2008-12-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/710482摘要：後生遺傳/上位遺傳(Epigenetics)乃一正常之生理現象，其在不影響DNA 序列的前提 下，經由DNA 甲基化、組蛋白(Histone)修飾及功能性RNA 之作用途徑，影響特定基因之表 現。此等上位遺傳對於基因體之修飾作用即為幹細胞之所以有別於已分化體細胞之差異所 在。後生遺傳於各類細胞間不同之印記是具有可逆性的。這種可逆性是謂幹細胞及再生醫學 研究之關鍵課題。 聯繫親代與子代之生殖細胞，尤其需要經由大規模之後生遺傳再程序化作用，以達到精 卵之全分化潛能。此等再程序化包含了在始基生殖細胞形成與遷徙初期之全基因組DNA 去甲 基化ˋ重新啟動失去活性的X 染色體ˋ去除上一代之基因印痕，及在精卵生成過程中全基因 組DNA 甲基化ˋ重新根據配子性別而設定親源特異性之基因組印痕標記等。由於生殖細胞形 成初期之數量相當稀少且採樣困難，對於上述後生遺傳再程序化之分子機制的探討一直是非 常大的挑戰。近年來藉助後生遺傳及生物科技之快速發展，以小鼠為動物模式對於上述課題 已有初步瞭解。然而此等試驗在人類不可能進行，因此於離體培養系統中模擬人類生殖細胞 形成與配子發育過程之模式的開發，即成為研究這些重要後生遺傳機制之最佳材料。 本子計畫將充分利用此整合型計劃之其他三個子計畫於體內及體外誘導分化前後之人類 胚幹細胞為材料，研究人類生殖細胞形成過程中之後生遺傳再程序化的調控機制。吾等將經 由分析誘導分化前後：基因印痕之再程序化現象，基因(coding gene)及功能性RNA（尤其是 microRNA）表達質與量之差別，DNA 甲基化，與各式組蛋白修飾(各個組蛋白不同胺機酸之甲 基化及乙醯化)之差異，深入探究誘導人類胚幹細胞分化為生殖細胞暨精卵之分子與後生遺 傳(Epigenetic)調控機制。此等研究之成果，可對後生遺傳機制維持各式幹細胞之多分化潛 能及調控幹細胞分化，帶來突破性之瞭解。這些知識勢必顯著提升以幹細胞為中心之再生醫 學成功率。<br> Abstract: Being the link between generations, germ cells have to undergo dramatic epigenetic reprogramming in order to obtain the pluripotency. These include genome-wide DNA demethylation followed by the re-establishment of DNA methylation marks with different timing in the two germ lines; the erasure of the parental specific genomic imprinting mark and re-establishment of new imprint marks according to the gender of the new generation, and reactivation of the inactivated X chromosome. The ambition of differentiating embryonic stem cells into oocytes or sperms in vitro is therefore the most challenging one. There have been intensive molecular and epigenetic studies of early germ cell determination as well as gametogenesis in mice over the last several years. However, similar experiments can not be performed in human. Establishing an in vitro model system that can recapitulate human germ cell development would therefore be an excellent tool to study the epigenetic programming/ reprogramming events from human germ cell formation and gametogenesis. Taking advantage of the in vitro model differentiating human ES cells into primordial germ cells, and the in vitro spermatogenesis and oogenesis attempted by Subproject 4, 2, 1 of this integrated program, respectively, we will carefully characterize the above mentioned epigenetic reprogramming events. We will also identify novel epigenetic regulatory pathways through the unbiased array based techniques. Based on the knowledge we learned from mice studies, we will also use the epigenetic landmark as a criteria to choose the best induction protocols for PGC formation and in vitro gametogenesis. Morover, we will artificially manipulate the epigenome of the differentiating ES cells in order to facilitate the formation of primordial germ cells. These include, but not limited to, the temporal usage of the DNA demethylating agent, 5-AZA, to mimic the demethylated state of PGCs when they enter the genital ridge. We shall be able to figure out many previously unidentified epigenetic regulatory pathways upon ES cell and GSC differentiation towards oocytes and sperm. We will also have the opportunity to reveal the genetic and epigenetic regulatory network from the initial epigenetic modification to the cascades that trigger stem cells to differentiate into different lineages. These analyses can significantly facilitate the establishment of the most efficient in vitro differentiation procedures attempted by researchers from the other projects. Moreover, the result from these studies will elucidate the mechanisms behind cell plasticity in general and will therefore provide a big breakthrough and can easily be applied to the fields of nuclear reprogramming, stem cell research and regenerative medicine.DNA 甲基化後生遺傳生殖細胞人類胚幹細胞DNA methylationEpigeneticsGerm cellsHuman Embryonic Stem cells人類胚胎幹細胞與生殖腺幹細胞發育為生殖細胞過程中之基因轉殖、分化與後生調節-子計畫三：離體分化生殖細胞之後生遺傳調控(2/3)