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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 生物科技研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16108
標題: 自然受精及體細胞核移置兔胚之基因表現與發育潛能探討
Characterization of Fertilized and Cloned Rabbit Embryos
作者: Chien-Hong Chen
陳建宏
指導教授: 宋麗英(Li-Ying Sung)
關鍵字: 著床前胚胎,複製兔,轉錄因子Oct-4,轉錄因子Cdx-2,組蛋白H4 lysine 5 乙醯化,組蛋白去乙醯&#37238,抑制劑,
Preimplantation embryos,Rabbit cloning,Oct-4,Cdx-2,H4K5ac,HDACi,
出版年 : 2012
學位: 博士
摘要: 本研究之目的為探討關鍵轉錄因子與後生遺傳修飾於著床前家兔胚胎中之時間及空間上的表現或分布,亦即利用免疫螢光染色法觀察胚胎中Oct-4及Cdx-2之表現及組蛋白H4 lysine 5 乙醯化(H4K5ac)之程度,並以此資訊運用於建立及改善家兔複製之效率。試驗一利用體外培養之兔胚,於不同胚期進行Oct-4 及 H4K5ac 染色,於兔胚中發現兩階段之 Oct-4動態表現模式,其訊號於受精後逐漸下降,於八細胞期達到最低,此後再次增加並於桑椹胚期達到最高。第二階段 Oct-4 動態變化發生於囊胚形成過程中,其訊號於早期囊胚期(eBL)開始下降, 並於擴大囊胚期(expBL)達到最低,爾後於孵化囊胚期(hBL) Oct-4訊號強度再次增加。此外,於孵化囊胚中,內細胞群(ICM)和滋養層細胞(TE) 均可表現Oct-4,但相較於TE,ICM具較高之Oct-4表現訊號。 顯著之H4K5ac訊號出現於受精卵之原核中,但其訊號在四細胞期後開始下降,於八細胞期達到達到最低,H4K5ac訊號於桑椹胚期後逐步恢復,於囊胚形成過程中, TE保持高程度之H4K5ac ,而較低程度之H4K5ac訊號則出現於eBL與expBL之ICM,但於 hBL期, ICM之H4K5ac 恢復其訊號強度並顯著高於TE 。試驗二中,Oct-4和H4K5ac於囊胚形成過程中之動態變化情形再次利用體內獲得之著床前兔胚獲得證實 ,此外,本研究首次於兔胚中觀察 Cdx-2之表現,其訊號可於早期囊胚(D4 eBL)之少數TE細胞核中發現,且逐漸增強但僅表現於TE細胞核中。試驗三比較不同卵子來源對家兔體細胞核移置效率之影響,研究發現從卵巢成熟濾泡收集之卵母細胞相較於從輸卵管收集之卵母細胞較具支持複製胚胎發育之潛能,並具有顯著較高之融合率及囊胚形成率,此外,利用卵巢成熟濾泡收集之卵母細胞已成功產製複製家兔。試驗四應用組蛋白去乙醯酶抑制劑(histone decaetylase inhibitor, HDACi) 改善家兔複製效率,結果顯示,經 tricostatin(TSA)或scriptaid(SCP)處理後,均可顯著增加複製胚胎發育至囊胚期,但囊胚細胞數仍顯著低於對照組之孤雌激活囊胚及受精囊胚,經免疫螢光染色發現,複製兔胚於hBL期具較低程度之H4K5ac及異常Oct-4 表現模式,而TSA處理並無法改顯著增加H4K5ac程度或改善複製胚胎表現 Oct-4之能力,然而SCP處理組則明顯增高H4K5乙醯化程度並具較正常之 Oct-4表現模式,值得注意的是,複製胚胎同時處理TSA和SCP後發現不僅提高囊胚發育能力,並改善 H4K5乙醯化程度及Oct-4 表現能力,其中囊胚細胞數量亦顯著增加至無異於對照組囊胚。本研究顯示 Oct-4、 Cdx-2及 H4K5ac 於著床胚兔胎中之動態變化可提供基礎資訊運用於家兔複製之研究。此外,卵巢濾泡卵母細胞具有較高之潛能支持體細胞核移置後之基因再程序化並成功複製家兔,而合併處理TSA 及SCP則顯著改善複製胚之品質及其發育能力。
The purposes of this study were to document the spatial and temporal distribution of key transcription factors and epigenetic events in preimplantation rabbit embryos derived in vitro or in vivo. The dynamic patterns of Oct-4, Cdx-2 and acetylated histone H4 lysine 5 (H4K5ac) were observed by immunochemistry staining and then used as fundamental information to establish rabbit cloning. In the first series of studies (Chapter 2 and 3), two waves of Oct-4 dynamics in the nuclei were revealed for the first time during rabbit preimplantation development. Oct-4 signal was present in zygotes, decreased gradually and reached its lowest level in 8-celled embryos and increased again in compact morulae (CM). A unique wave of Oct-4 expression was observed during rabbit blastocyst formation, which was down-regulated form CM to the early blastocyst (eBL) stage, bottomed at the expanded blastocyst (expBL) stage and then up-regulated to a level similar to that of the CM stage at the hatching blastocyst (hBL) stage. Differential Oct-4 intensity was observed in the inner cell mass (ICM) and the trophectoderm (TE) cells of hatching blastocysts, i.e. more intensive signal in the ICM than in the TE cells. The H4K5ac signal was initially found with high intensity in the pronuclei of zygotes, decreased at the 4-cell stage, bottomed at the 8-cell stage and gradually regained after the morula stage. During blastocyst formation, the TE cells maintained a high level of H4K5ac throughout the blastocyst stage, while the ICM cells showed a lower level of H4K5ac at eBL and expBL stages, and then resumed at the hBL stage. The dynamics of Oct-4 and H4K5ac patterns during blastocyst formation were confirmed by using in vivo embryos. Moreover, unlike the mouse embryo, Cdx-2 was detected after blastocyst formation and became evident only in the TE cells afterwards. The second series of studies (Chapters 4 and 5) were mainly focused on improving rabbit cloning efficiency using somatic cells as donor nuclei. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. In this study, we found that using follicular oocytes as recipient cytoplasts had greater fusion rate, morula development and blastocyst formation (P<0.05). In combination with the treatments of histone deacetylase inhibitors (HDACi), i.e. tricostatin A (TSA) and scriptaid (SCP), increased blastocyst rates (P<0.05) were observed after treatment of optimized HDACi concentrations. Notably, the synergic effects of TSA and SCP in cloned embryos appeared not only to improve blastocyst development, but also embryo quality indicated by the increased cell number and normalized Oct-4 expression pattern in hBLs. These studies provided fundamental information on the dynamic changes of crucial transcription factors and epigenetic modifications in preimplantation rabbit embryos. The present results also demonstrated that follicular oocytes possessed a higher reprogramming capacity to improve the development of cloned embryos, and the HDACi treatments apparently enhanced embryo quality by improving total cell number and Oct-4 expression.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16108
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