一、藉由蛋白質體學分析高糖環境下人類肝臟細胞內蛋白質體變化及其參與糖尿病誘導肝臟疾病之相關機制 二、藉由蛋白質體學分析具有pemetrexed抗藥性之人類肺腺癌細胞內蛋白質體變化及參與之相關機制

I. High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease II. Proteomic analysis of proteins responsible for the development of pemetrexed resistance in human lung adenocarcinoma

陳靖宜(Jing-Yi Chen)

指導教授 ： 詹鴻霖

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2014年)

摘要

研究一：藉由蛋白質體學分析高糖環境下人類肝臟細胞內蛋白質體變化及其參與糖尿病誘導肝臟疾病之相關機制

在高血糖下會造成肝臟細胞功能失調而引起許多代謝相關的疾病，其中包括了糖尿病型肝病。過去許多研究指出，高血糖會經由glycoxidation而導致肝臟細胞的損傷，然而，高血糖造成的這些效應的機制至今仍尚未被詳盡地研究出。在本研究中，我們將Chang liver cell分別培養在5.5 mM、25 mM 及 100 mM三種不同濃度葡萄糖培養液中，並利用甘露醇調整其滲透壓，進而利用蛋白質體學的方式研究這三種不同糖濃度培養下的細胞中其蛋白質表現差異及氧化還原的調節。在本研究結果中，我們鑑定到了在不同糖濃度培養下的細胞中，141個蛋白質具有表現量差異、29個蛋白質具有巰基變化。這些具有表現量差異的蛋白質參與了轉錄的調控、訊號傳遞、氧化還原的調控、細胞骨架的調控等功能，而具有巰基變化的蛋白質則是參與了蛋白質摺疊與基因調控等功能。我們進一步利用了臨床上的糖尿病病患與健康捐贈者的血清檢體確認galectin-3、GRP-78、GSTP1等蛋白質在血清中的表現量差異，以驗證蛋白質體學實驗中所得到的結果。總歸來說，本研究中我們利用完善的蛋白質體學平台分析高糖濃度下培養的肝臟細胞中，其蛋白質表現量差異以及巰基的變化，鑑定出了與糖尿病型肝病之病程發展相關的蛋白質，並且利用臨床檢體進行進一步的確認。我們認為，這些鑑定出的蛋白質未來在糖尿病型肝病的預測與診斷當中，將會成為非常有潛力的生物分子標靶。

研究二：藉由蛋白質體學分析具有pemetrexed抗藥性之人類肺腺癌細胞內蛋白質體變化及參與之相關機制

目前世界衛生組織統計資料中顯示，肺癌是全球十大死因之一，而在國內更是位居癌症死亡率之首，由此可見肺癌的治療與預防在現今的社會當中是相當迫切且不容小覷的。雖然目前肺癌用藥的研究與發展已相當蓬勃，但在臨床上的使用仍有很大的限制，其中一個主要的原因就是伴隨著療程時間越久而日趨嚴重的抗藥性問題。肺腺癌是所有肺癌種類中的最大宗，所佔的比例高達百分之四十，而pemetrexed是臨床上治療肺腺癌的第一線用藥之一。
在本研究中，我們使用A549與A549/PEM兩株分別對pemetrexed敏感以及具有抗性的細胞株，利用蛋白質體學的方式研究這兩種不同特性的細胞其蛋白質表現差異以及可能參與的抗藥性機制。在本研究結果中，我們鑑定到在A549處理pemetrexed之後，有81個蛋白質具有顯著的表現量差異，但在處理pemetrexed的A549/PEM中卻沒有顯著的變化；反之，在皆沒有處理pemetrexed的情況下，A549與A549/PEM間有72個蛋白質具有顯著的表現量差異，而這些具有表現量差異的蛋白可協助我們進一步探究造成pemetrexed抗藥性的相關機制。
此外，我利用siRNA干擾技術將Calreticulin、Flavin reductase以及Progesterone receptor component 1三個蛋白的表現抑制後，發現可以有效地抑制掉A549/PEM細胞所具有的抗藥性，降低細胞的存活率以及增加細胞凋亡。我們認為，這些鑑定出來在抗藥性細胞中具有差異表現的蛋白質，未來在pemetrexed抗藥性疾病的診斷當中，將會成為非常有潛力的生物分子標靶。

關鍵字

蛋白質體學

並列摘要

Project I: High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease

Hyperglycemia can cause a variety of abnormal disorders in liver cells, one of these abnormalities is diabetic liver disease. Previous study has shown that high glucose concentration in blood can damage liver cells via glycoxidation. However, the details of molecular mechanisms underlying the effects of high glucose concentration in blood in the development of diabetic liver disease have not to be elucidated. In this study, we incubated a liver cell line (Chang liver cell) in mannitol-balanced 5.5 mM, 25 mM and 100 mM glucose media and assessed protein expressional levels and redox-regulations. We identified 141 proteins that showed significant alterations in protein expression and 29 proteins that showed significant alterations in thiol reactivity in response to high glucose concentration. According to the proteomic results, several proteins involved in transcription-control, signal transduction, redox regulation, and cytoskeleton regulation have showed significant alterations in expression. However, proteins involved in protein folding and gene regulation have displayed significant alterations in redox regulations, related to the thiol reactivity. Further analyses by utilizing clinical plasma specimens confirmed that the proteins showed type 2 diabetic liver disease-dependent changes, such as galectin-3, GRP-78, GSTP1, etc. In conclusion, in this study we used a comprehensive hepatocyte-based proteomic approach to identify the high glucose concentration-induced alterations in protein expressional level and to identify redox-associated diabetic liver disease markers. Several identified proteins were validated with clinical samples and might serve as potential targets for the prognosis and diagnosis of diabetic liver disease.

Project II: Proteomic analysis of proteins responsible for the development of pemetrexed resistance in human lung adenocarcinoma

Lung cancer occupies the top 7 leading causes of death in the world and is the top one deadliest cancer in domestic. However, cancer drug resistance is one of the major reasons which cause the failure of chemotherapy in lung cancer. To comprehend the more detailed mechanisms of drug resistance for lung cancer, we used pemetrexed-sensitive-A549 cells and pemetrexed-resistant A549/PEM cells to examine the pemetrexed-resistance-dependent cellular responses and to identify the potential therapeutic targets for drug resistance. We combined two-dimensional differential gel electrophoresis (2D-DIGE) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to investigate the global protein expression alterations induced by pemetrexed treatment and pemetrexed resistance. A proteomic study indicated that pemetrexed-exposure changed the expressions of 81 proteins in A549 cells, whereas no significant response took place in treated A549/PEM cells, indicating these proteins are associated with drug specific resistance. Moreover, 72 proteins demonstrated differentially expressional levels between A549 cells and A549/PEM cells regarding as baseline resistance.
Further studies have used siRNA silencing to against calreticulin, flavin reductase and membrane-associated progesterone receptor component 1 (PGRMC1) proteins, to examine and evaluate their potency in the formation of pemetrexed resistance. The proteomic approach allowed us to identify numerous proteins which are involved in a variety of drug-resistance-forming mechanisms. In this study, we provide useful therapeutic candidates and diagnostic markers for the treatment of pemetrexed-resistant lung cancer.

並列關鍵字

Proteomics

參考文獻

[2] Gillery P. [Oxidative stress and protein glycation in diabetes mellitus]. Ann Biol Clin (Paris). 2006;64:309-14.

[3] Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation. 2006;114:597-605.

[4] Brownlee M. Advanced protein glycosylation in diabetes and aging. Annual review of medicine. 1995;46:223-34.

[7] Yang H, Jin X, Kei Lam CW, Yan SK. Oxidative stress and diabetes mellitus. Clinical chemistry and laboratory medicine : CCLM / FESCC. 2011;49:1773-82.

[8] Nakajima K, Yamauchi K, Shigematsu S, Ikeo S, Komatsu M, Aizawa T, et al. Selective attenuation of metabolic branch of insulin receptor down-signaling by high glucose in a hepatoma cell line, HepG2 cells. The Journal of biological chemistry. 2000;275:20880-6.