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研究生:陳舜華
研究生(外文):Shun-Hua Chen
論文名稱:利用蛋白質體學研究卵巢癌腫瘤組織間質液中之特異蛋白
論文名稱(外文):Proteomic analysis of ovarian cancer specific proteins in tumor interstitial fluid
指導教授:王子豪王子豪引用關係
指導教授(外文):Tzu-Hao Wang
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:87
中文關鍵詞:卵巢癌蛋白質體學腫瘤組織間質液
外文關鍵詞:ovarian cancerCA125TIFNIFSTIP1TPI1
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卵巢癌為長庚醫院林口醫學中心第二好發的婦癌,由於卵巢癌的早期診斷不易,造成預後及存活率不佳,目前在臨床上所使用的 CA125 並不能有效的早期診斷卵巢癌,而尋找其他腫瘤標記的研究中,大多使用病人血清做為檢體來源,但血清中有大量的共通蛋白質,而作為腫瘤標記的蛋白質往往在血清中的濃度極低,不容易被發現。所以在2004年時 Celis 研究團隊認為,這些腫瘤標記蛋白是由腫瘤細胞所釋放出來到腫瘤間質液中,再經由這些間質液進入血液循環中。Celis 團隊也成功的分析乳癌腫瘤組織間質液 (tumor interstitial fluid) ,並且利用蛋白質體學技術找到了169個特異蛋白,也證明了上述想法。在本研究中,我們將上述原理應用於人類卵巢癌間質液的蛋白質體學鑑定,合併蛋白質體學、西方墨點測試、免疫組織切片染色及酵素連結免疫吸附法 (ELISA) 的實驗方式,企圖尋找到能增加卵巢癌診斷的特異蛋白,甚而在臨床上能有所幫助。經過蛋白質體學的實驗結果及統計軟體,在總共596個點中找到8個在兩群組間表現量有顯著差異的蛋白點:stress-induced-phosphoprotein 1 (STIP1) , triosephosphate Isomerase (TPI1) , leucine aminopeptidase (LAP3) , type I keratin 16、macrophin 1 isoform 2 (MACF1) , brain-derived neurotrophic factor isoform b preproprotein (BDNF) , transferrin與 ubiquitin carboxyl-terminal esterase L1 (UCHL1)。在TPI1、STIP1 ELISA實驗中,卵巢癌症病人血清中的表現確實高於正常人血清,特別是STIP1也可以分辨卵巢癌血清及子宮內膜異位症 (Endometriosis) 血清,所以我們認為,STIP1及TPI1有作為卵巢癌的腫瘤標記的潛力。
Ovarian cancer is the second prevalent gynecological cancer at Lin-Kou Medical Center of Chang Gung Memorial Hospital. Worldwide, patients with ovarian cancers are often associated with poor prognosis and limited survival rates because their cancers were not detected at earlier stages. Currently, CA125 is the most commonly used tumor marker for ovarian cancer, but it only results in 78% sensitivity, 82 to 95% specificity, and 82% positive predictive value. Therefore, more tumor markers for detecting ovarian cancer are warranted. Readily accessible blood is a convenient source of tumor markers. However, screening for new tumor markers from blood is difficult because tumor markers, usually at low concentrations in blood, are easily obscured by serum abundant common proteins. In 2004, Celis proposed that tumor markers are secreted by tumor cells first into tumor interstitial fluid (TIF) before their eventual appearance in circulating blood. As a proof-of-principle, his group identified 169 breast cancer-specific proteins in TIF using proteomics approaches. In this study, we attempted to identify cancer-specific proteins from TIF derived from human ovarian cancer using proteomics, Western blot analysis, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). Using statistical analysis of 596 protein spots in two-dimensional gels between the TIF of ovarian cancer and normal interstitial fluid (NIF) of normal ovaries, we identified the following 8 differentially expressed proteins: stress-induced phosphoprotein 1 (STIP1), triosephosphate isomerase (TPI1), leucine aminopeptidase 3 (LAP3), type I keratin 16, macrophin 1 isoform 2 (MACF1), brain-derived neurotrophic factor isoform b preproprotein (BDNF), transferrin, and ubiquitin carboxyl-terminal esterase L1 (UCHL1). Subsequent ELISA experiments revealed the serum levels of STIP1 and TPI1 were significantly higher in ovarian cancer patients than in healthy controls. Our results suggest the potential of STIP1 and TPI1 as tumor markers for human ovarian cancer.
目錄

指導教授推薦書………………………………………………………….i
口試委員會審定書………………………………………………………ii
授權書…………………………………………………………………...iii
中文摘要………………………………………………………………...iv
英文摘要………………………………………………………………...vi
誌謝…………………………………………………………………….viii
目錄………………………………………………………………………x
圖表目錄………………………………………………………………..xii
第一章 前言……………………………………………………………...1
第二章 實驗動機與目的……………………………………………….10
第三章 實驗設計與流程……………………………………………….11
第四章 實驗方法……………………………………………………….12
一、 組織處理……………………………………………………….13
二、 Modified protein precipitation procedure………………………14
三、 IEF focusing……………………………………………………14
四、Two-dimensional polyacrylamide electrophoresis………………15
五、SYPRO Ruby stain………………………………………………16
六、Image analysis……………………………………………………17
七、In gel digestion and protein identification……………………….17
八、O.C.T frozen section……………………………………………..18
九、Western blot analysis…………………….....................................19
十、Immunohistochemistry………………………………...................20
十一、ELISA.........................................................................................21
十二、統計方法...................................................................................
第五章 實驗結果……………………………………………………….24
第六章 討論…………………………………………………………….29
參考文獻………………………………………………………………...36
附錄(18個表,11個圖)………………………………………………...46










圖表目錄

Table 1. Rehydration buffer…………………………………..................46

Table 2. 0.5 % agarose…………………………………………………..47

Table 3. 12.5 % two-dimensional polyacrylamide gel………………….48

Table 4. Equilibrium buffer……………………………………………..49

Table 5. 4 X resolving buffer…………………………………………50

Table 6. 5 X SDS running buffer……………………………………...51

Table 7. Fix/de stain solution……………………………………………52

Table 8. Matrix…………………………………………………………..53

Table 9. 4 X sample buffer……………………………………………54

Table 10. 10 % SDS-PAGE……………………………………………..55

Table 11. 5 X running buffer for western blot………………………...56

Table12. 1 X transfer buffer…………………………………………...57
Table 13. 1 X TBST…………………………………………………...58

Table 14. ELISA實驗藥劑配方………………………………………...59

Table 15. 1 X PBS…………………………………………………….60

Table 16. Protein ID……………………………………………………..61

Table 17. STIP1 and TPI1 ELISA quality control………………………62

Table 18. ELISA統計結果簡圖………………………………………...63

Figure 1. TIF表現量大於NIF的蛋白點……………………………….64

Figure 2. NIF表現量大於TIF的蛋白點……………………………….70

Figure 3. Transferrin ELISA…………………………………………….72

Figure 4. STIP1 western blot結果………………………………………73

Figure 5. TPI1 western blot結果………………………………………..75

Figure 6. UCHL1 western blot 結果……………………………………77
Figure 7. LAP3 western blot結果………………………………………79

Figure 8. Immunohistochemistry………………………………………..81

Figure 9. 三組群體 (卵巢上皮癌病患、正常婦女血清及子宮內膜異位症病患血清) ELISA比較……………………………………82

Figure 10. 子宮內膜異位症合併健檢婦女血清與卵巢癌病患血清
ELISA結果………………………………………………….84

Figure 11. ROC curve分析…………………………………………......86
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