吳世雄臺灣大學：生化科學研究所姚正良Yao, Jheng-LiangJheng-LiangYao2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52785肺癌是目前世界上最常發生的癌症，而且它具有高度的致死率。平均肺癌患者五年存活率約八到十四百分比。如此低的存活率主要是來自於肺癌診斷不易，通常發現時已經有相當的一段時間。因此在癌症形成初期有效診斷對於提升存活率是相當重要的，也可以增加臨床治療的成功率。 近年來許多報告指出在癌症中會有醣化差異的存在，例如在肝癌、肺癌、卵巢癌等等。這些差異中目前被提出主要跟癌症相關的有岩藻醣化(fucosylation)、唾液酸醣化(sialylation)以及高度分支化的產生。目前研究的方向主要可以分為基因層次的探討以及醣基與蛋白質層次的探討。在基因層次主要是研究醣轉移酶的表現程度、在醣基與蛋白質層次主要是研究醣基上的差異以及量上的差異。 本篇論文主要目的是希望能夠建立一個方法，找尋肺癌患者血清中的生物指標(biomarker)。在過去許多的研究中，大多數討論醣蛋白差異的報告並未明確的指出此差異是來是蛋白質表現量上的差異或是醣基上本身的差異。因此我們將外源凝集素(Lectin)的應用與蛋白質體學方法做結合，去尋找血液中具有成為生物指標潛能的醣蛋白。即便找尋到的差異可能來自於蛋白質表現量上的差異，也有可能當作癌症檢測指標。若此差異來自於醣基本身，則可進一步探討其差異的位置，以及其背後是否具有生理意義。 實驗中首先利用各種外源凝集素篩選血清，發現在刀豆球蛋白(Con A)篩選的結果中有顯著差異存在。因此進一步利用刀豆球蛋白進行親和層析，分離出可以與刀豆球蛋白結合的蛋白質，接著以二維電泳(2D-electrophoresis)進行展開或直接將層析後的蛋白質利用質譜進行鑑定。 最後結果發現利用此方法找到許多發炎相關的蛋白質，例如：haptoglobin、Complement C3c、C4A、serum amyloid，這些蛋白質在其他病毒感染的癌症例如肝癌或是細菌感染的癌症例如胃癌中曾經被討論過，這樣的結果是否也代表著肺癌可能是由於抽菸、油煙或其他刺激因子長期引發肺部的發炎而導致，仍值得深思。此外也鑑定到Fibrinogen有差異，在過去文獻中曾指出抽菸會造成此蛋白在血液中含量上升。由於實驗中所利用的刀豆球蛋白可以與大部分帶有N-glycosylation的蛋白質進行結合，所以找到有差異性的蛋白質大多屬於蛋白質量上的差異，因此若利用其他的外源凝集素以此方式進行操作，或許就能更容易找到具有醣基差異的蛋白質。另外在本次實驗中也發現在肺癌血清中補體(complement) C3的切割片段有差異性存在，這是否來自於癌細胞所造成的仍值得探討。Lung cancer is the most common cancer in the world and has the highly lethal ratio from cancer. The survival rates of 5-year patient are very low at about 8-14%. The low survival rate is caused by that disease diagnosis is usually late, and prognosis is poor. To diagnose effectively in the early stage of cancer formation is important for increasing survival rate. Detection in the early stage of lung cancer is critical for successful clinical therapy. Presently, many reports discuss the glycosylation changes in several cancer cases such as liver cancer, lung cancer, ovarian cancer etc. The differences are including fucosylation, sialylation, and high branches formation. They approach the subject matter from gene level to discuss the glycosyltransferase expression, and from the glycan and protein level to discuss the glycoform difference. The aim of our study is to setup methods and attempts to identify serum biomarker of lung cancer. According to several reports, they did not discuss that the difference in glycoprotein resulted from protein level or glycan level. In the experiment, we combine the application of lectin and the method of proteomics for researching in serum biomarker of glycoprotein. Even if the difference is in protein level, it also has the potential to be a biomarker for cancer diagnosis. If the difference is in glycan level, we can discuss the different site and the effect in physiology. First, we use several kinds of letin to screen serum. There are some differences in the result of Con A lectin staining. The affinity column of Con A was used to separate the protein which can be bound by Con A. Then the bound protein was separated by 2-D electrophoresis or direct identified by mass. In the result, we find that several identified proteins were in immune response and were reported before in other cancer like liver cancer or gastric cancer. It is needed to discuss that the possibility of smoking and some factors inducing lung inflammation lead to lung cancer. We use Con A in the experiment. Because Con A can bind almost all proteins with N-glycosylation, the differences of identified proteins were in protein level. If we use other kinds of lectin, we can identify protein differences in glycan level. In the other side, we find the difference in the fragments of complement C3. It is needed to discuss what causes the result, cancer or not.口試委員會審定書 i 誌謝 ii 中文摘要 iii 英文摘要 iv 目錄 v 縮寫表 vii 第一章 導論(Introduction) 1 1.1 肺癌 1 1.2醣蛋白 3 1.3醣化與癌症的關係 4 1.4 生物指標 6 1.5 補體系統 7 第二章 實驗材料(Material) 8 第三章 實驗方法(Methods) 11 3.1外源凝集素之篩選與染色 11 3.2外源凝集素管柱親合層析 15 3.3 二維電泳 17 3.4 蛋白質鑑定 19 3.5 西方點墨法試驗 20 第四章 實驗結果(Results) 22 4.1 外源凝集素的染色結果 22 4.2 Complement C3c之鑑定與分析 22 4.3應用外源凝集素找尋醣蛋白中生物指標之方法建立 24 4.4利用西方點墨法比較血清中差異蛋白質之存在情況 25 第五章 討論(Discussion) 26 5.1 Complement C3c 差異之原因探討 26 5.2 實驗中鑑定之蛋白質探討 27 5.3應用外源凝集素找尋醣蛋白中生物指標之方法討論 28 5.4總結與未來展望 29 第六章 圖表 30 參考文獻 48 附錄 534484091 bytesapplication/pdfen-US肺腺癌醣蛋白生物標記蛋白質體學補體C3lung adenocarcinomaglycoproteinbiomarkerproteomicscomplement C3[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52785/1/ntu-96-R94b46037-1.pdf