臺灣博碩士論文加值系統

近年來樟芝(Antrodia camphorata)已成為健康食品之來源，其生長於台灣特有之牛樟樹上，具抗癌、抗腫瘤及免疫調節等作用。本研究分別以樟芝的濾液與菌絲多醣體連續餵食BALB/c小鼠不同時間點，觀察是否具有抗發炎及增強免疫力之功效。分析小鼠服食樟芝的兩種多醣體對其免疫細胞，如巨噬細胞、T細胞、樹突狀細胞所產生之作用。研究結果顯示，長期餵食小鼠樟芝的兩種多醣體，再腹腔注射LPS使其產生發炎反應，經流式細胞儀測定分析，發現脾臟巨噬細胞上之Trem-1HI的表現受到抑制而下降，證實樟芝具有抗發炎的效果；樹突狀細胞上CD86呈現下降的現象；樹突狀細胞上之CD40HI、CD8αHI的表現均有增加趨勢；巨噬細胞上CD8αHI的表現也增加。其他協同刺激分子，如T細胞上ICOS的表現量增加與抗原呈獻細胞上CD86的表現下降，主要原因可能是CTLA-4參與調控平衡而抑制之。以RT-PCR觀察促發炎因子，發現細胞激素如TNF-α、IFN-γ、IL-4、IL-6、IL-10、IL-12有被抑制的現象。此外，利用Real-time PCR偵測Trem-1、IL-1β、iNOS的細胞激素之含量變化；Western blot觀察Trem-1的蛋白質表現，發現長期餵食後均有不錯的抗發炎功效。本論文研究結果顯示服食樟芝的兩種多醣體具有提升免疫力及抗發炎的功效，並從中探討各種免疫分子間相關之作用機轉。未來可考慮將所使用的細胞分子表面標記作為藥物作用篩選的標的物，進一步測試其他具有潛力的中草藥。

Antrodia camphorata grows on characterized Cinnamomum kanehirai in Taiwan, it has become the source of health food in recent years. A. camphorata has anticancer, antitumor and immunomodulatory properties. In this study, BALB/c mice were fed with polysaccharides obtained from fermented culture broth and mycelia of A. camphorata at different time points to investigate the anti-inflammatory action and the immunomodulatory effect. When mice were administrated with two kinds of polysaccharides for a period of time and then injected LPS into mice, we found that Trem-1HI expression on macrophages is inhibited measured by flow cytometry. This result indicates that A. camphorata has anti-inflammatory action. The expressions of CD40HI and CD8αHI markers on DCs and CD8αHI marker on macrophages increased apparently. However, the CD86 marker expressed on DCs decreased sharply, this result may regulated by the activation of CTLA-4 and ICOS from T cells. The TNF-α、IFN-γ、IL-4、IL-6、IL-10 and IL-12 mRNA cytokines inhibited obviously after feeding with A. camphorata by RT-PCR. In addition, we use real-time PCR to detect Trem-1、IL-1β and iNOS cytokine content and profile; also, to observe the expression of Trem-1 protein with western blot. The results demonstrated that two kinds of polysaccharides from A. camphorata possesses effects on both immunomodulation and anti-inflammation. These markers may serve as targets for further understanding the molecular basis and efficacy of A. camphorata and other Chinese herbal medicines in the future.

目 錄
頁次
目錄………………………………………………………………… I
表目錄………………………………………………………………III
圖目錄………………………………………………………………IV
中文摘要……………………………………………………………VII
英文摘要……………………………………………………………VIII

第一章 緒論……………………………………………………… 1
一、樟芝兩種萃取物成份…………………………………...1
二、生物體之免疫調節機制………………………………..2
三、發炎作用指標………………………………………………...5
四、研究動機……………………………………………………...8
第二章 材料與方法……………………………………………….9
一、實驗材料……………………………………………………...9
(一) 樟芝萃取物之製備………………………………..9
(二) 實驗動物……………………………………………….9
二、實驗設計…………………………………………………….10
三、實驗方法…………………………………………………….10
(一) 小鼠脾臟細胞之取得………………………………...10
(二) 以流式細胞技術分析偵測小鼠脾臟免疫細胞
表面標幟………………………………………….11
(三) 以反轉錄聚合酶連鎖反應（Reverse Transcription-Polymerase Chain Reaction ；RT-PCR）和即時定量聚合酶連鎖反應(Real-time quantitative PCR；Real-time PCR)分析相關細胞
激素之mRNA表現量…….....………...………..13
(四) 以西方轉漬法測定專一性蛋白表現…………...19
第三章 結果......................................................................................24
一、樟芝多醣體對小鼠脾臟免疫細胞之調控…………...24
(一) 免疫細胞表面標記測定…………………….…...24
(二) 以RT-PCR分析細胞激素之mRNA表現……….…32
(三) 以Real-time PCR分析細胞激素之mRNA表現.….34
(四) 小鼠脾臟細胞Trem-1 Protein之表現……...….36
第四章 討論…………………………….………………………….37
參考文獻……………………………………………………………..46














表 目 錄
頁次
表一、細胞激素寡核苷引子…………………………………….….59
表二、樟芝萃取物成分分析(%)…………………………………....60
表三、傳統PCR與即時定量PCR之比較………………………...61



















圖 目 錄
頁次
圖一、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中DC細胞上CD11c＋的表現百分比，以
流式細胞儀分析後之結果……………………………………62
圖二、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中DC細胞上CD40的表現百分比，以流
式細胞儀分析後之結果………………………………………64
圖三、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中DC細胞上CD8α的表現百分比，以流
式細胞儀分析後之結果………………………………………66
圖四、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中DC細胞上CD86的表現百分比，以流
式細胞儀分析後之結果………………………………………68
圖五、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中巨噬細胞上CD11b的數量表現百分比，
以流式細胞儀分析後之結果…………………………………70
圖六、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中巨噬細胞上CD8α的表現百分比，以流
式細胞儀分析後之結果………………………………………72
圖七、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中巨噬細胞上Trem-1的表現百分比，以流
式細胞儀分析後之結果………………………………………74
圖八、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中T細胞上表現CD4＋的細胞數量百分比，
以流式細胞儀分析後之結果………………………………….76
圖九、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中T細胞上表現CD8α＋的細胞數量百分比，以流式細胞儀分析後之結果……………………………….…78
圖十、 餵食樟芝發酵培養液與菌絲體後於各時間點犧牲BALB/c
小鼠之脾臟細胞中T細胞上ICOS的表現百分比，以流式
細胞儀分析後之結果…………………………………….…...80
圖十一、 以RT-PCR分析BALB/c小鼠經餵食樟芝發酵培養液各
時間點，腹腔注射LPS，16~18小時後犧牲，其脾臟細胞mRNA之表現………………………………………………..82
圖十二、 以Image quant 分析比較陽性對照組與餵食樟芝發酵培
養液各時間點之TNF-α、IFN-γ、IL-12 mRNA表現量….83
圖十三、 以RT-PCR分析BALB/c小鼠經餵食樟芝發酵培養液各
時間點，腹腔注射LPS，16~18小時後犧牲，其脾臟細胞mRNA之表現………………………………………….…….84
圖十四、 以Image quant分析比較陽性對照組與餵食樟芝發酵培
養液各時間點之IL-4、IL-6、IL-10 mRNA表現量……....85
圖十五、 以RT-PCR分析BALB/c小鼠經餵食樟芝菌絲體各時間
點，腹腔注射LPS，16~18小時後犧牲，其脾臟細胞
mRNA之表現…………………………………………..……86
圖十六、 以Image quant 分析比較陽性對照組與餵食樟芝菌絲體各
時間點之TNF-α、IFN-γ、IL-12 mRNA表現量…….……..87
圖十七、以RT-PCR分析BALB/c小鼠經餵食樟芝菌絲體各時間
點，腹腔注射LPS，16~18小時後犧牲，其脾臟細胞
mRNA之表現………………………………………………..88
圖十八、以Image quant分析比較陽性對照組與餵食樟芝菌絲體各
時間點之IL-4、IL-6、IL-10 mRNA表現量………………89
圖十九、以real-time PCR分析BALB/c小鼠經餵食樟芝發酵培養液
和菌絲體不同時間點後，腹腔注射LPS，16~18小時後犧牲，
其脾臟細胞Trem-1 mRNA之表現量……….……………….90
圖二十、以real-time PCR分析BALB/c小鼠經餵食樟芝發酵培養液
和菌絲體不同時間點後，腹腔注射LPS，16~18小時後犧牲，
其脾臟細胞IL-1β mRNA之表現量………………………….91




圖二十一、以real-time PCR分析BALB/c小鼠經餵食樟芝發酵培養
液和菌絲體不同時間點後，腹腔注射LPS，16~18小時後犧牲，其脾臟細胞iNOS mRNA之表現量……………….92
圖二十二、以Western blot分析餵食樟芝發酵培養液，經腹腔注射
LPS 16~18小時後犧牲之BALB/c小鼠脾臟細胞中
Trem-1 protein之表現………………………………………93
圖二十三、以Western blot分析餵食樟芝菌絲體，經腹腔注射
LPS 16~18小時後犧牲之BALB/c小鼠脾臟細胞中
Trem-1 protein之表現………………………………………95

附錄一、Real-time PCR之相關數據………………………………97
附錄二、圖示APC與T細胞之間的調控機制………………………...98

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