臺灣博碩士論文加值系統

不同種類靈芝對大腸癌細胞(HT-29)的影響
研究生:孫聖傑 指導教授:王海龍 博士
元培醫事科技大學醫學檢驗生物技術碩士班
摘要
靈芝是一種食用中藥，在亞洲地區長期使用為天然保健食品，其中含有許多藥理特性。許多研究證實靈芝可調節免疫功能、抑制腫瘤生長、抗氧化、保護肝臟、降血糖、抗過敏等功能，且無毒性。本研究目的在分析新竹新埔地區採集之野生靈芝菌株，分別代名為靈芝1號菌、靈芝2號菌、靈芝A菌、靈芝B菌、靈芝N菌、靈芝新埔菌、標準菌株赤芝CCRC36021等菌株發酵產物之生物活性。研究方法將7株靈芝株分別以液態培養28天，用分子篩分離不同分子量(大於100KDa、介於30kDa至100kDa、小於30kDa)之培養液，以不同濃度加入大腸癌細胞(HT-29)中培養。以MTT試驗測定細胞存活率及流式細胞儀觀察細胞週期。結果顯示，靈芝株分子量大於100KDa與對照組相比，36021標準菌株對HT-29大腸癌細胞的生長抑制效果較為顯著。在分子量介於30kDa至100kDa與對照組比較，靈芝N菌和標準菌株赤芝CCRC36021的培養液較其他菌株對HT-29大腸癌細胞株的生長抑制較為顯著效果。
以靈芝N菌靈芝株培養液和標準菌株赤芝CCRC36021對 HT-29大腸癌細胞株進行細胞週期實驗，結果顯示：靈芝N菌靈芝株培養液在分子量大於100KDa與分子量介於30kDa至100kDa大小之培養液與對照組相比，在G1期均有明顯停滯HT-29大腸癌細胞株生長的效果。
標準菌株赤芝CCRC36021培養液在分子量大於100kDa的培養液中，對於HT-29大腸癌細胞株之細胞週期在G1期有增加的效果；分子量介於30kDa至100kDa之培養液與對照組相比，在G1期有明顯停滯HT-29大腸癌細胞株生長的效果。
本研究結果顯示新竹新埔地區採集之野生靈芝N菌株多醣體，更有抑制癌細胞生長的效果，在G1期有明顯停滯HT-29大腸癌細胞株生長的效果。


關鍵字:靈芝、多醣體

The interference of different Ganoderma lucidum in colorectal cancer cell(HT-29)

Student: Sheng-Chieh Sun Adviser:. Hai-Long Wang
Graduate Institute of Medical Laboratory Science and Biotechnology,
Yuanpei University of Medical Technology

Abstract

Ganoderma lucidum is a type of edible herbs that has been used as natural health-promoting food in Asia for a long time, with several pharmacological features. Many researches have proven that Ganoderma lucidum is nontoxic and can regulate the immunological functions, inhibit tumor growth, increase resistance to oxidation, protect the liver, decrease blood glucose and combat allergies. This study aimed to analyze the biological activities of Ganoderma strain fermented products derived from wild Ganoderma lucidum strains collected in the Xinpu region of Hsinchu County, and code named Ganoderma No. 1, Ganoderma No. 2, Ganoderma A, Ganoderma B, Ganoderma N, Ganoderma Xinpu and Standard Ganoderma CCRC36021. In this study, the seven types of Ganoderma lucidum strains were respectively subject to liquid culture for 28 days. Molecular sieves were used to separate the culture solution of different molecular weights (those greater than 100kDa, between 30kDa and 100kDa, and those smaller than 30kDa), and then different concentrations of the culture solution were added to the colon cancer cells (HT-29) for culturing. MTT assay was adopted to determine the cell survival rate, while a flow cytometer was used to observe the cell cycle. The results showed that Standard Ganoderma CCRC36021 exhibited the most significant inhibition of HT-29 colon cancer cell growth in the 100kDa+ group versus Control comparison; while Ganoderma N and Standard Ganoderma CCRC36021 had the most significant inhibition of HT-29 colon cancer cell growth compared to the other strains in the 30kDa to 100kDa group versus Control comparison.
In the cell cycle experiment involving the culture solution of Ganoderma N and Standard Ganoderma CCRC36021 on HT-29 colon cancer cells, the results showed that the culture solution of Ganoderma N with molecular weight greater than 100kDa and that between 30kDa and 100kDa significantly retarded the growth of HT-29 colon cancer cell strains in G1 phase, compared to the Control group.
The culture solution of Standard Ganoderma CCRC36021 with molecular weight greater than 100kDa showed the effect of increasing the cell cycle of HT-29 colon cancer cell strains in G1 phase, while the culture solution with molecular weight between 30kDa and 100kDa significantly retarded the growth of HT-29 colon cancer cell strains in the G1 phase, compared to the Control group.
This study shows that the polysaccharides of wild Ganoderma N strains collected in Xinpu Township, Hsinchu County, have better inhibition performance in relation to the growth of cancer cells and obviously retarded the growth of HT-29 colon cancer cell strains in the G1 phase.


Keywords: Ganoderma lucidum, polysaccharides

口試論文審定書....................................................................................... Ⅰ
誌謝............................................................................................................Ⅱ
中文摘要.................................................................................................... Ⅲ
英文摘要.................................................................................................... Ⅳ
目錄.............................................................................................................Ⅵ
圖目錄........................................................................................................ Ⅸ
表目錄………………………………………………………………………………………………..……..XI
第一章 前言.................................................................................................1
1.1 靈芝簡介................................................................................................1
1.2 靈芝的生物活性成分及藥理特性........................................................1
1.2.1 多醣體.................................................................................................1
1.2.2 三帖類.................................................................................................2
1.2.3 核酸.....................................................................................................3
1.2.4 LZ-8 蛋白............................................................................................3
1.2.5胺基酸...................................................................................................3
1.3大腸癌......................................................................................................4
1.3.1大腸癌的病因.......................................................................................4
1.3.2大腸癌的治療.......................................................................................5
1.3.3大腸癌的預防.......................................................................................6
1.4研究動機..................................................................................................6
第二章 研究方法及材料.............................................................................7
2.1實驗材料..................................................................................................7
2.2研究方法與步驟......................................................................................8
2.2.1靈芝培養...............................................................................................8
2.2.2多醣體萃取方法...................................................................................8
2.2.3多醣體分子量分離...............................................................................8
2.2.4液態多醣體含量測定....................................8
2.3大腸癌細胞..............................................................................................9
2.4 MTT Assay..............................................................................................9
2.5流式細胞儀............................................................................................10
第三章 研究結果.......................................................................................11
3.1七株靈芝之靈芝培養液中多醣體含量................................................11
3.2不同濃度之靈芝培養液對HT-29細胞株MTT細胞活性表現.............11
3.2.1靈芝培養液30%濃度之細胞存活率變化..........................................11
3.2.2靈芝培養液20%濃度之細胞存活率變化..........................................11
3.2.3靈芝培養液10%濃度之細胞存活率變化..........................................12
3.2.4靈芝培養液1%濃度之細胞存活率變化............................................12
3.3不同濃度靈芝培養液對HT-29細胞株於細胞週期的表現..................13
第四章 結論與討論....................................................................................14
第五章 圖表與說明....................................................................................15
參考文獻......................................................................................................31




























圖目錄
頁次
圖一: 不同靈芝株在濃度30%對細胞存活率的變化...............................15
圖二: 不同靈芝株在濃度20%對細胞存活率的變化...............................15
圖三: 不同靈芝株在濃度10%對細胞存活率的變化...............................16
圖四: 不同靈芝株在濃度1%對細胞存活率的變化.................................16
圖五: 不同濃度N菌株培養液對HT-29細胞株之細胞週期細胞變化.........................................................17
圖六: 不同濃度N菌株培養對HT-29細胞株之G1期細胞變化..................................................................................................................18
圖七: 不同濃度N菌株培養液對HT-29細胞株之G2期細胞變化..................................................................................................................18
圖八: 不同濃度N菌株培養液對HT-29細胞株之S期細胞變化..................................................................................................................19
圖九: 不同濃度N菌株培養液對HT-29細胞株之細胞週期細胞變化..................................................................................................................20
圖十: 不同濃度N菌株培養液對HT-29細胞株之G1期細胞變化..................................................................................................................21
圖十一: 不同濃度N菌株培養液對HT-29細胞株之G2期細胞變化..................................................................................................................21
圖十二: 不同濃度N菌株培養液對HT-29細胞株之S期細胞變化..................................................................................................................22
圖十三: 不同濃度36021標準菌株培養液對HT-29細胞株之細胞週期細胞變化..................................................................................................................23
圖十四: 不同濃度36021標準菌株培養液對HT-29細胞株之G1期細胞變化..................................................................................................................24
圖十五: 不同濃度36021標準菌株培養液對HT-29細胞株之G2期細胞變化..................................................................................................................24
圖十六: 不同濃度36021標準菌株培養液對HT-29細胞株之S期細胞變化..................................................................................................................25
圖十七: 不同濃度36021標準菌株培養液對HT-29細胞株之細胞週期細胞變化..................................................................................................................26
圖十八: 不同濃度36021標準菌株培養液對HT-29細胞株之G1期細胞變化..................................................................................................................27
圖十九: 不同濃度36021標準菌株培養液對HT-29細胞株之G2期細胞變化..................................................................................................................27
圖二十: 不同濃度36021標準菌株培養液對HT-29細胞株之S期細胞變化..................................................................................................................28
表目錄
頁次
表一: 不同靈芝株培養28天多醣體含量(mg/ml) .....................................29
表二:實驗流程與架構.................................................................................30

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