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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張震東 | |
dc.contributor.author | Ya-Yeh Ho | en |
dc.contributor.author | 何雅燁 | zh_TW |
dc.date.accessioned | 2021-06-16T03:01:56Z | - |
dc.date.available | 2020-07-20 | |
dc.date.copyright | 2015-07-20 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-02 | |
dc.identifier.citation | 1. Harvey, A.L., et al., Current strategies for drug discovery through natural products. Expert Opinion on Drug Discovery, 2010. 5(6): p. 559-568.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54522 | - |
dc.description.abstract | 天然藥物源於自然界中存在有藥理活性的天然產物。多種從中草藥萃取出的天然藥物都有抗發炎或抗癌的功效;且大部分的天然物帶有親電子性官能基,可與目標蛋白質共價修飾其親核基側鏈。天然物andrographolide萃取自爵床科穿心蓮 (Andrographis paniculata Nees),其有廣泛性的生藥活性,如抗發炎、抗癌、抗菌等。再者研究報告指出andrographolide以中草藥萃取物或其衍生物均為安全性用藥;其在抗發炎方面功效雖被廣泛探討,然詳細藥理機制尚未明朗。NF-κB轉錄因子在細胞增生與凋亡中,是為重要調控者角色;NF-κB在過度活化的狀態下,會造成慢性發炎而提高癌症風險,且調控多種基因表現,所以此訊號傳導路徑常被視為治療疾病標靶對象。我們分析andrographolide的分子結構並推測其可能為一種共價藥物,藉麥可加成反應方式,andrographolide可被半胱胺酸之側鏈攻擊進行麥可加成反應之位置,與蛋白質形成共價鍵結,進而修飾蛋白質。我們以新的方法學,運用抗andrographolide多株抗體,發現以andrographolide處理細胞後,NF-κB會被andrographolide所修飾。利用子宮頸癌細胞以腫瘤壞死因子 (tumor necrosis factor-α) 來誘導NF-κB活化模式中,我們發現先以andrographolide處理過的細胞,NF-κB從細胞質移轉至細胞核會被抑制。因此,我們推測NF-κB上的某些半胱胺酸側鏈被andrographolide修飾之後,可能會影響NF-κB移轉進核以及活化,也推測其它被修飾的蛋白質也一起影響NF-κB活化。另外,我們也偵測到YB-1亦可被andrographolide所修飾;在肺腺癌細胞中,以順鉑 (cisplatin) 引起基因毒性誘導YB-1活化之模式,發現YB-1進核效果亦受andrographolide所抑制。且以轉化生長因子-β1 (transforming growth factor-β1) 去誘導YB-1表現及上皮間質轉化(epithelial-mesenchymal transition) 之模式,andrographolide能以抑制YB-1表現量且調降上皮間質轉化相關因子表現,得以發現andrographolide有抑制癌細胞移行的潛力而有抗癌功效。 | zh_TW |
dc.description.abstract | Natural compounds are small molecules that are produced by bio-organisms. Many natural products are endowed with electrophilic functional groups that covalently modify nucleophilic residues in specific protein targets. The natural product, andrographolide, is the major active principle isolated from the plant Andrographis paniculata Nees, has been shown with a broad range of biological activities, such as anti-inflammatory, anticancer, and antibacterial. Moreover, both the use of andrographolide in traditional medicine and toxicity data in animals suggest the safety in andrographolide for diversiform clinical conditions. Although some of its anti-inflammatory effects have been investigated, little is known about the mechanisms underlying the pharmacological action of andrographolide. NF-κB transcription factors are critical regulator of apoptosis and proliferation. NF-κB is frequently constitutively activated in patients with chronic inflammatory conditions such as cancer. Accordingly, NF-κB controls multiple genes involved in human diseases rendering the NF-κB signaling pathway a target for therapy. We analyzed the structure of andrographolide and predicted that andrographolide may be a covalent drug. Andrographolide can be attacked by the cysteine residues of the protein through Michael addition to form the covalent bonds. We developed a new methodology by generating anti-andrographolide polyclonal antibody for target identification. Then, we demonstrated that the NF-κB could be modified by andrographolide after the cells were treated with andrographolide. In the TNF-α induced NF-κB activation model, we found that the NF-κB translocation from the cytoplasm to the nucleus can be inhibited in cervical cancer cells. Therefore, we surmised that some cysteine residues of the NF-κB are crucial for the NF-κB translocation and activation. On the other hand, we also showed that the YB-1 could be modified by andrographolide after the cells were treated with andrographolide. In the cisplatin induced YB-1 activation model, we found that the YB-1 translocation from the cytoplasm to the nucleus can be inhibited in pulmonary adenocarcinoma cells. In the TGF-β1 induced YB-1 activation model, overexpression of YB-1 could be inhibited and EMT-regulating genes could be downregulated, when the cells treated with andrographolide. A potent motility inhibitory effect of andrographolide has been demonstrated in pulmonary adenocarcinoma cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T03:01:56Z (GMT). No. of bitstreams: 1 ntu-104-R02b46005-1.pdf: 3809214 bytes, checksum: 8d88e4d11d9f6bfd3530479ef6c053c6 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口委審定書....................................................................................................................... I
誌謝.................................................................................................................................. II 中文摘要......................................................................................................................... III 英文摘要.......................................................................................................................... V 縮寫表............................................................................................................................ VII 目錄................................................................................................................................ IX 第一章 緒論.................................................................................................................. 01 1.1 天然藥物................................................................................................... 01 1.1.1 天然藥物的歷史............................................................................ 01 1.1.2 天然藥物的發展............................................................................ 01 1.2 天然物Andrographolide........................................................................... 02 1.3 小分子藥物之標靶鑑定........................................................................... 03 1.3.1 小分子藥物的發展........................................................................ 04 1.3.2 標靶鑑定方法................................................................................ 04 1.4 共價藥物................................................................................................... 07 1.5 半胱胺酸................................................................................................... 10 1.6 麥可加成反應........................................................................................... 11 1.7 NF-κB訊息傳遞路徑............................................................................... 12 1.7.1 NF-κB轉錄因子............................................................................ 12 1.7.2 TNF-α誘導NF-κB活化之模式................................................... 14 1.7.3 NF-κB訊息傳遞阻斷.................................................................... 15 1.7.4 發炎反應與癌症之關聯性............................................................ 15 1.8 YB-1調控路徑......................................................................................... 16 1.8.1 YB-1調控因子............................................................................... 16 1.8.2 誘導YB-1活化之EMT模式....................................................... 18 1.9 研究動機................................................................................................... 19 第二章 材料與方法...................................................................................................... 20 2.1 材料........................................................................................................... 20 2.2 細胞實驗方法........................................................................................... 20 2.2.1 細胞培養........................................................................................ 20 2.2.2 細胞繼代........................................................................................ 21 2.2.3 藥物處理細胞................................................................................ 21 2.2.4 以TNF-α處理細胞....................................................................... 22 2.2.5 以Cisplatin處理細胞.................................................................... 22 2.2.6 以TGF-β1處理細胞..................................................................... 22 2.3 蛋白質實驗方法....................................................................................... 23 2.3.1 細胞蛋白質樣本製備.................................................................... 23 2.3.2 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳........................................ 23 2.3.3 蛋白質轉印 (Protein Transfer)..................................................... 25 2.3.4 西方墨點法 (Western Blot).......................................................... 25 2.3.5 免疫沉澱 (Immunoprecipitation,IP).......................................... 26 2.3.6 細胞核質分離 (Cytoplasmic/Nuclear Fractionation) .................. 27 2.3.7 抗體................................................................................................ 27 2.3.8 藥物抗原製備................................................................................ 27 2.4 質譜儀分析andrographolide反應產物................................................... 28 2.5 製備p50與p50 C62S蛋白質................................................................. 30 2.5.1 聚合酶連鎖反應............................................................................ 31 2.5.2 表現質體pET21b-p50與pET21b-p50 C62S製備...................... 32 2.5.3 質體抽取........................................................................................ 33 2.5.4 質體轉型作用................................................................................ 33 2.5.5 蛋白質表現與純化........................................................................ 34 2.6 細胞存活率分析 (MTT assay)............................................................... 34 2.7 免疫螢光染色法 (Immunofluorescence staining)................................... 35 2.8 細胞侵襲分析 (Cell invasion assay) ..................................................... 36 第三章 結果.................................................................................................................. 37 3.1 Andrographolide對HeLa細胞存活率影響........................................ 37 3.2 Andrographolide對蛋白質的訊號修飾會隨著pH值變動.................. 37 3.3 Iodoacetamide修飾蛋白質對andrographolide之標靶影響................. 38 3.4 Andrographolide於HeLa細胞中修飾蛋白質之訊號模式................... 38 3.5 Andrographolide可以對p65與p50進行修飾....................................... 39 3.6 Andrographolide對TNF-α誘發NF-κB進細胞核之現象影響............ 39 3.7 Andrographolide共價修飾p50 C62...................................................... 40 3.8 質譜分析法找尋andrographolide修飾的蛋白質................................. 41 3.9 Andrographolide對cisplatin誘發YB-1進細胞核之現象影響............ 42 3.10 Andrographolide對TGF-β1誘發細胞之EMT現象影響..................... 42 第四章 討論.................................................................................................................. 45 4.1 藥物的專一性......................................................................................... 45 4.2 以抗體搜尋藥物的目標蛋白................................................................. 46 4.3 Andrographolide抑制NF-κB訊號傳導路徑的機制........................... 47 4.4 Andrographolide抑制YB-1所調控的EMT機制............................... 47 第五章 結語.................................................................................................................. 49 第六章 實驗結果圖表.................................................................................................. 50 圖1 不同濃度的andrographolide對HeLa細胞之存活率影響.................. 50 圖2 Andrographolide在有或無胎牛血清培養液中處理細胞之差異性..... 52 圖3 Andrographolide修飾蛋白質之訊號會隨pH值升高而增加............... 53 圖4 Iodoacetamide阻礙andrographolide修飾蛋白質................................ 54 圖5 將andrographolide對HeLa細胞處理不同濃度與時間的訊號模式.. 55 圖6 NF-κB的兩個次單元體p65、p50均可被andrographolide修飾....... 58 圖7 隨著andrographolide處理細胞濃度提升而明顯抑制 NF-κB進入細胞 核............................................................................................................. 60 圖8 p50 C62為 andrographolide重要的修飾位置...................................... 63 圖9 藉質譜分析法找尋andrographolide修飾的蛋白質............................. 65 圖10 藉andrographolide處理PC14細胞而明顯抑制 YB-1進入細胞核.....67 圖11 藉andrographolide處理PC14細胞濃度提升而抑制EMT現象........ 69 表1 藉質譜分析法找尋andrographolide修飾的蛋白質............................. 72 表2 藉質譜分析法找尋andrographolide修飾p50的胺基酸位置............. 76 第七章 參考文獻.......................................................................................................... 77 第八章 附錄.................................................................................................................. 89 附圖1 天然藥物的來源與作用機製............................................................ 89 附圖2 Andrographolide 結構與可被親和基攻擊的位置........................... 90 附圖3 以親和性為基礎 (affinity-based target identification) 之蛋白質鑑 定分析方法………………………………………………………… 91 附圖4 以活性為基礎 (activity-based proteome profiling,ABPP) 之蛋白質 鑑定分析方法……………………………………………………… 92 附圖5 小分子藥物標靶鑑定方法:正向選擇性與負向選擇性之差異性...93 附圖6 Michael reaction之反應機制……………………………………… 94 附圖7 NF-κB家族的5個成員與其所含的domain……………….…….... 95 附圖8 NF-κB之兩種主要訊號傳遞路徑………………………………… 96 附圖9 誘導NF-κB活化的訊號因子以及NF-κB活化的基因…..……….. 97 附圖10 Andrographolide的使用劑量與安全性…………………………… 98 附圖11 NF-κB由兩個次單元體p65、p50所組成,可被Andrographolide 修飾………………………………………………………………… 99 附圖12 Andrographolide處理細胞而明顯抑制NF-κB進入細胞核…...... 101 附圖13 Andrographolide處理細胞而明顯抑制細胞侵襲能力………….. 103 | |
dc.language.iso | zh-TW | |
dc.title | 探討天然物穿心蓮內酯對癌細胞之影響 | zh_TW |
dc.title | The Effects of A Natural Product Andrographolide in Cancer Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳宏文,張茂山,張?仁 | |
dc.subject.keyword | 穿心蓮內酯,共價藥物,麥可加成反應,NF-κB,YB-1, | zh_TW |
dc.subject.keyword | Andrographolide,covalent drug,Michael addition,NF-κB;YB-1, | en |
dc.relation.page | 103 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-02 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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