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標題: | 基因體技術於藥物作用與基因調控之應用 Application of Genomic Approaches on Discovering Drug Effects and Gene Regulation |
作者: | Pin-Hao Ko 柯品豪 |
指導教授: | 賴亮全(Liang-Chuan Lai) |
關鍵字: | 基因體技術,品質管控,基原辨識,藥物作用,基因調控, Genomic Approaches,quality control,plant species identification,drug Effects,gene regulation, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 基因體技術係指可大量偵測生物體中基因序列、表觀或表現之技術,並有多方面的應用。而藥物的研發程序可由天然植物開始,爾後到成分分離、再到純化物的下游機轉。在天然植物過去多以外觀及組織辨識,此判定方式較為主觀;天然植物煎煮液的品質則以層析法判定特定指標成分,此判定方式無法代表處方中的全部成分;藥物作用在生物體後還會引發很多下游路徑;而純化物的作用另牽涉目標蛋白辨識,這些方面基因體技術多可有所助益。因此本篇論文的主軸概念,是要以基因體技術的方式,建立上述項目的應用程序。同時特定基因的下游機轉也可以基因體技術加以入手,故一併討論。
首先,在植物藥方面,為建立準確品質管控,植物基原辨識以DNA序列為依據,利用植物內轉錄間隔區(internal transcribed spacer, ITS)區段作為標的。本論文以黨參、黃耆等生藥為例作為檢測目標,並定序ITS區段以確認這些生藥基原正確。其次,因生藥煎煮液中含有多種成分,雖然層析法可將其成分分離,但卻無法對全部訊號做解讀,因此本論文利用主成分分析(principal component analysis)程式將訊號降維並視覺化,以確認水煎劑型的品質穩定。最後,這些生藥水萃物於肝癌細胞株可抑制生長,但牽涉的下游路徑很多,因此本論文採用微陣列技術加以判斷,並判定這些藥物的效果是牽涉於細胞生長等路徑。 其次,在小分子化合物方面,因為目標蛋白是藥物作用的重要步驟,而目前對目標蛋白的辨識並沒有公認快速良好的方式。因此本論文與沈雅敬教授實驗室合作,先進行有機合成取得一系列純化合物,並選定其中對細胞生長抑制效果佳的1-(9'-methyl-3'-carbazole)-3,4-dihydro-β-carboline (MCDC)做進一步測試。因此項化合物為全新合成藥物,故先以電腦虛擬鍵合程式idTarget預測可能標的蛋白為macrophage migration inhibitory factor (MIF)。其次,在轉換到細胞模式中進行測試時,需選擇內生性低表現MIF的細胞株來實驗,因此本論文利用篩選平台CellExpress篩選U251細胞株進行胞內鍵合測試。最後,用微陣列技術則判斷該藥物下游的機轉牽涉於細胞週期、細胞存亡與細胞組裝。 基因體技術在本論文中的第三個運用是研究基因的作用機轉。之前我們實驗室發現SEMA5A在非吸菸女性的肺癌組織中的表現量較低,然而以基因造成此肺癌的機轉仍不明,因此本論文中以此基因為例子進行檢測。在轉殖SEMA5A的肺癌細胞株中,微陣列被用來檢測並發現該基因牽涉於細胞生長等路徑;而焦磷酸定序技術的實驗則說明SEMA5A在A549及H1299等肺癌細胞株中的甲基化程度較MRC-5及BEAS-2B等正常肺細胞株來的明顯。 總結來說,本篇論文將一部分基因體技術應用於生藥、合成藥物及特定基因的研究,或可增加相關領域之進展。 Genomic technologies are those can detect gene sequences, epigenetic appearances or gene expressions on a genome-wide scale. Drug development typically starts with investigation of natural plants and a separation of its inclusions, and then the definition of the bio-mechanisms thereof. However, natural plants are typically identified subjectively by their appearance or histological characteristics. The quality of natural plant decoction is determined by chromatography with few specific indicators, which are not able to represent all components in the decoction. To improve on these issues, genomic technologies, that is, technologies that can detect gene sequences, epigenetic appearances or gene expressions on a genome-wide scale, can be applied. These technologies can also be used to identify target proteins for a specific compound or discover downstream pathways in living organisms after drug treatment. This dissertation describes the application of genomic technologies on these issues, from the identification of nature plants to the functionality of genes. First, the quality control of pharmacologic mechanism in botanic research is complicated, because plant contains numerous inclusions. For plant species identification, sequences of the internal transcribed spacer (ITS) regions were used to validate the identification of Codonopsis pilosula, Astragalus membranaceus and other plants. Next, since principal component analysis (PCA) could visualize all signals in high performance liquid chromatography (HPLC), quality control of aqueous extracts from these herbs was carried out through HPLC-PCA. Lastly, microarray was used to examine the expression of downstream genes in HepG2 cells applied with the aqueous extracts of these crude drugs. The functions of the genes expressed were involved in cellular development, growth, and proliferation. Next, regarding discovering the effects of small molecule drugs, the most crucial but difficult step is to identify target protein whose biological functions has been initiated. In this dissertation, a new compound, 1-(9'-methyl-3'-carbazole)-3,4-dihydro-β-carboline (MCDC) was synthesized by cooperating with Prof. Ya-Ching Shen’s lab. The macrophage migration inhibitory factor (MIF) was predicted as a potential target protein by the idTarget program. Next, in order to choose suitable cells for intracellular bonding test, the CellExpress system was used, which identified U251 cells with low endogenous expression levels of MIF as the cell of choice. Lastly, the expressions of downstream genes caused by MCDC were examined by microarray, and the expressive genes were found to be involved in cell cycle, cell death and cellular assembly. The third application of genomic approach in this dissertation was to explore the related pathways caused by the gene, SEMA5A. Previous studies in our lab showed that SEMA5A had a lower expression in lung cancerous tissues from non-smoking women. However, the detailed mechanism of this phenomenon is still unknown. Therefore, in this dissertation, SEMA5A regulated genes were analyzed by microarray and their functions were shown to be involved in cell signaling and gene expression. Furthermore, pyrosequencing revealed that methylation status of SEMA5A in lung cancer cell lines, A549 and H1299, was higher than that of normal cell lines, MRC-5 and BEAS-2B. In summary, by using genome technologies, this dissertation illustrates three applications: identification of the mechanisms and cellular functions in Codonopsis pilosula, Astragalus membranaceus, identification of the target protein and cellular functions of genes of MCDC, as well as the roles that the genes SEMA5A have intra cellularly. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74922 |
DOI: | 10.6342/NTU201904138 |
全文授權: | 有償授權 |
顯示於系所單位: | 生理學科所 |
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