- 學位論文
探索小鼠視覺皮質層中星形膠質細胞的印記體
Determination of Dynamic Imprintome in the Astrocytes of Mouse Visual Cortex
摘要
基因組印記是一個表觀遺傳的機轉，經由此模式，基因的表達會受制於父方或母方來源的染色體之影響。基因組印記主要發生在神經系統，其機制上的異常，會造成神經或精神上的疾病。然而，基因組印記在腦中的作用，至今尚未釐清。印記基因的表達是相當動態的。不同時間、空間、外在環境以及細胞型態都會調控其表現情形。此外，星形膠質細胞在哺乳類腦中佔據近半成的體積，並在神經系統扮演重要的角色。縱然如此，基因組印記的研究一直以來是以神經元為主，而其在星形膠質細胞的表現則完全未知。為了瞭解基因組印記的動態特性，又其對星形膠質細胞的影響，視覺系統（視網膜、視叉上核、視覺皮質層）對光的依賴性及可塑性強的特色，提供了合適的研究目標。本研究立足於不同的環境調控、以及發育時間點，探討動態基因組印記在視覺系統及視覺皮質層內的星形膠質細胞的表現情形。利用次世代定序技術，我們首先找出老鼠視覺系統在正常環境飼養(12小時光照、12小時黑暗)以及無光線飼養(24小時全暗)的情況下特有的印記體，並找出其中受光線調控的印記基因。在細胞層次，利用基因轉殖鼠及單細胞採集的方法，我們也發現了一些潛在星形膠質細胞中的印記基因。為了更進一步了解基因組印記背後的分子機制，我們依據定序結果及後續確認作業，深入探討Trappc9的不同基因亞型的印記現象。最後，我們擴大研究範圍到人類大腦，建立了一從大體腦組織篩選印記基因的平台。綜上所述，本研究強調了基因組印記在不同組織、細胞形態、環境刺激、基因亞型，及物種的多形性。這些分析結果提供了為什麼大腦要演化出基因組印記這種特殊機制的線索。更重要的，人類印記基因相關疾病或許可從當中找出一些新的治療標的及策略。
並列摘要
Genomic imprinting is an epigenetic process which causes monoallelic gene expression in a parent-of-origin-specific manner. It predominately occurs in the nervous system. Dysregulation of imprinted genes is involved in various neurological and psychiatric disorders, but their roles in the brain are still unclear. Genomic imprinting is spatiotemporally dynamic, and also varies between different experiences and cell types. Moreover, astrocytes occupy near half of the volume of adult mammalian brains and play significant roles in the nervous system. However, a neuron-centric mentality has dominated this field of research, and the genomic imprinting status in astrocytes is totally unknown. In order to investigate the dynamic traits of genomic imprinting and its effects in astrocytes, visual system (visual cortex, suprachiasmatic nucleus, and retina), whose maturation heavily depends on light experience, provides an ideal model to study. In the present study, we determined the dynamic imprintome in the mouse visual system and specific in the visual cortical astrocytes with temporal and experience-regulated resolution. Using deep sequencing, we first profiled the imprinting status in mouse visual system under dark-reared (24hr dark) and normal light-reared (12hr/12hr, dark/light) environment. The region specific imprintomes and some experience-regulated imprinted genes were identified. Next, taking advantage of engineered mice and single cell collecting methods, we discovered some potential imprinted genes expressed in the mouse visual cortical astrocytes. To better understand the molecular mechanisms behind genomic imprinting, we further examined an interesting isoform-specific imprinting effect of Trappc9, based on our RNA-Seq result and follow up validation. Last, we expanded the research horizon from mouse to human brain, and built up a reliable platform to determine the imprintome in the human postmortem brain. In summary, the present study highlights variation in genomic imprinting between tissues, cells, experiences, isoforms, and species. The results of these profiling works may give clues about why brains develop genomic imprinting during evolution. Most importantly, the knowledge gained here may identify new molecular targets and provide therapeutic strategies for genomic imprinting disorders.