蛋白表現的效應，意指GNES可透過增加HO-1蛋白來減少NFATc1蛋白調控的蝕骨細胞生成。關於調控HO-1蛋白表現的訊息路徑，我們發現GNES會誘導磷酸化Akt蛋白表現，且同時伴隨Nrf2蛋白進入細胞核及受ARE序列驅動的冷光酵素活性，這些結果顯示GNES可藉由調控 Akt-Nrf2-HO-1 路徑降低細胞內活性氧生成，抑制NFATc1核轉位及蝕骨細胞分化。

Although several current therapeutic drugs for postmenopausal osteoporosis are available, there is a need for alternatives with minimal side effects. The aim of this study was to evaluate the effects of water extract of Ganoderma neo-japonicum (GNES) on RANKL- induced osteoclast differentiation and investigated the underlying molecular mechanism(s) of action. GNES not only inhibited osteoclast formation of RAW 264.7 mouse macrophages, but also inhibited resorption pit formation in a dose-dependent manner. Mechanistically, GNES increased heme oxygenase-1 (HO-1) expression, and attenuated intracellular ROS production and nuclear translocation of nuclear factor of activated T-cells NFATc1, the master transcription regulator of osteoclast differentiation. Inhibition of HO-1 expression with HO-1 siRNA could reverse the inhibitory effect of GNES on osteoclast differentiation, ROS production and nuclear translocation of NFATc1, suggesting that HO-1 contributed to the attenuation of NFATc1-mediated osteoclastogenesis by GNES. In regarding to the signaling pathways regulating HO-1 expression, we found that GNES could induce Akt1 phosphorylation at Ser473, coincidentally with nuclear translocation of Nrf2 and enhancement of antioxidant response element (ARE)- driven luciferase activity. These results indicated the inhibitory effect of GNES on osteoclast differentiation is due to the decrease of ROS production, via the enhancement of Akt-Nrf2-HO-1 pathway, which ultimately suppresses NFATc1 translocation to nucleus.

目錄
論文審定書 i
誌謝 ii
摘　要 iii
Abstract iv
目錄 vi
圖次 ix
表次 x
附錄 xi
第一章 文獻回顧 1
第一節 骨母細胞與蝕骨細胞在維持骨質平衡上的角色 1
第二節 骨質疏鬆 1
2-1 簡介 1
2-2 臨床治療骨質疏鬆之藥物及副作用 2
第三節 RANKL 誘導蝕骨細胞的訊息傳遞 3
3.1轉錄因子NFATc1 3
3.2 活性氧(ROS)在RANKL誘導蝕骨細胞生成的角色 4
第四節 第一型血紅素氧化酶(heme oxygenase-1, HO-1)調控蝕骨細胞生成的角色 4
第五節 靈芝組成對HO-1蛋白及蝕骨細胞生成的效應 4
第六節 研究動機與目的 5
第二章 研究方法 6
第一節 儀器及藥品 6
一、 細胞 6
二、 儀器相關設備 6
三、 藥品試劑 7
第二節 實驗方法 8
一、 新日本靈芝水萃物的製備 8
二、 細胞培養 9
三、 蝕骨細胞之分化 9
四、 細胞存活率分析 9
五、 過氧化物質(ROS)含量分析 9
六、 細胞轉染 (Transfection) 10
七、 細胞蛋白萃取 11
八、 西方墨點法分析 (Western blotting) 12
九、 細胞免疫螢光染色 13
十、 mRNA 表現量測定 14
十一、 酒石酸具耐受性酸性磷酸酵素染色法 15
十二、 酒石酸具耐受性酸性磷酸酵素活性測定 15
十三、 重吸收試驗 (Resorption assay) 16
十四、 MMP-9酵素活性染色圖譜 (Gelatin Zymography) 16
十五、 統計分析 16
第三章 結果 17
第一節 GNES合併 RANKL 處理對 RAW 264.7 細胞的毒性效應 17
第二節 GNES對 RANKL 誘導蝕骨細胞生成的抑制效應 17
第三節 GNES對蝕骨細胞骨基質在吸收功能的影響 18
第四節 GNES 抑制蝕骨細胞MMP-9活性的能力 18
第五節 GNES抑制蝕骨細胞基因表現的能力 18
第六節 GNES對RANKL誘導RAW 264.7 細胞NFATc1蛋白轉位的影響 19
第七節 GNES處理對RANKL誘發RAW 264.7細胞活性氧生成的影響 19
第八節 單獨GNES及合併RANKL處理對RAW264.7細胞HO-1蛋白表現的影響 19
第九節 HO-1蛋白在調控GNES抑制蝕骨細胞生成的角色 20
第十節 GNES誘發細胞Nrf2蛋白轉錄活化 20
第十一節 探討MAP kinase, PI3K-Akt訊息傳遞在GNES調控Nrf 2轉錄活化的角色 21
第四章 問題與討論 22
參考文獻 24
圖次 29
表次 47
附錄 49

 
圖次
圖 一、新日本靈芝Ganoderna neojaponicum 29
圖 二、新日本靈芝水萃物製備流程 30
圖 三、評估GNES合併處理RANKL對RAW264.7小鼠巨噬細胞的毒性效應 31
圖 四、GNES對RANKL誘導RAW 264.7細胞分化成蝕骨細胞的影響 32
圖 五、GNES抑制蝕骨細胞再吸收的能力 33
圖 六、GNES抑制蝕骨細胞MMP-9蛋白活性 34
圖 七、GNES對RANKL誘導RAW 264.7細胞分化再吸收功能基因的表現 35
圖 八、GNES對RANKL誘導RAW 264.7 細胞NFATc1蛋白分布的影響 36
圖 九、GNES在抑制RANKL誘發RAW 264.7細胞活性氧生成的效應 37
圖 十、GNES單獨及合併RANKL處理對RAW 264.7細胞HO-1蛋白及基因表現 38
圖 十一、HO-1蛋白在調控GNES抑制蝕骨細胞生成的角色 40
圖 十二、GNES誘發細胞Nrf2蛋白轉錄活化 44
圖 十三、MAP kinase, PI3K-Akt抑制劑對GNES調控HO-1蛋白生成的影響 45
圖 十四、GNES抑制蝕骨細胞分化假說圖 46

 
表次
表 一、siRNA sequence 47
表 二、semi qPCR primer 48

 
附錄
附錄 一 49
附錄 二 50

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