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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡丰喬(Feng-Chiao Tsai) | |
dc.contributor.author | Sin-Yu Liang | en |
dc.contributor.author | 梁芯瑜 | zh_TW |
dc.date.accessioned | 2021-06-17T02:27:08Z | - |
dc.date.available | 2025-08-17 | |
dc.date.copyright | 2020-09-01 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68605 | - |
dc.description.abstract | 頭頸部鱗狀上皮細胞癌在台灣有相當高的發病率及致死率,且容易復發及轉移,然而目前臨床治療上並沒有很大的突破。因此,找到更加有效的治療標的是必要的。纖維母細胞生長因子受器(fibroblast growth factor receptor, FGFR)參與了胚胎發育、血管新生、傷口癒合等重要的生理功能,且在細胞增生、分化以及移動上都扮演了重要的角色。不僅如此,近年來有研究顯示異常的FGFR和許多癌症的發生及其病程發展相關,但FGFR在頭頸癌病程上所扮演的角色目前仍然沒有被釐清。過去實驗室利用FGFR抑制劑AZD4547來觀察對口腔鱗狀上皮癌細胞遷移的影響,發現在沒有FBS並加入纖維母細胞生長因子(fibroblast growth factor 1, FGF1)作刺激的環境下給予AZD4547,細胞移動速度會顯著的下降;然而在有胎牛血清(fetal bovine serum, FBS)的環境下給予AZD4547,卻無法達到抑制細胞遷移的效果。進一步確認FBS中的何種物質導致此現象的發生,發現到在沒有FBS並加入表皮生長因子(epidermal growth factor, EGF)的環境下再給予AZD4547,能夠看到類似在FBS中的結果。藉此,我們推論表皮細胞生長因子受器(epidermal growth factor receptor, EGFR)和FGFR 訊息之間可能存在交互作用。 為了進一步解釋EGFR與FGFR 訊息傳遞之間的交互作用,我們透過單細胞追蹤的技術去探討其中一個受器結構與訊息傳遞的存在與否,對另一個受器在細胞遷移上造成何種影響。從實驗結果我們觀察到EGF可以回復FGFR抑制劑的抑制效果,卻無法回復敲減FGFR2的抑制效果,藉此推論FGFR受器結構的存在與否,對於和EGFR之間的交互作用是重要的。 我們提出三個可能的機轉模型來說明EGFR和FGFR之間的互動:receptor level model、internalization model以及transactivation model。Receptor level mode假設細胞中的FGFR平時可能透過某種機制去促進EGFR的表現或抑制EGFR的降解,但從qPCR 和Western blot實驗結果中看到,FGFR2 knockdown後EGFR的mRNA和蛋白質表現量並沒有減少的情形發生。我們所提出的另一個假說為Internalization model,細胞中的受體彼此之間會競爭進入endosome的機會,所以當FGFR2被knockdown時,就會增加EGFR進入endosome的機會。但從免疫螢光染色實驗可以觀察當FGFR2 knockdown時,進入細胞內的EGFR表現量會相對減少。這兩個假說都和我們的實驗結果不相符。 最後需要釐清的假說是transactivation model,了解FGFR2受體存在與否,能否改變EGFR的下游訊息傳遞。我們藉由免疫螢光染色實驗觀察EGFR下游訊息傳遞的目標: pERK (RAS/MPAK路徑)、pAKT(PI3K/AKT路徑)和pMLC2(PLC路徑),發現這三個路徑活化對於FGFR受器結構的存在具有不同的依存度,其中PI3K/AKT路徑最容易受到影響,在FGFR敲減時會減少AKT磷酸化,FGFR過度表現wild type或kinase-dead mutant,都會增加AKT磷酸化,證明FGFR受器結構對於EGFR下游訊息傳遞是重要的。不僅如此,我們還注意到,FGFR敲減會減少EGFR受器磷酸化,EGFR和FGFR的交互作用在受器階層就已經發生。 之前的實驗結果指出:當FGFR的訊息傳遞接頭蛋白(adaptor protein) FGFR substrate 2FRS2)被敲減時可以輕微減弱EGF在FGFR抑制劑上的回復效果,且從Western blot結果發現到敲減FRS2時可以讓細胞中的pERK表現量減少。接下來我們希望確認FGFR結構,是否會影響其帶來FRS2的量,藉此影響EGFR的下游訊息傳遞。而我們仍需再透過其他實驗來釐清並加強此部分的推論,期望能對EGFR和FGFR交互作用機制有更充分的瞭解後,對頭頸癌的治療提供指引與方向。 | zh_TW |
dc.description.abstract | Head and neck squamous cell carcinoma (HNSCC) cause high metastatic rate, recurrence rate and mortality in Taiwan, but there is still no breakthrough in clinical therapy. Subsequently, we aimed toward finding new therapeutic targets for HNSCC and focused on the fibroblast growth factor receptor (FGFR), which is important in the malignancy cell proliferation, differentiation, and migration. When we utilized FGFR inhibitor AZD4547 to treat SAS (HNSCC cell line) in cell migration assays, fibroblast growth factor (FGF1)-stimulated SAS migration was repressed. However, AZD4547 failed to inhibit SAS migration when fetal bovine serum (FBS) was present. Besides, the presence of EGF (epidermal growth factor) also abolished the inhibitory effect of AZD4547 on SAS migration. Taken together, we suggested that EGFR (epidermal growth factor receptor) and FGFR signaling interaction occurred during cell migration. To explain the cross talk between EGFR and FGFR signaling, we used single-cell tracking to clarify how FGFR or EGFR inhibition modulates migration behavior when the EGFR or FGFR signaling take place. We discovered that EGF rescued the FGFR inhibition of HNSCC cell migration, but EGF could not rescue the effect of FGFR2 knockdown. Thus, we confirmed that interaction between EGFR and FGFR happens through physical interactions between receptors. To clarify how EGFR interacts with FGFR signaling, we proposed three hypothetical models: receptor level model, internalization model and transactivation model. For receptor level model, FGFR in cells may somehow increase EGFR expression. But FGFR2 knockdown did not decrease EGFR levels in qPCR and western blot. In internalization model, receptors will compete with each other for entering endosome, so when FGFR2 was knocked down, it will increase the chance for EGFR to enter endosome. We observed that the distribution of EGFR to the endosome decreased in FGFR2 knockdown group in immunofluorescence staining results. The above data showed these two models were not correct. The transactivation model was verified by understanding whether or that the physical presence of the FGFR2 receptor changed the EGFR downstream signaling. We examined EGFR signaling by immunofluorescence staining of pERK (RAS/MPAK pathway), pAKT (PI3K/AKT pathway), and pMLC2 (PLC pathway). And we found that the activation of these three pathways had different extent of dependence to the FGFR receptor structures. PI3K/AKT pathway was the most susceptible, because FGFR knockdown reduced AKT phosphorylation, and FGFR wild type or kinase-dead mutant overexpression increased AKT phosphorylation. These results proved the structure of FGFR receptor was important for EGFR downstream signaling. Not only that, we also noticed that FGFR knockdown reduced EGFR receptor phosphorylation, and the interaction between EGFR and FGFR occurred at the receptor level. Previous data showed that FRS2 knockdown suppressed the rescue effect of EGF on FGFR suppression. Furthermore, FGFR2 knockdown but not FGFR inhibitor reduced ERK phosphorylation, these clues supported the transactivation model. In the future, we hope to confirm whether the FGFR physical structure will affect the amount of FRS2, thereby affecting the downstream signaling of EGFR. Further investigations are in progress to approve our hypotheses, with the hope to increase our understanding of EGFR-FGFR interactions and to improve the present HNSCC treatment. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:27:08Z (GMT). No. of bitstreams: 1 U0001-1708202011395600.pdf: 4231333 bytes, checksum: 67bf79eff334b5320b40d36bbb6b02b1 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 致謝 1 摘要 2 目錄 6 圖目錄 11 表目錄 14 第一章、緒論 15 1. 頭頸部鱗狀上皮細胞癌(Head and neck squamous cell carcinoma, HNSCC) 15 1-1 頭頸癌概論及流行病學 15 1-2 頭頸癌病程發展與其現有臨床治療 15 1-3 EGFR在頭頸癌細胞中過度表現而產生相關治療策略 16 1-4 影響頭頸癌中EGFR抑制效果的潛在機制 17 2. 纖維母細胞生長因子受器(Fibroblast growth factor receptors,FGFRs)會影響EMT與癌症移轉,可能成為頭頸癌治療新標的 18 2-1頭頸癌的進展與上皮-間質的轉換相關 18 2-2 FGFR為有潛力的頭頸癌治療標的 18 2-2-1 FGFR的構造與訊息傳遞 18 2-2-2 FGFRs的表現與疾病進展相關 18 2-2-3 FGFRs在頭頸癌中有高度表現 19 3. EGFR和FGFR 兩者在細胞遷移實驗中顯示出交互作用 20 4. EGFR和FGFR交互作用的三種可能機制 23 4-1 Receptor level model 23 4-2 Internalization model 25 4-3 Transactivation model 27 5. 研究目標: 釐清EGFR和FGFR之間的交互作用 29 第二章、材料與方法 30 1.細胞培養 30 2.細胞蛋白質萃取(Protein extraction)與西方墨點法(Western blotting) 30 2-1 細胞蛋白質萃取 30 2-2 膠體製備及電泳 30 2-3 電泳及蛋白質轉漬 31 2-4 一級抗體與二級抗體免疫結合 31 2-5 ECL呈色與定量分析 32 2-6 Western blot buffer 成分配方 32 3. 慢病毒基因靜默技術(Lentivirus shRNA knockdown) 33 3-1 將基因靜默細菌株包成病毒 33 3-2 MTS assay 34 3-3 慢病毒感染流程 (Virus infection) 35 4. 慢病毒過度表現技術(Lentivirus-based overexpression) 35 4-1製作pEYFP-P2A-FGFR2IIIb質體 35 4-2製作pEYFP-P2A-FGFR2IIIb-A649T FGFR2 kinase-dead質體 35 4-3將FGFR2過表現質體包成病毒 35 4-4慢病毒感染流程 (Virus infection) 36 5.免疫螢光染色 (Immunofluorescence assay) 36 5-1 固定細胞 36 5-2 破膜 (Permeabilization) 及去除非特異性結合 (Blocking) 36 5-3 一級抗體與二級抗體免疫結合 36 5-4 影像分析 36 6.免疫共沉澱 (Co-Immunopreciptation assay) 37 6-1 樣品處理 37 6-2 預清洗(pre-clean)與抗體結合 38 6-3 收集免疫沉澱結合物(Immunopreciptaion complex) 38 第三章、結果 39 1. 觀察FGFR2結構存在與否對EGFR之下游訊息傳遞造成影響 39 1-1 EGFR之下游訊息傳遞路徑 39 1-2 FGFR2結構存在與否對EGFR之下游訊息傳遞RAS/MAPK 路徑之影響 40 1-2-1 FGFR2 knockdown對pERK變化量之影響 40 1-2-2 FGFR2 overexpress對pERK變化量之影響 45 1-3 FGFR2結構存在與否對EGFR之下游訊息傳遞PI3K/AKT 路徑之影響 52 1-3-1 FGFR2 knockdown對pAKT變化量之影響 52 1-3-2 FGFR2 overexpress對pAKT變化量之影響 58 1-4 FGFR2結構存在與否對EGFR之下游訊息傳遞PLC路徑之影響 65 1-4-1 FGFR2 knockdown對pMLC2變化量之影響 65 1-4-2 FGFR2 overexpress對pMLC2變化量之影響 69 2. 確認EGFR和FGFR兩者交互作用的層級 76 2-1 尋找合適的EGFR受體活化指標 76 2-2 FGFR2 knockdown對EGFR受體活化之影響 78 第四章、討論與結論 81 1. FGFR2 結構存在與否對EGFR下游路徑各自帶來不同影響 81 2.了解EGFR和FGFR產生互動是在哪個作用層級 84 3. EGFR和FGFR彼此產生交互作用的方式 84 4.確認FRS2扮演串連EGFR和FGFR交互作用的角色 86 5. 改用其他cell line以改善SAS細胞中過高pERK baseline 對實驗結果之干擾 88 6.細胞中的異質性以及分析方法的完善 88 第五章、參考資料 92 第六章、附錄 95 1. PLASMID MAP 95 2.縮寫表 96 3. MATLAB SCRIPT 97 3-1 螢光板校正 97 3-2 pERK分析 101 3-3 pAKT, pMLC2分析 108 3-4 合併參數 132 | |
dc.language.iso | zh-TW | |
dc.title | 上皮細胞生長因子受器與纖維母細胞生長因子受器在頭頸癌細胞中的交互作用 | zh_TW |
dc.title | EGFR - FGFR interactions in head and neck cancer cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳青周(Ching-Chow Chen),張玉芳(Yu-Fong Chang),鄭乃禎(Nai-Chen Cheng),涂熊林(Hsiung-Lin Tu) | |
dc.subject.keyword | 頭頸部鱗狀細胞癌,表皮細胞生長因子受器,纖維母細胞生長因子受器,纖維母細胞生長因子受器受質2,細胞遷移,訊息傳遞, | zh_TW |
dc.subject.keyword | HNSCC,EGFR,FGFR,FRS2,cell migration,signal transduction, | en |
dc.relation.page | 132 | |
dc.identifier.doi | 10.6342/NTU202003704 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-18 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥理學研究所 | zh_TW |
顯示於系所單位: | 藥理學科所 |
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