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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周綠蘋 | |
| dc.contributor.author | Wen-Ching Ho | en |
| dc.contributor.author | 何玟瑾 | zh_TW |
| dc.date.accessioned | 2021-06-16T10:19:49Z | - |
| dc.date.available | 2018-09-24 | |
| dc.date.copyright | 2013-09-24 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60498 | - |
| dc.description.abstract | 肝細胞瘤是全球致死率極高的癌症之一， 現行肝癌治療臨床標靶藥物Sorafenib抑制細胞中酪胺酸激酶的活性消滅癌細胞，但從臨床上成效有限的病例顯示，Sorafenib對癌細胞Raf pathway的長期抑制誘發了其他替代訊息傳遞的活化，造成了Sorafenib的後天抗藥性。
為了探討Sorafenib抗藥性的問題，實驗室先前將肝癌細胞Huh7經過Sorafenib長期的藥物篩選，建立一株Sorafenib藥物敏感度低的細胞株Huh7R，發現其AKT相較於原生態Huh7高度活化。並且從先前差異蛋白質體學分析(Stable Isotope Labeling by Amino acids in Cell culture ，SILAC)，發現一小分子量14kDa凝集素在Huh7R中表現較多。由於Huh7R中AKT的高度活化，與14kDa凝集素的高表現量，我們推測AKT可能是14kDa凝集素的上游調控因子。透過PI3K 抑制劑LY294002的抑制，隨著AKT活化程度的降低可以看到14kDa凝集素的減少，進一步我們加入mTOR抑制劑rapamycin，同樣看到14kDa凝集素的表現下降，另外透過加入氯化亞鈷誘發轉錄因子HIF1α的上升，發現14kDa凝集素基因表現增加。以上結果顯示Huh7R細胞高度活化的AKT pathway 經由mTOR/HIF1α增加14kDa凝集素的基因表現。 為了更進一步了解14kDa凝集素在Huh7R細胞中扮演的角色，我們在Huh7R細胞抑制生成14kDa凝集素，以及在Huh7細胞促進表現14kDa凝集素，發現在Huh7R細胞中減少14kDa凝集素會使Sorafenib藥物感受性增加，細胞遷移與侵襲的能力降低。從以上實驗我們發現Sorafenib抗藥性細胞中誘發高度活化的PI3K/AKT pathway，經由mTOR與HIF1α調控14kDa凝集素的高表現，並影響癌細胞抗藥性與轉移能力。 | zh_TW |
| dc.description.abstract | Hepatocellular carcinoma (HCC) is one of the most serious health problems which results in cancer-related death worldwide. The targeted drug sorafenib has improved the survival of patients with advanced liver cancer. However, accumulatively clinical evidences have suggested that the blockade of Raf signaling might result in unexpected molecular events.
To understand the sorafenib unrevealed mechanism, our lab established a sorafenib-resistant HCC cell line , Huh7R, from Huh7. From SILAC-base (Stable Isotope Labeling by Amino acids in Cell culture) quantitative proteomic analysis, we found a 14kDa-lectin in Huh7R cells. In order to investigate the correlation between sorafenib resistance and the up-regulated lectin in Huh7R, we compared the protein expression levels between Huh7 and Huh7R cells, which established by long term exposure to sorafenib, and observed a dramatic increase of a 14kDa-lectin in Huh7R by Western blotting. We also found that the 14kDa-lectin is an AKT downstream effector which was up-regulated in Huh7R. After treatment with PI3K inhibitor LY294002, we observed the reduction of the protein expression along with the decrease of phosphorylated-AKT. Furthermore, to uncover the signaling pathway, we treated Huh7R cells with mTOR inhibitor rapamycin and hypoxia-mimetic agent cobalt chloride, and we detected the 14 kDa-lectin decreased as mTOR inhibited by rapamycin and the lectin increased after HIF1α was induced. We hypothesized that AKT/mTOR may regulate expression of the 14kDa-lectin through the induction of HIF1α. To further examine the role of 14kDa-lectin in Huh7R, we knockdowned the endogenous 14kDa-lectin in Huh7R and overexpressed 14kDa-lectin in Huh7. Functional analysis indicated that the overexpression of the 14kDa-lectin in Huh7R contributes to sorafenib resistance, cell migration and invasion. In conclusion, 14kDa-lectin could be mediated by PI3K/AKT signaling pathway, and be involved in the acquired resistance to Sorafenib in HCC. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T10:19:49Z (GMT). No. of bitstreams: 1 ntu-102-R00442024-1.pdf: 5449953 bytes, checksum: b12c02cb528570715019dd4ba2de86b6 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 摘要 I
Abstract II 縮寫 IV 第一章 導論 1 第一節 肝癌 1 1.1 肝癌之臨床病理及特徵 1 1.2 肝癌之流行病學 1 1.3 肝癌治療之臨床進展 1 1.3-1 肝癌的分期 1 1.4 Sorafenib之作用機轉和療效 3 1.5藥物的後天抗藥性機制 4 1.6 Sorafenib抗藥性 5 1.7癌細胞侵襲與轉移機制 6 第二節 半乳糖凝集素(Galectin)之研究 8 2.1 Galectin family 8 2.2 Galectin-1之特性 9 第三節 研究動機 11 第二章 實驗材料 12 第一節 肝癌細胞株 12 第二節 藥品 12 第三節 大腸桿菌與質體 14 第四節 抗體 14 第五節 重要儀器 15 第三章 實驗方法 17 第一節 肝癌細胞的培養 17 第二節 蛋白質分析法 17 第三節 細胞生存能力測試(MTT assay) 22 第四節 細胞傷口癒合分析 23 第五節 細胞遷移與侵襲實驗 (transwell assay) 23 第六節 反轉錄反應聚合酶連鎖反應(RT-PCR) 24 第七節 重組蛋白質體之建立 26 第八節 質體轉染(transfection) 30 第九節 類病毒載體感染技術(lentivirus Infection) 30 第十節 細胞核質分離 32 第四章 實驗結果 33 第一節 Huh7及Huh7R細胞之特性分析 33 第二節 Huh7及Huh7R細胞差異表現之蛋白 33 2.1 galectin-1與galectin-3在Huh7與Huh7R之差異表現 33 2.2 galectin-1在細胞內之分佈 34 第三節 Huh7及Huh7R細胞移動侵襲之能力 34 3.1細胞移動及侵襲能力測試 34 3.2移動標誌蛋白在Huh7與Huh7R的表現差異 34 第四節 galectin-1上游調控路徑 35 4.1探討galectin-1是否為PI3K/AKT調控下游蛋白 35 4.2探討galectin-1是否為mTOR調控下游蛋白 35 4.3 探討HIF1α是否為galectin-1上游因子 36 第五節 抑制galectin-1表現以分析其功能 36 5.1 shRNA之篩選與確認 36 5.2抑制galectin-1後之功能性分析 37 5.3抑制galectin-1改變移動標誌蛋白表現 38 第六節 促進galectin-1表現以分析其功能性 38 6.1 galectin-1重組蛋白之建立與確認 38 6.2 galectin-1並未影響Huh7藥物敏感度 39 第五章 討論 40 第一節 Sorafenib抗藥性之探討 40 1.1 PI3K/AKT pathway參與Sorafenib抗藥性 40 1.2 JAK-STAT pathway參與Sorafenib抗藥性 40 1.3缺氧微環境誘發Sorafenib抗藥性 41 1.4細胞膜受器與Sorafenib抗藥性之關係 41 1.5 Epithelial-mesenchymal transition(EMT)與Sorafenib抗藥性之關係 41 第二節 AKT調控之抗藥性機制 42 第三節 HIF1α上游調控機制 43 第四節 galectin-1之上游調控機制 43 第五節 galectin-1於Huh7R之角色 44 5.1 galectin-1之分佈與功能分析 44 5.2 galectins之演化與功能代償 45 第六節 galectin-1 於癌症醫療之應用 46 6.1癌症生物標誌 46 6.2 galectin-1作為生物標誌之可能性 47 第六章 參考文獻 48 圖 54 附錄 74 | |
| dc.language.iso | zh-TW | |
| dc.subject | Sorafenib | zh_TW |
| dc.subject | 肝細胞瘤 | zh_TW |
| dc.subject | 抗藥性 | zh_TW |
| dc.subject | AKT | zh_TW |
| dc.subject | 凝集素 | zh_TW |
| dc.subject | Hepatocellular carcinoma | en |
| dc.subject | Sorafenib | en |
| dc.subject | drug resistance | en |
| dc.subject | lectin | en |
| dc.subject | AKT | en |
| dc.title | 探討受AKT調控之下游表現蛋白於Sorafenib抗藥性之肝腫瘤細胞中所扮演之角色 | zh_TW |
| dc.title | To study AKT downstream effector proteins involved in acquired sorafenib resistance in Huh7 cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中,楊雅倩 | |
| dc.subject.keyword | 肝細胞瘤,Sorafenib,抗藥性,凝集素,AKT, | zh_TW |
| dc.subject.keyword | Hepatocellular carcinoma,Sorafenib,drug resistance,lectin,AKT, | en |
| dc.relation.page | 78 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-16 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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