- 學位論文
腫瘤生物標誌的特色與利用奈米微粒改良聚合酶連鎖反應
Characterization of Cancer Biomarkers and Development of Nanoparticle Based PCR Platform
摘要
現今許多腫瘤已逐漸發展出腫瘤篩選、檢測和預測腫瘤發展等相關生物標記。利用觀察基因突變，染色體易位，基因表達圖譜等，尋找出與癌症相關的可能生物標記。然而，目前可作為臨床檢測的癌症生物標記仍相當有限，原因是尚未有良好的敏感性與特異性。本研究主要目標，希望在不同的腫瘤中，包含良性前列腺增生症，前列腺癌，膽管癌和胃腸道間質瘤，藉基因分析評估，盼可進一步定義新的生物標記。另外，為提高研究技術聚合酶鏈鎖反應效率，利用奈米級二氧化鈦作為改良反應效能的材料進行反應測試試驗。 論文一中，利用雄激素受體轉染前列腺增生上皮細胞株-1基因表達微陣列分析，篩檢與雄激素刺激相關基因，其中許多與細胞生長、凋亡和增殖等途徑有關的相關基因。 論文二，以免疫組織化學分析法，發現在前列腺癌中基因PRAC的表達異常。其中更發現，PRAC表達是受甲基化影響，且雄激素非依賴型前列腺癌受PRAC表達所調控。 論文三，利用互補去氧核醣核酸微陣列晶片基因表達分析法比較膽管癌與正常膽管組織的差異性。其中，排列前50名的異常表達基因以逆轉錄聚合酶鏈式反應加以驗證結果反應一致。高表達基因包括CLCA3，COL1A2，DCN，GLIPr2和NID1，CYP2C7和低表達基因SLC10A1。這些基因的調控經分析與細胞增殖，凋亡，代謝和細胞週期有關。 論文四中發現ezrin 蛋白在胃腸道間質瘤中普遍均有表達現象。此外，ezrin 蛋白氨 基酸殘基Thr567磷酸化的情況更與KIT活性有相對關係，此可能是觸發某些下游訊息傳導使腫瘤產生的重要因子之一。 論文五，為改善傳統聚合酶鎖反應，利用0.2奈米的二氧化鈦，可有效獲得較高產量的去氧核醣核酸產物。因此，二氧化鈦奈米顆粒可被用作高效添加劑於聚合酶連鎖反應系統中。總結，本論文藉由各種高科技分子基因分析法，定義出許多相關於良性前列腺、前列腺癌、肝膽癌和胃腸道間質瘤新的生物標記，及有效提高去氧核醣核酸產物的改良技術，希望可以進一步提供發展新的研究與治療策略。
並列摘要
The development of different types and forms of biomarkers for screening, detection, and prognostication has revolutionized the management of various tumors. Besides changes in various biochemical entities, genetic mutations, chromosomal aberrations, changes in gene expression patterns have generated a plethora of candidate cancer biomarkers. However, the value of a list of existing candidate cancer biomarkers could be limited by the general lack of sensitivity and specificity exhibited by most of the cancer biomarkers found to date. The overall aim of this thesis is to apply the functional genomic approach to identity the aberrant expression patterns of candidate genes in different solid tumors including benign prostatic hyperplasia (BPH), prostate cancer (PCa), cholangiocarcinoma (CCA) and gastrointestinal stromal tumor (GIST), and to assess the possibility of finding in new biomarkers. On the other part, effect of titanium dioxide (TiO2) nanoparticles in improving the efficiency of polymerase chain reactions (Conventional PCR, qRT-PCR) was explored. In paper 1, array based gene expression analysis in BPH epithelial cell lines identified many androgen responsive genes which provides the base for further understanding of androgen role in BPH. In paper II, immunohistochemical analysis revealed the aberrant expression patterns of PRAC in PCa samples. This identification indicates that PRAC protein could be used as a biomarker for the disease prognosis. Moreover, methylation effects on PRAC gene expression and also androgen-independent regulation of PRAC expression in PCa cells was demonstrated. In paper III, cDNA expression profiling in rat models identified several dysregulated genes in CCA compared with the non-cancerous liver tissue and differentially expressed genes were found to involve in various pathways such as cell proliferation, apoptosis, metabolism and cell cycle. In addition, further validation of up-regulation of CLCA3, COL1A2, DCN, GLIPr2 and NID1, and down regulation of CYP2C7 and SLC10A1 were consistent with microarray expression profiling data. Further studies on these gene sets may provide the basis for understanding the role of candidate genes in the carcinogenesis of disease. In paper IV, expression analysis revealed that GIST samples exhibiting expression of phosphorylated Thr567 in the ezrin protein were highly associated with immunoactivities of KIT and merlin expression respectively which indicates that GISTs with activated KIT protein may induce phosphorylation of the downstream protein ezrin at certain residues, thereby triggering subsequent signal transduction cascades and driving downstream pathways of tumor progression. In paper V, for conventional PCR, the results showed that in the presence of 0.2 nM of TiO2 a significant amount of target DNA could be obtained even with the less initial template concentration. Contrary to expectation, TiO2 NPs were unable to enhance the efficiency of qRT-PCR. Therefore, TiO2 NPs may be used as efficient additives to specifically improve the conventional PCR system. In conclusion, the identified differentially expressed genes in this thesis have generated novel hypothesis regarding the molecular mechanisms involved in the development of BPH, PCa, CCA, and GISTs. Importantly, gene expression profiling identified by cDNA microarray can be further studied to provide important novel biomarkers for the prognosis and for the development of novel therapeutic strategies.