2007-12-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/700438摘要：體細胞核移殖(somatic cell nuclear transfer，SCNT)技術具有將已分化之細胞核再程序化(reprogramming)至全能性(totipotency)階段；而胚幹細胞 (embryonic stem cells, ESC)，具有無限潛能發展為個體各個部位之細胞型態， 結合此等概念而衍生之治療性複製(therapeutic cloning)為研究各類疾病治療模型之利器，為未來建立病人特異性胚幹細胞(patient-specific embryonic stem cells)及再生醫學帶來無限希望。現階段在體細胞移殖研究上，應用新鮮未受精或受精後之成熟減數分裂中期卵(metaphase II oocytes)為目前最有效率之受體細胞來源，然而目前此等研究所遭遇最大的瓶頸亦為受體卵母細胞來源之侷限，以及取卵之道德與成本問題。是故，尋找可利用的卵源即為發展治療性複製研究當下最迫切之問題。 一般在不孕症中心，不孕症患者經過超級排卵(superovlation)後，會將過多之卵母細胞冷凍保存以備未來之使用。目前臨床試驗報告指出，冷凍卵子可成功地被應用為人之不孕症治療。經治療成功後，患者普遍接受捐贈此等過剩的冷凍卵子作為研究之使用。若在法規範圍允許並取得患者之同意捐贈後，此等冷凍卵子不但為治療性複製研究最佳之卵源，且取卵之道德問題亦迎刃而解。然而此等冷凍卵子使否具有再程序化體細胞進而建立胚幹株細胞的能力，仍待進一步釐清。因此，本計畫擬由小鼠卵之玻璃化(vitrification)冷凍技術，首先探討卵之冷凍暨解凍後存活率，再以此等冷凍暨解凍復甦之卵子經由孤雌激活(parthenogenetic activation)，或利用皮膚纖維母細胞（skin fibroblast cells）進行體細胞複製，進而探討此等冷凍卵子之體外(in vitro)胚胎發育、體細胞複製及建立胚幹細胞株之潛能及其可行性。透過本計畫之執行，期將此等技術於小鼠模式中確立，於基礎研究上，除有助於國內在體細胞複製及胚胎幹細胞研究邁向卓越外，於臨床醫學研究上，並可提供不孕症冷凍技術改進及相關試驗研究之基礎，為未來建立病人特異性胚幹細胞株-實現治療性複製之研究提供了最佳動物模式。 <br> Abstract: Nuclear reprogramming by somatic cell nuclear transfer (SCNT) to derive patient-specific embryonic stem cells (ntESCs), which is known as therapeutic cloning, remains a very promising possible medical therapy for many fatal human diseases. At present, the most convincing approach for reprogramming somatic nuclei to the totipotent state is through SCNT using fresh unfertilized or fertilized oocytes; however, a major obstacle for human applications is that volunteer donation of fresh human oocytes is uncertain, but utilization of leftover cryopreserved oocytes donated with informed consents by infertile couples undergoing in vitro fertilization (IVF) who do not wish to keep frozen oocytes anymore for future replacement is possible with minimal ethic concerns. Recent clinical reports showed that cryopreserved oocytes could be successfully used for human fertility treatment. After fertility treatment is successful, patients are more likely to donate their excess leftover cryoperserved oocytes for research. These oocytes may be made available to generate patient-specific ntESCs. However, it is still not clear if the frozen and thawed oocyte function like fresh ones and contain its nuclear reprogramming activity in the cytoplasm. Before applying to human, it is important to have an animal model to exam the feasibility of SCNT and ES cell derivation by using frozen and thawed oocytes. The aim of this study is to test the validity and efficiency of deriving ESCs from cryopreserved mouse oocytes via parthenogenetic activation (self therapy with no NT involved), or carrying out SCNT using the skin fibroblast cells (for any patient). This study will perform a proof-of-principle experiment in mice may serve as a new source for SCNT and ES research in the upcoming future.卵之玻璃化冷凍體細胞核移殖核之再程序化胚幹細胞oocyte cryopreservationnuclear reprogrammingsomatic cell nuclear transfer (SCNT)embryonic stem cell (ESC)