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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 周綠蘋(Lu-Ping Chow) | |
| dc.contributor.author | Ming Jiun Tsai | en |
| dc.contributor.author | 蔡銘駿 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:33:54Z | - |
| dc.date.available | 2020-09-13 | |
| dc.date.copyright | 2017-09-13 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68757 | - |
| dc.description.abstract | 肝癌是世界上最常見的癌症之一。其中有百分之七十五的肝癌屬於肝細胞癌 (hepatocellular carcinoma, HCC)。在臺灣，肝癌的盛行率為所有癌症的第二名，僅次於肺癌。目前臨床上已有許多生物標記可用來診斷與評估肝癌治療後的預後，也有許多具潛力的生物標記正在被研究。根據 Barcelona-clinic liver cancer staging (BCLC staging)，晚期的肝癌病人常使用唯一的口服肝癌標靶藥物 sorafenib 治療。Sorafenib 是一種多重激酶抑制劑，能藉由 Raf 抑制 MAPK 細胞傳遞路徑，且是目前唯一美國 FDA 核可上市的口服肝癌標靶藥物，再加上其副作用相對和緩而被用來治療肝細胞癌後期之病人。然而，目前並沒有一個好的方法能用以預測 sorafenib 治療肝癌病人後的預後。近來雖然有許多的研究著力於 sorafenib 治療效果相關生物標記的探討，並沒有找到適當的生物標記能應用到臨床上以評估預後。因此找到能用以預測肝癌在 sorafenib 治療後之療效之生物標記是刻不容緩。
以定量蛋白質體學 (quantitative proteomics) 的方法，細胞培養中標記穩定同位素胺基酸 SILAC (stable isotope labeling of amino acids in culture) 之技術，分析HuH-7 在 sorafenib 治療前與治療後，細胞內及分泌性蛋白質表現量的差異，並以質譜儀 LC MS/MS 鑑定這些蛋白質，再以生物資訊分析軟體IPA (ingenuity pathway analysis) 對這些胞內與分泌性的蛋白質做疾病與功能上的分析。分別在細胞裂解液與細胞培養液中鑑定到2611個與1022個蛋白質，且分別有1091個與145個蛋白質的表現量是下降的。再將這些蛋白質以生物資訊分析軟體 TMHMM、SecretomeP 以及 SignalP 分析，發現在細胞裂解液與細胞培養液中共有103個蛋白質屬於分泌性蛋白質。這些蛋白質皆與細胞凋亡、細胞增生、血管生成與癌症有關，並從中篩選出四個有潛力之生物標記 CTGF、Galectin-3、Glypican-3 及 HMGB1。再分別以活體外之細胞實驗及活體內小鼠模式動物進行驗證，這四個蛋白質的表現量都會因sorafenib的治療而下降，且在活體內的腫瘤生長也會因sorafenib 的治療而受到明顯抑制。並也探討這些蛋白質所參與的細胞傳遞路徑，發現這些蛋白質皆屬於 MAPK/ERK 下游之表現蛋白質。另外也觀察細胞的生長情形，將蛋白質GPC3的表現抑制後，細胞的生長也會受到抑制。根據我們的研究結果，我們找到四個具有潛力的生物標記可以用來預測肝癌病人經 sorafenib 治療後之預後狀況。這些生物標記需更多臨床上相關的研究及驗證，從而了解其價值，並應用到臨床。 | zh_TW |
| dc.description.abstract | Hepatocellular carcinoma, HCC, is the most common liver cancer in the world. Clinically, there are many biomarkers which can be used to diagnose HCC and predict prognosis. According to the Barcelona-clinic liver cancer staging (BCLC staging), sorafenib is the only anticancer drug with proven prognostic efficacy for treatment of HCC. Sorafenib is a multi-kinase inhibitor and inhibits the MAPK pathway by inhibiting Raf. Because sorafenib is the only approved drug for advanced HCC and exhibits relatively mild adverse effect, biomarkers which can be used to predict sorafenib efficacy can assist in identifying the patients who are likely to benefit from the treatment. Many studies have attempted to investigate biomarkers of sorafenib efficacy by analyzing the associations between potential markers and patients’ outcomes. However, there are no appropriate biomarkers can be used to estimate prognosis. Therefore, it is necessary to find biomarkers which can be used to predict the prognosis of HCC patients after sorafenib treatment.
We applied the quantitative proteomics method (Stable Isotope Labeling of Amino acids in Culture, SILAC) to analyze the differences of secretory and cytosolic proteins levels between HuH-7 and sorafenib-treated HuH-7 cells. We further used the LC MS/MS approach to identify the above proteins. The Ingenuity Pathway Analysis (IPA) was performed to analyze the difference of functions between secretory and cytosolic proteins of HuH-7 and sorafenib-treated HuH-7 cells. According to our results, we identified 2611 proteins in cell lysate and 1022 proteins in conditioned medium of sorafenib-treated HuH-7 cells. Of these proteins, there are 1091 and 145 proteins down-regulated in cell lysate and conditioned medium respectively after sorafenib treatment. Furthermore, we analyzed these two groups by bioinformatics software, TMHMM, SecretomeP and SignalP, and totally 103 proteins were classified as the secretory proteins. These proteins were found to be relating to apoptosis, proliferation, angiogenesis and cancer. In which, we selected four candidates, CTGF, Galectin-3, Glypican-3 and HMGB1, which may be potential biomarkers for predicting prognosis of HCC patients after sorafenib treatment. We validated these potential biomarkers by western blot in vitro and immunohistochemistry in vivo and found that the expressions of these four proteins are inhibited by sorafenib. In addition, the tumor volume was also decreased in vivo after sorafenib treatment. We further studied the involved cell signaling of these potential biomarkers with several inhibitors and found that these four proteins are regulated by MAPK/ERK pathway. Moreover, proliferation was also inhibited in GPC3-knowdown HuH-7 cells. In conclusion, we found that these four proteins are potential biomarkers for predicting prognosis of HCC patients after sorafenib treatment. It is expected to make these potential biomarkers be valuable in clinical use. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:33:54Z (GMT). No. of bitstreams: 1 ntu-106-R04442019-1.pdf: 3121178 bytes, checksum: 4a7a05fd30650e18ce43adf2dfd02e4e (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員審定書 i
謝誌 ii 中文摘要 iii Abstract v 縮寫 vii 目錄 ix 圖目錄 xii 表目錄 xiii 附錄目錄 xiv 第一章 導論 1 第一節 肝癌 1 1.1 肝癌之流行病學 1 1.2 肝癌的危險因子 1 1.3 肝癌的診斷 2 1.4 肝癌的分期 2 第二節 臨床治療 3 2.1 肝臟切除術 (Liver resection) 3 2.2 肝臟移植 (Liver transplantation) 3 2.3 局部消融術 (Local ablative therapy) 3 2.4 肝動脈化學栓塞療法 (Liver Transarterial Chemoembolization, TACE) 3 2.5 標靶藥物蕾莎瓦 (Sorafenib) 3 第三節 肝細胞癌的生物標記 (Biomarkers for HCC) 4 3.1 生物標記的定義 (Definition of biomarkers) 4 3.2 癌症生物標記 (Cancer biomarkers) 4 3.3 現行的肝細胞癌生物標記 (Current biomarkers for HCC) 4 3.4 血清生物標記 (Serum biomarkers) 5 第四節 蛋白質體學 6 4.1 蛋白質體學定義 (Definition of proteomics) 6 4.2 定量蛋白質體學 (Quantitative proteomics) 6 4.3 蛋白質體學在癌症生物標記研究上的應用 7 第五節 分泌蛋白體 (Secretome) 7 第六節 肝細胞癌之訊息傳遞路徑 (Cell signaling of HCC) 8 第七節 蕾莎瓦 (Sorafenib) 8 第八節 研究動機 9 第二章 實驗材料 10 第一節 細胞株 10 第二節 抗體 10 第三節 藥品 11 第四節 試劑組 12 第五節 生物材料 13 第六節 重要儀器 13 第七節 酵素 14 第八節 軟體及資料庫 14 第三章 實驗方法 15 第一節 肝癌細胞的培養 15 1.1 細胞的培養 (Cell culture) 15 1.2 細胞的計數與細胞的存活率 (Cell counting and cell viability) 15 第二節 蛋白質分析法 15 2.1 蛋白質濃度測定 (BCA protein assay) 15 2.2 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 16 2.3 電泳膠體的染色 (Protein staining) 18 2.4 西方點墨法 (Western blot) 19 第三節 Sorafenib影響HuH-7細胞蛋白質表現差異之鑑定 21 3.1 細胞培養中標記穩定同位素胺基酸 (Stable Isotope Labeling of Amino acids in Culture, SILAC) 21 3.2 膠體內水解 (In-gel digestion) 21 3.3 液相層析偶合串聯式質譜儀分析 (NanoLC-MS/MS analysis) 23 3.4 生物資訊學 (Bioinformatics) 23 第四節 異種移植小鼠 (Xenograft mice) 24 第五節 免疫組織化學染色 (Immunohistochemistry) 25 5.1 免疫組織化學染色溶液之配置 (Preparation of buffer for IHC)/ 25 5.2 免疫組織化學染色之步驟 25 第六節 訊息傳遞的研究 26 6.1 細胞加藥處理 (Drug treatment) 26 6.2 裂解細胞 (Cell lysis) 26 6.3 細胞裂解液的配置 (Preparation of RIPA cell lysis buffer) 27 第七節 小髮夾RNA (shRNA) 抑制目標基因 (gene knockdown) 27 7.1 菌株培養 27 7.2 中量shRNA質體DNA萃取 28 7.3 帶有shRNA慢病毒 (Lentivirus) 之製備 28 7.4 以帶有shRNA慢病毒感染肝癌細胞 29 7.5 抗生素篩選 (Screening) 29 第四章 實驗結果 30 第一節 分析細胞中分泌性蛋白質之表現量差異 30 1.1 細胞培養中標記穩定同位素胺基酸之定量 30 1.2 Mascot軟體之蛋白質鑑定及定量 30 1.3 分泌性蛋白質之分析 30 1.4 生物資訊學對蛋白質之疾病與功能分析 31 第二節 候選蛋白之驗證 31 2.1 HuH-7 細胞處理 sorafenib 後蛋白質表現量差異之驗證 31 2.2 動物模式中以 sorafenib 治療活體內動物腫瘤之驗證 32 第三節 CTGF、Galectin-3、Glypican-3 及 HMGB1之調控路徑 32 3.1 MAPK 路徑與 CTGF、Galectin-3、Glypican-3 及 HMGB1之表現量關係 32 3.2 ERK 路徑與 CTGF、Galectin-3、Glypican-3 及 HMGB1 之表現量關係 33 3.3 p-38 路徑與 CTGF、Galectin-3、Glypican-3 及 HMGB1 之表現量關係 33 第四節 Glypican-3對細胞株HuH-7生理功能分析 33 第五節 結論 34 第五章 討論 35 第一節 蛋白質體學與生物資訊學在分泌蛋白體之應用 35 第二節 候選蛋白質對肝癌細胞的調控 36 第三節 肝癌生物標記 38 第四節 精準醫療 (Precision medicine) 39 第六章 參考文獻 41 圖 47 表 56 附錄 63 | |
| dc.language.iso | zh-TW | |
| dc.subject | Galectin-3 | zh_TW |
| dc.subject | 肝細胞癌 | zh_TW |
| dc.subject | 生物標記 | zh_TW |
| dc.subject | Glypican-3 | zh_TW |
| dc.subject | HMGB1 | zh_TW |
| dc.subject | CTGF | zh_TW |
| dc.subject | 分泌蛋白體學 | zh_TW |
| dc.subject | HMGB1 | en |
| dc.subject | biomarker | en |
| dc.subject | secreted protein | en |
| dc.subject | CTGF | en |
| dc.subject | Galectin-3 | en |
| dc.subject | Glypican-3 | en |
| dc.subject | Hepatocellular carcinoma | en |
| dc.title | 以蛋白質體學方法鑑定蕾莎瓦治療人類肝細胞癌後之預後生物標記 | zh_TW |
| dc.title | Identification of Potential Prognostic Biomarkers for Sorafenib Efficacy in Hepatocellular Carcinoma Cells by Proteomic Approaches | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 龔秀妮(Hsiu-Ni Kung),黃楓婷(Feng-Ting Huang) | |
| dc.subject.keyword | 肝細胞癌,生物標記,分泌蛋白體學,CTGF,Galectin-3,Glypican-3,HMGB1, | zh_TW |
| dc.subject.keyword | Hepatocellular carcinoma,biomarker,secreted protein,CTGF,Galectin-3,Glypican-3,HMGB1, | en |
| dc.relation.page | 69 | |
| dc.identifier.doi | 10.6342/NTU201703750 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-18 | |
| 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 |
| Appears in Collections: | 生物化學暨分子生物學科研究所 | |
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| File | Size | Format | |
|---|---|---|---|
| ntu-106-1.pdf Restricted Access | 3.05 MB | Adobe PDF |
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