- 學位論文
幼年期睡眠剝奪對褪黑激素生合成、代謝狀態與認知功能之長期影響：離子影像學、生化學、形態學與行為科學之分析研究
Persisted Effects of Early-Life Sleep Deprivation on Melatonin Biosynthesis, Metabolic Activity and Cognitive Functions: Ionic Imaging, Biochemical, Morphological, and Behavioral Studies.
摘要
幼年期睡眠剝奪 (early-life sleep deprivation, ESD) 是相當鉅大的生活壓力，會導致生物體出現嚴重的代謝障礙與心智失常等問題。已知松果腺 (pineal gland) 之主要分泌產物－褪黑激素 (melatonin) 在代謝狀態與認知功能的調節上，扮演相當重要的角色，緣此，本研究擬深入探討松果腺中職司褪黑激素生合成 (melatonin biosynthesis) 的相關分子路徑 [經正腎上腺素媒介之鈣離子湧入 (norepinephrine-mediated Ca2+ influx) 與胞內蛋白激酶A (protein kinase A, PKA) 活化暨下游環單磷酸腺苷反應元件結合蛋白的磷酸化 (phosphorylation of cAMP-response element binding protein, pCREB)]，是否會因幼年期睡眠剝奪的處理而誘發顯著的改變。研究使用剛斷奶之幼年大鼠 (weaning rats)，將之慢性睡眠剝奪後，置於正常環境下 (光照：黑暗之日週期為12：12) 飼育至成年。飛行式二次離子質譜儀 (time-of-flight secondary ion mass spectrometry, TOF-SIMS)、免疫點墨分析 (immunoblotting)、免疫組織化學法 (immunohistochemistry) 與質譜光度分析法 (spectrometric assay) 分別用來偵測松果腺細胞內鈣離子、正腎上腺素受體 (adenoreceptors)、PKA、pCREB 與血清中褪黑激素濃度的潛在改變。松果腺細胞之生化活性 (bio-energetics) 與生物體之代謝狀態則以細胞色素氧化酶 (cytochrome oxidase, COX) 暨血清中葡萄糖 (glucose)、三酸甘油脂 (triglyceride)、胰島素 (insulin) 與高密度/低密度脂蛋白 (high- and low-density lipoproteins) 之濃度高低加以評估。幼年期睡眠剝奪對認知功能的影響則以生物體在曠場試驗 (open field) 與高腳十字迷津 (elevated-plus maze) 等行為工具的表現加以測試。實驗結果顯示，正常鼠 (normal untreated group) 之松果腺細胞中，其胞內鈣離子與參與褪黑激素生合成的相關蛋白活性表現皆較強，細胞呈現較強的生化能量，血液中褪黑激素的濃度亦比較高，實驗鼠整體呈現正常的代謝狀態與認知功能。然經幼年期睡眠剝奪處理後 (ESD treated group)，其松果腺細胞內不僅鈣離子表現下降，胞內參與褪黑激素生合成之相關蛋白活性表現亦較少，細胞生化活性明顯降低，血液及行為分析檢測都顯示歷經幼年期睡眠剝奪處理之實驗動物，其成年時期之代謝狀態與認知功能皆出現長期且不可逆的異常損傷與反應。考量到松果腺細胞內經鈣離子調控之訊息傳遞路徑抑低 (depressed Ca2+-mediated signaling pathway)，及其導致之褪黑激素分泌不足 (reduced melatonin biosynthesis) 可能與代謝或認知功能失常有正向關連，於孩童時期輔以褪黑激素，或可對改善或避免因幼年期睡眠剝奪所引致之代謝障礙與認知功能失常有顯著貢獻。
並列摘要
Early-life sleep deprivation (ESD) is a serious condition with severe metabolic and mental sequelae. The pineal hormone melatonin plays an important role in homeostatic regulation of metabolic and cognitive function. Considering norepinephrine-mediated Ca2+ influx and subsequent protein kinase A (PKA) activation is responsible for downstream cAMP-response element-binding protein (CREB) phosphorylation and melatonin biosynthesis, the present study determined whether Ca2+ expression, together with the molecular machinery participated in melatonin production would significantly alter after ESD. Weaning rats subjected to chronic ESD and maintained naturally (light:dark cycle = 12:12) to adulthood were processed for time-of-flight secondary ion mass spectrometry (TOF-SIMS), immunoblotting, immunohistochemistry together with spectrometric assay to detect the Ca2+ signaling, adrenoreceptors, PKA, phosphorylated CREB (p-CREB) as well as the serum level of melatonin, respectively. Pineal bio-energetics and metabolic function were determined by measuring the cytochrome oxidase activity and serum level of glucose, triglyceride, insulin, high- and low-density lipoproteins, respectively. The potential changes of cognitive expression following ESD was assessed by behavioral performances in both the open field and elevated-plus maze testing. Results indicated that in normal rats, strong Ca2+ signaling along with intense adrenoreceptors, PKA, and pCREB activities were all detected in pinealocytes. Enhanced Ca2+ imaging and signaling pathway corresponded well with intact bio-energetics, normal melatonin production and standard metabolic and cognitive activities. However, following ESD, not only Ca2+ but also pineal signaling activities were all significantly decreased. Blood and behavioral analyses showed a positive correlation between reduced melatonin level and impaired metabolic / cognitive functions after ESD. As depressed Ca2+-mediated signaling pathway and melatonin biosynthesis are positively correlated with the development of metabolic and cognitive dysfunction, supplementary use of melatonin in childhood may thus serve as a practical way to prevent or counteract the ESD-induced metabolic or cognitive deficiencies.