陳玉如Chen, Yu-Ju吳景斌Wu, Jing-Bin2023-12-082027-08-152023-12-082022https://etds.lib.ntnu.edu.tw/thesis/detail/5b910b22d2cdec324c81638f1dd5f5a4/http://rportal.lib.ntnu.edu.tw/handle/20.500.12235/120919消化道癌症在世界各地都有很高的發生率在台灣亦同，他們對病人及健保體系都造成相當程度的負荷。發展更具效率的診斷工具勢必能夠緩解此負擔。血清澱粉蛋白A(SAA)為一種急性反應蛋白並與多種癌症皆有所關聯。我們團隊於先前發現了24種SAA的等位基因異構體，在健康個體與胃癌病人中其多變複雜的表現模式能夠運用於胃癌病人的診斷。然而，SAA異構體產生的來源至今仍尚未明瞭。為了進一步了解SAA異構體產生的機制和異構體的癌症特異性與否，我們想藉由癌症細胞模型來模擬病人中SAA的分泌與修飾來了解血清中的SAA異構體的特性，並比較病人及細胞的結果解析在不同癌症群體中是否具有癌症特異性的異構體。在與高雄醫學大學吳登強醫師的合作下，我們使用兩種癌症細胞:肝癌(Huh7、HepG2、Hep3B)、胃癌(AGS、N87)及正常細胞(GES-1)並設計以細胞激素 (IL-6, IL-1β, TNF-α) 加以刺激來進行實驗。藉由半自動化的奈米探針純化方法並結合基質輔助雷射脫附游離飛行時間質譜儀 (MALDI – TOF – MS)來分析細胞液中的SAA異構體形式。由於細胞液中所含有的SAA濃度很低，我們運用了濃縮過濾的方法，在其濃縮過程能留住目標蛋白也能達到除鹽功效。首先，我們根據美國食品藥物管理局的指南進行方法確效，建立5 -200 ng的校正曲線，且經對數轉換亦有良好的線性(r2 = 0.9859)，最低點5 ng其異日間變異數為14.1%。肝癌細胞(HepG2)經過IL-6, IL-1β刺激後透過西方點墨法(Western blotting)我們能觀察到SAA而(MALDI – TOF – MS)卻無信號產生；而肝癌細胞(Huh7、HepG2、Hep3B)在IL-6, IL-1β, TNF-α共同刺激下SAA分泌顯著上升並且Western blot與MALDI – TOF – MS皆可偵測。在正常胃細胞(GES-1)具11683 (SAA1α)、11655 (SAA1γ(R−))、11491(SAA2β(R−))、11404 (SAA2β (RS−))等異構體而胃癌(AGS、N87)中卻無任何異構體的表現。在肝癌細胞中(Huh7、HepG2、Hep3B)只有找到11683 (SAA1α)、11629 (SAA2γ) 等完整型異構體。比較肝癌細胞與肝癌病人中SAA的表現形式，兩者可以找到相同的完整型異構體，而在細胞中無截斷型異構體存在。結果推測SAA可能在病人檢體與細胞中具不同的修飾作用，而人體血清中可能存在一些與癌症相關的蛋白酶作用並涉及N端、C端截斷等修飾作用。簡而言之，我們改善的方法能夠運用於細胞且在肝癌細胞及正常胃細胞中能夠檢測到SAA的表現。未來，我們想透過細胞共培養系統,來更進一步驗證假說中受傷的細胞與正常細胞間的交互作對SAA造成的影響。The high incidence of gastrointestinal (GI) cancers worldwide and in Taiwan continues to cause burden to patients and the healthcare system. Effective diagnosis tools are thus necessary to ease this burden. Serum amyloid A (SAA), is an acute phase protein, and its overexpression has been implicated in many different cancers. In particular, our group has previously discovered 24 allelic variants of SAA and its heterogeneous pattern among gastric cancer patients and healthy control could be used for gastric cancer diagnosis. However, the source of SAA variants is still not fully understood. To further study the secretion mechanism of SAA and its cancer type specificity, we characterize the SAA isoforms in patient serum and cancer cell line model, which may mimic the SAA secretion or modification from the cancer patient. We compared with patient and cell result to uncover the specific variant patterns for various cancer groups. By collaboration with Dr. Deng-Chyang Wu at Kaohsiung Medical University, two cancer models, including liver cancer cell line (Huh7、HepG2、Hep3B) and GC gastric cancer cell line (AGS、N87), and normal cell (GES-1) were designed with and further stimulation of cytokines (IL-6, IL-1β, TNF-α). By semi-automated nanoprobe-based affinity purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detected SAA patterns of in cell. An additional ultrafiltration step was performed to concentrate the low concentration of SAA in cell lysates. First, the method was validated following the United States Foods and Drugs Administration guidelines. The method has linearity (r2 = 0.9859) in the range of 5-200 ng after log2 transformation and limit of detection (5.0 ng) with interday CV of 14.1%. After IL-6, IL-1 treatment, SAA was detectedin Western blotting but not in MALDI-TOF-MS in liver cancer cell (HepG2). By treatment with IL-6, IL-1β, TNF-α together, SAA secretion could be increased and detected by both WB and MALDI-TOF-MS in liver cancer cell (Huh7、HepG2、Hep3B). We detect 11683 (SAA1α), 11655 (SAA1γ), 11491 (SAA2β (R−)) and 11404 (SAA2β (RS−)) in GES-1, but not in gastric cancer cells (AGS, N87). In liver cancer cells (Huh7, HepG2 and Hep3B), only intact variants 11683 (SAA1α) and 11629 (SAA1γ) were found. Compared to SAA pattern in liver cancer patients and liver cancer cell line the same intact variants can be found, but no truncated forms were found in the cell lines. The results suggest different SAA modification in patient and cell and cancer-associated proteases may involve in N- or C-terminal truncation in human serum. In summary, we developed a method for SAA analysis in cell lines and have detected SAA variant. in further verify the association of SAA and cancer type specificity. In future study, it is potential to explore the mechanistic role of SAA in normal cell and cancer cell lines through co-culture system.消化道癌症血清澱粉A異構體質譜癌症細胞Gastrointestinal cancersSerum amyloid ASAAVariantsIsoformsMass spectrometryCancer cell lineetd