- 學位論文
以蛋白質體學方法鑑定與人類肝細胞癌相關之生物標記
Identification of Human Hepatocellular Carcinoma-related Biomarker by Proteomic Approaches
摘要
肝癌是全球發生率及死亡率極高的癌症之一，在台灣肝癌的發生率也有逐年增加的趨勢。目前臨床方面則是藉由肝癌的分期來選擇治療的方式，然而現行並沒有一個良好、快速且經濟的非侵入性診斷方法，能檢測肝癌的疾病進展與及時提供臨床早期判斷治療療效。因此，本研究由較易取得的血液檢體中，尋找適當的生物標記，來及時追蹤疾病進展與預後狀況之評估，產生極大的興趣。 近幾年，藉由蛋白質體學方法尋找有潛力之生物標記研究的興起，本研究利用細胞培養中標記穩定同位素胺基酸 (Stable Isotope Labeling of Amino acids in Culture, SILAC) 之差異蛋白質體學方法與離線二維液相層析質譜法 (Off-line 2D LC MS/MS)，分析在肝癌細胞 (HuH-7) 中加入肝癌的唯一標靶藥物蕾莎瓦 (Sorafenib) 模擬達到治療效果後肝癌的分子機轉，鑑定到蛋白質中有427個蛋白質因為蕾莎瓦的處理而表現量下降。經由訊息傳遞資料庫 (Ingenuity Pathway Analysis, IPA) 分析肝癌細胞內分子與訊息傳遞的變化，發現表現量下降的蛋白質皆與抑制癌細胞的增生和促進癌細胞的死亡有關，再經過分泌性蛋白質軟體TMHMM、SignalP和 SecretomeP分析後，共預測有131個表現量下降的蛋白質具有分泌性，並從中篩選31 kDa凝集素為一個有潛力的生物標記。藉由西方點墨法驗證，肝癌細胞中與細胞外31 kDa凝集素確實會在蕾莎瓦的處理下表現量受到抑制。因此再深入探討肝癌細胞中訊息傳遞路徑與31 kDa凝集素表現量的關係，得知MAPK路徑中的ERK與p-38路徑的活化與31 kDa凝集素生成量有關。最後運用酵素連結免疫吸附法 (Enzyme-link Immunosorbent Assay, ELISA) 驗證肝癌病人血清中31 kDa凝集素的表現量比健康人高，而經過肝動脈化學栓塞療法 (Liver Transarterial Chemoembolization, TACE) 與標靶藥物蕾莎瓦治療後的病人血清中，31 kDa凝集素表現量與治療前有差異，表示31 kDa凝集素可能是肝癌中疾病進展或療效與藥效有關的生物標記，能有助於臨床判斷、治療選擇和抗癌藥物的開發。
並列摘要
Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers. In Taiwan, it ranks on top three deadly cancers for many years. In clinic, the Barcelona-Clinic Liver Cancer (BCLC) staging classification links the stage of the disease to a specific treatment strategy. However, there has no fast and economic efficiently method for diagnosis the progression and prognosis of the liver cancer. Serum biomarkers secreted by a tumor can be used to evaluate cancer progression and to reflect the early effects of cancer therapeutics. Recently, the proteome-based approaches have been widely applied in searching potential cancer biomarkers. According to the proteomic analysis, we can find out a tumor biomarker whose function may involve in the disease progression and prognosis. Moreover, the biomarker also can be a potential therapeutic indicator. Until now, Sorafenib is the only drug of molecular targeted therapy for HCC. We applied the quantitative proteomics method (Stable Isotope Labeling of Amino acids in Culture, SILAC) to analyze the differences of protein expression levels between HuH-7 and Sorafenib-treated HuH-7 cells. We further used the off-line 2D LC MS/MS approach to identify the above proteins. The Ingenuity Pathway Analysis (IPA) was performed to analyze the difference function between HuH-7 and Sorafenib-treated HuH-7 cells. According to the our results, 2611 quantified proteins were identified from the SILAC experiments, and 427 proteins expression level were decreased after Sorafenib treatment. We found that most proteins were related to the survival and proliferation of cancer cells. By using TMHMM, SignalP and SecretomeP softwares, a total of 131 proteins were classified as the secreted proteins, and the non-classical secreted protein. We found 31 kDa-lectin may correlate to cancer proliferation and survival. Furthermore, we used the Western blotting to validate the candidates and find the 31 kDa-lectin regulated by the MAPK signaling pathway. ELISA analysis further showed that 31 kDa-lectin expression levels were up-regulated in HCC patients, and the expression levels of 31 kDa-lectin were reduced in HCC patients’ serum after TACE and Sorafenib treatment. According to the above results, we conclude that 31 kDa-lectin is a potential biomarker of disease progression and therapy efficacy, and it is expected to become valuable in routine clinical care and anticancer drug development.
參考文獻
延伸閱讀
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