CFHiTEA, SyBBi, College of Science, NCU

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實驗室主要發展方向

  1. 提供高通量實驗分析核心設施-基因微陣列核心實驗室(DNA Microarray Core, CFHiTEA, SyBBi, NCU)之操作晶片與分析服務。
  2. 建置疾病發展預測或診斷之基因圖譜,提供臨床醫師治療與用藥建議。
  3. 發展個人化醫療,提供病人最有利的治療方式。
  4. 藉助微陣列整合平台尋找並快速篩選新的治療藥物。
  5. 協助臨床基礎醫學研究實質結合,提供醫師轉譯醫學諮詢與建教合作。


Research Interests

肺腺癌 (Lung Adenocarcinoma)

肺癌是目前全球也是台灣男性和女性癌症死亡的首要原因。其中,非小細胞肺癌是最常見的一種。到目前為止,對非小細胞肺癌的治療仍以手術是最常見的治療首選。但手術治療的結果仍然不能令人滿意。因此,尋找並揭示腫瘤標記是最有效的診斷和治療。因此本實驗室已在台北榮民總醫院組織庫收集了臨床樣本並利用 Affymetrix 微陣列晶片觀察肺腺癌的整體基因表現,並以 block bootstrap 方法作配對實驗之統計分析。

基因微陣列晶片系統(Microarray)和即時定量聚合酶連鎖反應(Quantitative Real-time Polymerase Chain Reaction)的分析顯示罹患肺癌的病人跟肺癌有關的基因。再用細胞學實驗去解開這些基因跟癌症的關聯。例如,在生物訊息分析和驗證下,FLJ10540 在肺癌中是一種新的癌症相關的基因。且 FLJ10540 基因跟 PI3K/Akt 的訊息傳遞路徑是有關的。此外,我們也證實了另外兩種蛋白 COX-2 和 mPGES-1,在 119 例患者經蛋白質表現量的分析結果後發現 COX-2 和 mPGES-1 種蛋白共同過度表現的患者要比單一 COX-2 或 mPGES-1 過度表現的患者之癒後情況更糟糕

總之,需要進一步結合臨床和基礎醫學研究作為癌症治療目標。期許發展出開創性轉譯醫學研究。

Lung cancer is currently the leading cause of cancer death in both men and women worldwide as well as in Taiwan. Among them, non-small-cell lung cancer is the most common one. So far, surgery is still the first choice of treatment for localized non-small-cell lung cancer; however, the result of surgical treatment remains unsatisfactory. It is, therefore, imperative to reveal markers for efficient diagnosis or therapy. There were 60 clinical samples collected in tissue bank of Taipei Veterans General Hospital. Recently, we have applied Affymetrix microarray to monitor global gene expression of lung adenocarcinoma and adapted block bootstrap for using with a paired design to circumvent the within-pair dependence.
 
Systematic microarray and Q-RT-PCR analyses successfully reveal that the proposed re-sampling technique of block bootstrap suits paired design experiments and adequately detects genes with minimal variation in our microarray dataset. These lung cancer related candidates were further analyzed through systematical global measurements using shRNA followed by functional characterization with the goal to unravel their roles in the carcinogenesis. In particular, bioinformatics analysis and empirical validation indicate that FLJ10540 is a novel oncogene and invasion enhancer in lung cancer. FLJ10540-elicited cell migration and invasion are mediated by activation of the PI3K/AKT pathway. Beside that, we also validated another two candidates, COX-2 and mPGES-1 by Western blotting and the 119 patients’ results were showed that the patients with COX-2 and mPGES-1 protein co-expression were present much worse prognostic relevance than the patients with only COX-2 or mPGES-1 over-expression, whether the overall or disease-free survival curve analysis.
 
In summary, further clinical and basic medical studies are needed to evaluate the potential of these discriminators as targets for cancer therapy. The long-term goals are to identify novel targets and cellular pathways involved in lung adenocarcinoma with the self-expectation to make seminal biomedical research advances.
 
J Pathol. 2010; 222 (4): 367-79.
J Thorac Oncol. 2010; 5 (8): 1167-1174.
PLoS ONE. 2009; 4 (4): e5052.
 

肝纖維化 (Liver Fibrosis)

在肝病的形成過程中,先期常發生發炎與肝纖維化,而後期轉變成肝硬化或是肝癌。這是亞洲最常見的疾病。基因微陣列晶片的發展可監控全基因組的轉錄,協助提升對疾病的全面了解。

過去,我們使用二甲基亞硝胺(DMN)誘導大鼠出血性細胞壞死與發炎反應以及肝纖維化。經過6週的時間後,不論是病理組織學,生物化學或即時定量聚合酶連鎖反應定量分析皆證實此誘導肝臟受損之大鼠模式可引發出血性細胞壞死與發炎反應,並形成肝纖維化。藉助基因微陣列晶片實驗,成功鑑定出256個與肝損傷相關的基因。使用資料庫將基因表現分群,再利用基因功能特性及特徵資料庫進一步分類出細胞代謝、細胞生長及外力影響相關基因組。在這些基因中,根據組織病理學,我們鑑定出44出血性細胞壞死與發炎反應相關和62個肝纖維化相關潛在基因與可能之靶標藥物相關研究於20092011年獲得日本、新加坡與美國之學術專利

總之,我們相信此研究中對於出血性細胞壞死與發炎反應和肝纖維化提供了新的生物前景,進而發展出早期肝功能損害的分子標記,可提供基礎研究和臨床應用。

The development of hepatocellular carcinoma (HCC) is generally preceded by cirrhosis, which occurs at the end stage of fibrosis. This is a common and potentially lethal problem of chronic liver disease in Asia. The development of microarrays permits us to monitor transcriptomes on a genome-wide scale; this has dramatically speeded up a comprehensive understanding of the disease process.
 
Here we used dimethylnitrosamine (DMN), a nongenotoxic hepatotoxin, to induce rat necroinflammatory and hepatic fibrosis. During the 6-week time course, histopathological, biochemical, and quantitative RT-PCR analyses confirmed the incidence of necroinflammatory and hepatic fibrosis in this established rat model system. Using the Affymetrix microarray chip, 256 differentially expressed genes were identified from the liver injury samples. Hierarchical clustering of gene expression using a gene ontology database allowed the identification of several stage-specific characters and functionally related clusters that encode proteins related to metabolism, cell growth/maintenance, and response to external challenge. Among these genes, we classified 44 potential necroinflammatory-related genes and 62 potential fibrosis-related markers or drug targets based on histopathological scores. We also compared the results with other data on well-known markers and various other microarray datasets that are available.
 
In conclusion, we believe that the molecular picture of necroinflammatory and hepatic fibrosis from this study may provide novel biological insights into the development of early liver damage molecular classifiers than can be used for basic research and in clinical applications.
Gene Expr. 2006; 13(2): 107-32.
 

系統生物學在傳統中國醫學的應用 (
Traditional Chinese Medicine Application by Systems Biology)
  
肝纖維化是台灣慢性肝病共同潛在致命的疾病,同時影響人口比例超過 4%。然而慢性肝炎會越來越多發展成肝硬化及肝纖維化,但治療的方法卻明顯缺乏。為追求確定且有效的藥物治療,運用肝纖維化的分子標記協助尋找改善患者肝纖維化的藥物是一項重要任務。千百年來,中國人已使用草藥治療各種疾病,目前在西方國家也有日益增長此類治療的趨勢,替代了只靠傳統西方醫學治療的單一方法。在護肝作用的關鍵治療藥物搜尋過程中,中國傳統草藥(GP)在經由組織病理學、血液生化學與基因微陣列晶片分析後被發現極具潛力的候選藥物。結果顯示,GP治療對肝臟發炎或肝纖維化都有明顯的抑制效果。此外,這些實驗的結果在我們的研究當中,能夠幫助我們開啟新的視野與了解人類的疾病,並且可以提供為疾病的治療與診斷之標的基因與未來可發展成治療藥物之預測基因組。相關研究於2006年獲得美國專利,目前也以食品形式於市場銷售

Progression of hepatic fibrosis is a common and potentially lethal problem in chronic liver disease in Taiwan, affecting more than 4% of the population. Incidence of cirrhosis is growing as a result of the widespread occurrence of chronic hepatitis, as well as the evident lack of an established therapy for hepatic fibrosis. The quest for the identification of an effective drug treatment to improve the outcome of patients with liver fibrosis and to develop molecular markers for liver fibrosis diagnosis is an important task. Herbal medicines have been used in Chinese population for thousands of years and there has been a growing trend in Western countries to use as alternative medicine to treat a wide range of diseases, such as inflammatory diseases and chronic liver diseases (including hepatitis and fibrosis). In the course of search for key therapeutic agents for hepatoprotective effect, a traditional Chinese medicine (GP) begins to emerge as the winner from histopathologic and biochemical to microarray data. The results show that treatment of GP can significantly prevent DMN-induced hepatic inflammation and fibrosis. In addition, forty-four necroinflammary related and 62 genes not only set the stage for a functional dissection by our previous study, but also opened up a new perspective on several uncharacterized novel genes linked to the human disease and provided potential targets for the rational development of therapeutic drugs (for example, GP). Finally, we had built a free and publicly accessible website to allow the scientific community to use this information. 

eCAM. 2012; 2012: 256561.  
PLoS ONE. 2013; 8(1): e53988. 


實驗技術改進 (Technological Improvement) 

即時定量聚合酶連鎖反應

微陣列晶片的發展,使我們能夠有機會監測全基因組的轉錄表現量。為了驗證基因微陣列晶片,定量反轉錄聚合酶鏈反應是最常用的方法之一。基因定量分析的是如何在這些研究中正確地納入統計分析是一重要需解決的問題。而在統計分析領域中,利用不同方法做正常化時,可能尋找出的基因標記就會不同。藉助基因微陣列晶片實驗數據,利用 statistical re-sampling 方法減少微陣列基因間的基因表現差異。進而發現 DDX5 是個新的肺癌相關實驗之定量反轉錄聚合酶鏈反應的校正基因。

  

Q-RT-PCR

Background: The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements. Results: Sixty-six Affymetrix microarray slides using lung adenocarcinoma tissue RNAs were analyzed by a statistical re-sampling method in order to detect genes with minimal variation in gene expression. By this approach, we identified DDX5 as a novel internal control for Q-RT-PCR. Twenty-three genes, which were differentially expressed between adjacent normal and tumor samples, were selected and analyzed using 24 paired lung adenocarcinoma samples by Q-RT-PCR using two internal controls, DDX5 and GAPDH. The percentage correlation between Q-RT-PCR and microarray were 70% and 48% by using DDX5 and GAPDH as internal controls, respectively. Conclusions: Together, these quantification strategies for Q-RT-PCR data processing procedure, which focused on minimal variation, ought to significantly facilitate internal control evaluation and selection for Q-RT-PCR when corroborating microarray data.

BMC Genomics. 2007; 8:140.
 
Microarray Probe Redefinition