臺灣博碩士論文加值系統

近十年來菇類廣泛的被研究，發現許多的菇蕈類具有生物活性的成分。硫磺菌（Laetiporus sulphureus）是ㄧ種已知的食用菇蕈，生長在根部腐爛的落葉林或針葉樹上，其分佈泛世界分佈，而在台灣常發現於低海拔地區之闊葉樹幹上，為褐腐型木材腐朽菌，所以是ㄧ種容易培養的菇蕈類。在我們實驗室中已確立硫磺菌最佳的生長條件和具有抑制腫瘤生長的生物活性，因此其他的生物活性如抑制轉移、抗氧化及免疫調節的活性將是此篇研究的重點。此外也利用二維電泳的技術分析硫磺菌在不同培養基條件下其菌絲體蛋白質的不同點。
結果顯示硫磺菌的水萃物可以抑制腫瘤細胞（如HeLa）的增生，更進一步使用流式細胞儀偵測硫磺菌引導HeLa細胞進行細胞凋亡的機制，發現隨著濃度的增加與處理時間的加長可提高HeLa細胞進行細胞凋亡的比例，但硫磺菌水萃物對倉鼠卵巢正常細胞CHO-K1較沒影響。另一方面此篇研究發現硫磺菌可以輔助DOX誘導HeLa細胞進行細胞凋亡的效果，但對正常的倉鼠細胞CHO-K1卻沒有明顯的增加。利用二維電泳的技術分析硫磺菌使用PM及SC兩種培養基之菌絲體，發現不同培養基成長的菌絲體其胞內蛋白質明顯的不同，而此兩種培養基所得的菌絲體水萃物對抑制腫瘤細胞的活性也有明顯差異，換句話說，硫磺菌使用不同培養基培養所得菌體其生物活性有顯著的差異。
在抗轉移試驗中，使用gelatin zymograph analysis及RT-PCR研究硫磺菌對於HT1080細胞分泌金屬蛋白酶MMP-2及MMP-9的影響，結果顯示硫磺菌水萃物（PM培養基）可以顯著地抑制MMP-9的蛋白質的分泌與mRNA的表現。在免疫調節試驗，同樣使用RT-PCR技術探討硫磺菌對於主要免疫系統中的cytokines（IL-1、IL-6、TNF-α、iNOS）的影響，結果發現硫磺菌確有調節Raw264.7分泌這些cytokine的功效，但是SC培養基之發酵液可以增加IL-1、IL-6、TNF-α、iNOS的表現但卻無法抑制腫瘤細胞的成長，表示硫磺菌在不同培養條件下其生物活性功能並不一樣。

在抗氧化活性方面發現使用FASCAN來偵測HeLa細胞氧化程度，結果硫磺菌水萃物可抑制H2O2對HeLa細胞內產生氧化的傷害。綜合上述的結果證實硫磺菌確實具有一些生物活性，希望這些發現可對之後研究硫磺菌活性有所助益。

In recent decades, mushrooms have been widely consumed for their favor and nutritive values. However, some of them also have medicinal values. Laetiporus sulphureus is an edible mushroom known to cause heart-rot disease in deciduous trees and conifers. This fungus is widespread and grows in large clusters on live and dead trunks of hard wood species, and is therefore easily cultivated. The optimum cultural medium and some bioactivities such as antitumor for Laetiporus sulphureus had been studied previously. Therefore, other bioactivities, such as anti-metastasis, antioxidant and immuno-modulatory activities, will be further investigated in this study. Moreover, two-dimension electrophoresis analysis was also used to distinguish the different bioactivities of L. sulphureus between different cultural conditions such as different types of nutrients.
The results showed that the growth of tumor cells, such as HeLa cell, can be significantly inhibited by the water extract of mycelium from L. sulphureus, and the mechanism of the growth-inhibition was due to apoptosis pathway evaluated by flow cytometer. On the other hand, the study demonstrated that L. sulphureus significantly enhanced DOX-induced growth inhibitory effect of HeLa cells. However, L. sulphureus did not affect the DOX-induced inhibitory effect on CHO-K1 cells. In addition, two-dimension electrophoresis analysis was used to distinguish the different bioactivities of L. sulphureus in different nutrition sources. The results showed that there were significantly variant protein profiles between high and low anti-tumor activities. The anti-metastasis was conducted by getalin zymograph analysis and RT-PCR assays and the results showed that the secretion of protein and mRNA expression in matrix metalloproteinase (MMP-9) were obviously suppressed while HT1080 cells were treated with the water extract of mycelium from L. sulphureus cultured in PM. The immuno-modulation of L. sulphureus was also evaluated and this study was conducted by RT-PCR assay. The results showed that L. sulphureus cultured in SC can significantly enhance the mRNA expression of IL-1、IL-6、TNF-α and iNOS. Finally, the antioxidant activity from L. sulphureus was assayed by FASCAN and the result showed that the water extract of mycelium from L. sulphureus has higher antioxidant activity than fermentation broth.
All results, in this study, suggested L. sulphureus indeed possess some biology activities. Hopefully, these findings are useful to further studies on bioactivity of L. sulphureus.

中文摘要……………………………………………………………………...5
Abstract…………………………………………………………………….....7
前言…………………………………………………………………………....9
文獻整理……………………………………………………………………...10
ㄧ、硫磺菌…………………………………………………………………...10
二、硫磺菌其生物活性……………………………………………………...10
2.1多醣體…………………………………………….. …………………..10
2.2酚類……………………………………………….. …………………..11
2.3黃酮類…………………………………………….. …………………..11
三、癌症……………………………………………………………………...12
3.1腫瘤……………………………………………….. …………………..12
3.2細胞程式凋亡及細胞壞死……………………….. …………………......12
3.3基質蛋白酵素……………………………………... ………………….13
3.4 Doxorubicin……………………………………….. ………………….14
四、自由基……………………………………………….. …………………15
4.1自由基如何產生……………………………………………………….15
4.2自由基對細胞造成的傷害…………………………………………….16
4.3 抗氧化物質………………………………………... …………………16
五、免疫調節……………………………………………... ………………...17
5.1發炎反應……………………………………………. ………………...18
5.2 Interleukin-1……………………………………….. …………………18
5.3 Interleukin-6……………………………………….. …………………18
5.4 Tumor necrosis factorα……………………………. …………………18
5.5 Inducible nitric oxide synthase……………………. …………………19
5.6. 菇類多醣與免疫系統……………………………... …………………19
研究動機…………………………………………………... …………………21
實驗架構…………………………………………………... …………………22
研究方法…………………………………………………... …………………23
ㄧ、實驗材料……………………………………………... …………………23
(一) 硫磺菌…………………………………………….. …………………..23
(二) 實驗細胞株……………………………………….. …………………..23
(三) 實驗藥品………………………………………….. …………………..23
(四) 實驗儀器………………………………………….. …………………..26
二、實驗方法……………………………………………... ………………….27
(一) 液態發酵培養……………………………………... ………………….27
(二) 硫磺菌生長觀察…………………………………... ………………….27
(三) 樣品製備…………………………………………... ………………….28
(四) 細胞存活率測定…………………………………... ………………..28
(五) Matrix Metalloproteinases………………………...………………...29
(六) 電泳分析法…………………………………………………………...29
(七) 西方墨點法………………………………………. ………………….30
(八) 反轉錄-聚合酵素鏈鎖反應…………………………………………..31
(九) 二維電泳………………………………………….. ………………….33
(十) 流氏細胞儀……………………………………….. ………………….35
(十一) 抗自由基試驗………………………………….. ………………….36
(十二) 粗多醣測定…………………………………….. ………………….38
(十三) Nitrite Assay……………………………………. ………………….39
(十四) 統計分析……………………………………….. ………………….39
結果…………………………………………………... ……………………….40
一、硫磺菌生長條件……………………………………... …………………..40
二、抑癌試驗……………………………………………... …………………..41
2.1癌細胞存活試驗……………………………………. …………………..41
2.2硫磺菌對細胞凋亡之影響…………………………. …………………..42
2.3硫磺菌對DOX所引導的細胞凋亡之影響……….. …………………..42
2.4金屬蛋白酶…………………………………………. …………………..43
2.5硫磺菌在不同培養基其菌絲體在2D圖譜之差異.. …………………..44
三、免疫力試驗………………………………………………………………..45
3.1 Nitrite Assay……………………………………………………………..45
3.2 RT-PCR assay……………………………………….. ………………….45
四、抗氧化試驗………………………………………………………………...46
4.1硫磺菌捕捉DPPH之能力…………………………. …………………..46
4.2使用流式細胞儀偵測細胞內氧化程度…………….. …………………..47
4.3自由基和黑色素的關係…………………………….. …………………..47
討論………………………………………………………………………………49
ㄧ、硫磺菌發酵…………………………………………………………………49
二、硫磺菌細胞存活試驗………………………………………………………49
三、MMP活性試驗………………………………………. ……………………51
四、免疫力試驗………………………………………………………………….52
五、抗氧化試驗………………………………………………………………….53
結論………………………………………………………………………………55
參考文獻…………………………………………………………………………56

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