慢性緩和性壓力誘發神經異常之全基因體研究

Abstract

研究背景: 生活的壓力源自於複雜的社交及環境因子，負向壓力常會導致負向情緒的產生因而產生許多精神疾患，或影響到神經-內分泌-免疫系統的平衡造成其他病症。迄今為止，壓力所導致之神經系統之各項功能性變化的機制仍待釐清。研究方法:我們建立了四週的慢性緩和性壓力動物模式以模擬人類在日常生活中所受之壓力源，並引起老鼠的憂鬱焦慮行為。藉每週三項行為測試：轉輪、懸尾、強迫游泳測試來評量老鼠的憂鬱焦慮行為變化，並以核磁共振造影及小動物正子攝影觀察腦部變化。於實驗終點分別收取老鼠眼眶血探測活性氧化物及細胞激素；取尿液進行代謝體分析；取腦部之杏仁核、海馬迴、前額葉皮層、大腦皮質等四個部位，進行全基因體晶片分析及微核糖核酸晶片分析，資料再經GeneSpring GX 與Metacore 軟體加以探討可能參與壓力導致之神經疾患之訊息傳遞路徑。第二部份則藉由靜脈及皮下注射肺癌細胞株來探討情緒對癌症進程與轉移的影響。初步結果:四週後壓力組老鼠較對照組有顯著的體重減輕(9%，p<0.0001)，在行為測試中不動時間增長。腦部基因體的分析顯示，大於兩倍變化且有顯著差異(FDR<0.05)的基因，在前述四部位中各有505、272、51、331個；利用此1005 個基因進行階層式群集分析可明白區分出各腦區及實驗與對照組。大於兩倍變化且有顯著差異的微核糖核酸各有115、61、62、60 個。結果揭露不只大量的基因在龐大的訊息傳遞網路中參與壓力與情緒的調控，微核糖核酸也扮演重要角色。在Metacore 分析的結果中，許多有高倍變化的基因所調控的訊息路徑皆和神經疾病、神經系統發育、神經細胞生長、軸突神經導引、細胞訊息傳遞等功能相關。在癌症進程與轉移研究部份則因多次實驗結果變異太大尚無法下結論。結論:本研究提供了壓力誘發神經異常在分子病理機轉上的新觀點，針對壓力誘發之基因群進行研究，找出專一作用的分子與藥物，或有助於治療相關疾病。

Background: Stressful life events which consist of social or environmental distresses (negative stressors) commonly cause emotional change and thus lead to disorders on psychiatry or other neuro-endocrine-immune axis. The mechanisms of stress-caused functional changes in neuronal system remain unclear. Methods: We utilized 4-week CMS (Chronic Mild Stress) animal model mimicking the daily hassles from social or environments to provoke the unset of depression- or anxiety- like behaviors. Behavior evaluation (wheel-running, tail suspension, and forced swimming tests) were performed every week. MRI and microPET were also performed to observe the change of brain. At the endpoint, mice were sacrificed, the blood were collected for ROS analysis and cytokine array, while the urine were gathered for metabolomic assay, and the four parts including amygdala, hippocampus, prefrontal cortex and cerebral cortex of the brain were collected for whole genome gene and microRNA profiling. The microarray data were analyzed by GeneSpring, Metacore software to explore the potential pathways involved in neuro-pathologenesis under stress. Besides, we injected two lung cancer cell lines both intravenously and subcutaneously to see if emotion has any relations to cancer progression and metastasis. Results: Mice under CMS exhibits reduced motor activity and increased weight loss. The results of gene profiling show there were 505 genes with two-fold change in amygdala, 272 in hippocampus, 51 in prefrontal cortex and 331 in cerebral cortex while microRNA profiling showed 115, 61, 62and 60 microRNAs with two-fold change in these four parts respectively. These total 1005 genes with two-fold change are chosen for hierarchical clustering and can distinguish either the brain parts or treatment. The results reveal its tight connection to emotion regulation to the not only the genes but also the microRNAs. Most of CMS-affected genes are predicted to be involved in great networks related to neurological disease, nervous system development, cell growth, axon guidance, and transduction signaling, as well as many known or unknown genes that reportedly affect psychiatry. But there’s no enough evidence to conclude if emotion’s positively or negatively related to cancer progression and metastasis. Conclusion: These findings might provide insights into the molecular pathological mechanisms contributing to stress-induced neural malfunctions. Searching for compounds or drugs that can target the chronic stress-induced genes may be a potential therapy for related diseases.