牛樟芝萜類生理活性探討及其生合成相關基因之分析

Analysis of Antrodia cinnamomea biological activity and the terpinoid biosynthesis related genes

童凱鴻(Kai-Hong Tong)

指導教授 ： 曾顯雄

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02554

摘要

樟芝是台灣特有具抗癌功效之真菌，本實驗欲獲得與樟芝三萜類生合成相關的基因群組之資訊，藉以進行樟芝具抗癌活性三萜類的相關研究，因此使用樟芝菌絲體建構cDNA library，再以經過定序的cDNA序列與NCBI資料庫進行序列比對，並從中篩選出ergosterol biosynthesis protein，Putative mevalonate kinase，Diphosphomevalonate decarboxylase，Farnesyl cysteine carboxyl methyltransferase，squalene epoxidase及Lanosterol 14 α-demethylase等可能與三萜類生合成相關且具顯著相似性之六個基因序列，並以此設計專一引子對，以gDNA為模板進行PCR，所得產物經定序、比對驗証確為上述基因之片段後，以DIG標識作為探針，並與所建構之Fosmid Library進行菌落雜合（colony hybridization），篩檢Fosmid DNA library中可能含有三萜類生合成基因群組的菌株，經實驗證實，上述六個基因並無組成任何基因群組。為進一步了解具抗癌活性的三萜類其生合成途徑，利用含fluconazole、butenafin、compactin等可阻斷三萜類代謝途徑的藥物培養樟芝菌絲，並將菌絲以甲醇萃取以進行HepG2及A549等癌細胞毒性測試與HPLC及TLC之分析，實驗結果顯示，compactin處理並未對樟芝的抗癌產生顯著影響，藉由butenafine處理使樟芝三萜類產生顯著改變，但其抗癌功能仍無明顯變化，而使用fluconazolet處理則使樟芝三萜類及其抗癌活性有明顯下降情形，因此猜測，對HepG2具有特異毒殺作用的三萜類產物可能是位於lanosterol 14α-demethylase下游之代謝產物，為確認此一假設，目前正利用siRNA的技術進行lanosterol 14α-demethylase基因之靜默化，期望藉此確定對HepG2肝癌細胞具有抑制效應的三萜類成分及其生合成路徑。

關鍵字

牛樟芝 ； 三萜類

並列摘要

Antrodia cinnamomea is a fungus endogenous to Taiwan and thought to have anticancer activities. In current study we attempt to analyze and elucidate the genes that possibly involved with terpenoids biosynthesis. Six putative genes：ergosterol biosynthesis protein，Putative mevalonate kinase，Diphosphomevalonate decarboxylase，Farnesyl cysteine carboxyl methyltransferase,squalene epoxidase and Lanosterol 14 α-demethylase , were identified from the A. cinnamomea cDNA library after blastX with nr NCBI database, and primers were designed to amplify these specific genes using gDNA of A. cinnamomea as template by polymerase chain reaction. The obtained gene products were further sequenced, and their identies verified, labeled with DIG and used as probe to screen the constructed A. cinnamomea Fosmid library. The results from colony hybridization revealed these genes were not allocated in a cluster. In order to investigate further on the possibility of terpenoids in anticancer activity, we used flucanazole, butenafin, and compactin, which are thought to block terpenoids biosynthesis pathway at specific steps. The effect of these inhibitors on A. cinnamomea were analyzed using High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) in combination with the bioactivity assay using liver cancer cell Hep G2 and lung cancer cell A549. The results showed both the biosynthesis of terpenoids and the anticancer activity of A. cinnamomea were not significantly affected by compactin; with respect to butenafin, through the terpenoids varied greatly either qualitatively or quantitatively, the anticancer activity was not changed in a regular pattern; while flucanazole markedly reduced the biosynthesis of terpenoids as well as the anticancer activity. Consequently, it is suggested that the downstream terpenoids products mediated by lanosterol 14α-demethylase was mainly ascribed to the anticancer activity. Currently,we are conducting lanosterol 14 α-demethylase gene silencing by using siRNA to prove the actual role played by these gene products in suppression of the liver cancer cell Hep G2.

並列關鍵字

Antrodia cinnamomea ； tritenpene

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