- 學位論文
建立多重標的酪胺酸激酶抑制劑 PD153035之單一細胞層級檢測平臺
The Construction of Single-cell Level Detection Platform for Studying Multitargeted Tyrosine Kinase Inhibitor PD153035
摘要
根據行政院衛生署國民健康局癌症登記資料顯示，自民國七十一年起， 癌症即躍居國人十大死因第一位，其中，肺癌在男性癌症死因高居第二位(僅次於肝癌)，在女性癌症死因中更居於首位，因此未來對肺癌的研究將會是影響國人健康的重要指標。在臨床上主要分為小細胞肺癌(small-cell lung cancer; SCLC)與非小細胞肺癌(non-small-cell lung cancer; NSCLC)兩類，其中非小細胞肺癌發生率占85-88％。一般非小細胞肺癌的藥物治療方式主要以化學治療和標靶藥物治療為主。由於大約88-99% 的非小細胞肺癌有發生表皮生長因子受體（epidermal growth factor receptor; EGFR）的大量表現的情形，因此表皮生長因子受體酪胺酸酶抑制劑（epidermal growth factor receptor- tyrosine kinase inhibitor; EGFR-TKI）近年已成為非小細胞肺癌理想標靶藥物。由於胞攝作用(endocytosis)即是透過EGFR訊息傳遞路徑而開啟一連串動態反應，故表皮生長因子受體酪胺酸酶抑制劑可作為研究指標，而經由EGFR訊息傳遞路徑而開啟的胞攝作用，如何由細胞膜與細胞骨架所構成的立體空間網路進行運輸便成為近年研究的重點項目。微管(microtubule)、中間絲(intermediate filaments)、肌動蛋白(actin)是組成細胞骨架(cytoskeleton)的成份之一，其中肌動蛋白的主要功能為協助細胞的移動及細胞內的傳輸作用。近年已有研究利用雙色染色與即時螢光顯微鏡研究肌動蛋白在細胞內的分佈以及與細胞膜外基質及細胞膜形成的動態網絡，發現肌動蛋白參與胞攝作用(endocytosis)的過程，已證實在特定的位置才能表現出肌動蛋白對胞攝作用的重要性，以及與位於細胞膜下緣，肌動蛋白構成細胞骨架的複雜程度有關。本研究利用生物功能性量子點(biofunctionalized quntum dots)與生物單分子技術進行肺癌偵測及治療的應用，故標靶藥物治療的作用機制的驗證與肺癌治療可行性及新標靶藥物的評估為首要目標。藉由生物功能性量子點與EGF結合後，透過胞攝作用進入A431細胞後的不同階段，透過PD153035與cytochalasin D等藥物，分別進行erbB-2酪胺酸酶的抑制與防止肌動蛋白的聚合(polymerization)，由外而內地且分階段性探討胞攝作用透過不同路徑進入A431細胞內的時間與空間上的傳輸過程的差異性，並且評估胞攝作用在各個時間點的影響。透過此生物單分子層級的癌症治療檢測平台的建立，可以作為後續對標靶藥物訊息傳遞機制的驗證參考，希望與其他癌症治療檢測平台有進一步延伸性的互補，使癌症檢測能更趨完善。
並列摘要
According to the statistics from Bureau of Health Promotion, lung cancer was the leading cause of death in Taiwan since 1982. Among the cancer-related death in Taiwan, lung cancer was the second in male-group, and was the first in female-group, the research of lung cancer will become important marker for national health in the future. The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The occurrence of NSCLC accounts for as many as 85% to 88% of cases. The primary treatment for NSCLC usually involves chemotherapy and targeted therapy. Because the epidermal growth factor receptor (EGFR) is highly expressed in 88 to 99% of NSCLC, EGFR-tyrosine kinase has become a particularly promising targeted drug for treating NSCLC. Because the EGFR signaling pathway triggered a series of dynamic response of endocytosis, studies of epidermal growth factor receptor tyrosine inhibitor can be used as indicators. How the endocytosis triggering by EGFR signaling pathway through the three-dimensional network, which composed of the cell membrane and cytoskeleton will become the focus of recent research projects. Actin filaments constitute a major part of the cytoskeleton within cells, and the main function is related to the cell migration and intracellular trafficking. Recently, using dual labeling fluorescence microscopy revealed that actin participates in the endocytosis through a dynamic network of both the actin filaments and the extracellular matrix. In this study we utilize the biofunctionalized quntum dots conjugated with the epidermal growth factor (EGF) and QD-EGF is gradually transported into A431 cells through binding to EGF receptors. We further apply three drugs including PD153035 (inhibitor of erb-2 tyrosine kinase activity), cytochalasin D (inhibitor of actin polymerization) to quantify the efficiency of receptor-mediated endocytosis through actin filaments at different spatial-temporal stages. Hence, the EGFR targeted drug and the corresponding signal transduction pathway could be validated using this single-molecule approach.
參考文獻
延伸閱讀
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