Chemogenomic and Molecular Analysis of Signal Transduction Pathways in In Vivo and In Vitro Models

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Title:	Chemogenomic and Molecular Analysis of Signal Transduction Pathways in In Vivo and In Vitro Models
Authors:	林祐詩;Lin,Jack Yu-Shih
Contributors:	系統生物與生物資訊研究所
Keywords:	系統生物學;強啡肽原;京都百科全書基因和基因組路徑資料庫;絲裂原活化蛋白激酶;信號轉導路徑;Chemogenomic;Dynorphin;PC12 cell;Systems Biology;KEGG;inflammation
Date:	2016-01-26
Issue Date:	2016-03-17 20:35:39 (UTC+8)
Publisher:	國立中央大學
Abstract:	系統生物學方法與一般研究常規模式的差別，在於它具有能通過在不同層次上的組織而非單個分子和細胞要素之間的靜態交互運作，進而能瞭解這些分子和細胞要素在動態系統下展現出生物路徑的行為和機制的運作。在這項研究中，我建立了體內和體外模型來研究每個系統中的信號轉導路徑。在體內模型的研究中，我建立了大鼠模型，並在其新生兒後腳掌給予化學刺激後，研究神經元在非主體的週邊有害刺激下的靈敏度，評估其長大時在非主體的週邊給予同樣化學刺激後，生理和分子方面疼通的行為變化。在與受刺激腳掌同側之脊髓中的背根神經節，我們注意到強啡肽原（proDYN）基因的增加。從磷酸-ERK（pERK）活性的增加，進而瞭解到在非主體的週邊刺激發炎情況下，胞外信號調節激酶（ERK）路徑會明顯活化。這表示新生兒非主體的週邊刺激發炎可透過絲裂原活化蛋白激酶（MAPK /ERK）生物路徑中的強啡肽原進而改變疼痛處理的路徑。由於動物模型的複雜性，我們亦利用PC12細胞培養系統建立體外模型，來研究信號轉導機制。在該體外模型中，我利用分子生物學的思維方式，以及高通量的實驗和系統生物學分析，去研究在神經生長因子（NGF）的誘導下PC12細胞的神經元分化和強啡肽原基因的表現及生物路徑。我們利用京都百科全書基因和基因組路徑資料庫（KEGG），將我們的陣列實驗數據與該資料庫中的基因資料庫及生物路徑資料庫數據進行比對。交叉比對後產生了830個表現有改變的基因，其中395基因更有兩倍以上的明顯改變，另外也比對出191個有關聯的生物路徑。其中四個最活躍的途徑包括絲裂原活化蛋白激酶（MAPK）路徑，軸索引導通路 (Axon Guidance Pathway)，Wnt通路，以及神經營養因子路徑 (Neurotrophin Pathway)。因此，這些研究顯示，在NGF誘導的神經元分化和炎症引起的疼痛調節現象中，多個信號轉導路徑會被活化，其中MAPK / ERK路徑的表現為明顯。此外，就像在體外的PC12模型系統中，神經生長因子也可在體內動物模型系統中，調節非主體的週邊刺激發炎所引起的疼痛反應。;Systems biology methodologies have the advantages of supplementing the conventional mode of study by facilitating the understanding of biological pathways and mechanisms in terms of their dynamic system behavior on different levels of organization rather than the static interaction among the individual molecular and cellular elements. In this study, I set up an in vivo and in vitro models to study the signal transduction pathways in each system. In the in vivo model, I investigated neuronal sensitivity to a noxious stimulus in a rat model of neonatal hind-paw peripheral inflammation and assessed changes in pain behavior at the physiological and molecular levels after peripheral reinflammation in adulthood. After reinflammation, an increase in the expression of the prodynorphin (proDYN) gene was noted in the spinal cord ipsilateral to the afferents of the neonatally treated hind paw. The involvement of the activation of extracellular signal-regulated kinases (ERK) in peripheral inflammatory pain hypersensitivity was evident by the increase in phospho-ERK (pERK) activity after reinflammation. This indicates that peripheral inflammation in neonates can alter the pain processing pathway through the activation of the MAPK/ERK pathway through the expression of the dynorphin. Due to the complexity of the animal models, signal transduction mechanisms are also examined in the PC12 cell culture systems. In this in vitro model, I investigated through the molecular biology paradigm, as well as high throughput experiments and system biology analysis, the genes and pathways associated with Nerve Growth Factor (NGF) induced neuronal differentiation, as PC12 cells were also known to produce dynorphin after NGF stimulation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to match the data from microarray experiments to biologic networks in the KEGG database. Cross-matching to the KEGG gene database resulted in 830 genes. Among these, I identified 395 genes with their expressions significantly altered compared to the baseline expressions. I also identified 191 involved biologic pathways in the KEGG database. The most active pathways include the mitogen-activated protein kinase (MAPK) pathway, axonal guidance pathway, the Wnt pathway, and the neurotrophin pathway. Thus, these studies showed that in both the NGF induced neuronal differentiation and inflammation induced pain modulation, multiple pathways were activated including, most notably, the MAPK/ERK pathway. As in the in vitro PC12 model system, NGF may also play a role in the inflammation induced pain modulation in the in vivo animal model system.
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