臺灣博碩士論文加值系統

中文摘要
胚胎能否順利著床取決於著床前期胚胎和子宮內膜的發育狀況，血癌抑制因子(leukemia inhibitory factor)是著床及成功懷孕所必須的因子，但是血癌抑制因子在著床前期胚胎發育所扮演的角色並不十分清楚。本論文針對血癌抑制因子對胚胎發育的調控做研究，首先評估在正常培養狀況下，小鼠囊胚期胚胎型態與著床率之關聯，將在體外培養形成的囊胚依囊胚腔形成的大小以及是否孵化分成三組。分別植入假懷孕母鼠的子宮內，比較胚胎型態，包括囊胚直徑、囊胚細胞數目等與著床率的關聯性，我們的結果指出，囊胚腔較小則內細胞團(inner cell mass)、滋養外胚層細胞(trophectoderm)及內細胞團與滋養外胚層細胞比值皆明顯下降，導致胚胎著床率也下降，已孵化的胚胎會有最高的著床率。藉由體外培養及胚胎植入模式的建立繼續探討於雙原核時期顯微注射血癌抑制因子反意寡核苷酸(antisense)抑制胚胎內血癌抑制因子的表現，並觀察其對著床前期鼠胚發育的影響。注射0.25 fmol血癌抑制因子反意寡核苷酸後，胚胎的發育不受影響，注射0.5、1.0 fmol及2.0 fmol血癌抑制因子反意寡核苷酸後，胚胎繼續發育至桑甚胚期及囊胚期的比例則有意義的降低，注射4.0 fmol血癌抑制因子反意寡核苷酸則胚胎無法發育到四細胞期以上。測定胚胎中血癌抑制因子的免疫活性則有隨著血癌抑制因子反意寡核苷酸注入濃度升高而下降的趨勢。注射2.0 fmol血癌抑制因子反意寡核苷酸後，測量囊胚的直徑、胚胎細胞總數目、內細胞團(inner cell mass, ICM)及滋養外胚層(trophectoderm, TE) 的細胞數目以及內細胞團滋養外胚層比值皆有意義的降低，將注射1.0及2.0 fmol血癌抑制因子反意寡核苷酸後未孵化的囊胚植入的子宮中，各組囊胚著床率皆顯著的降低。添加50 ng/ml的血癌抑制因子於培養液時可以有效的改善注入血癌抑制因子反意寡核苷酸後胚胎受損的情形，本實驗結果指出血癌抑制因子在著床前期的胚胎正常發育上扮演相當重要的角色。

Good embryo development and receptive endometrium are essential factors for embryo implantation. Leukemia inhibitory factor (LIF) is an essential factor for implantation and establishment of pregnancy. However, its role in the development of preimplantation embryos remains controversial. The aim of this study is to assess the effects of LIF on development of preimplantation mouse embryo. We observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. Although there was no significantly different in the implantation rates between the grade III and grade II, grade I was significantly decreased as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE) as compared with grade III. These findings indicated that the expanded and haching blastocyst selections for embryo transfer in in vitro culture were evaluated with the high implantation rate. We successfully established the model of in vitro culture and blastocyst transfer for following experiments of LIF. Changes in preimplantation embryos were determined after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage. The 0.5- or 1.0-fmol treated groups had significantly lower percentages of embryos developed to the morula or blastocyst stage and the 2.0-fmol treated group had significantly lower percentages of embryos developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the 4.0-fmol treated group. Moreover, there was a decreasing trend in the levels of LIF immunoactivity with the increasing amount of LIF antisense oligonucleotide injected. The diameter of blastocysts in the 2.0-fmol treated group was significantly smaller than that in the untreated group. The blastocysts in this group had significantly lower numbers of blastomeres and cells in the ICM or TE and ICM/TE ratio. The 1.0- or 2.0-fmol treated groups had significant lower implantation rates than their corresponding control groups. In the 2.0-fmol groups with supplementing exogenous LIF, significantly lower percentages were also observed in the four-cell, morula, and blastocyst stages. However, blastocysts treated with 50 ng/ml LIF had a significant higher percentage than those in the LIF gene impaired group without LIF supplement. These results indicate that LIF is a critical factor for the normal development of embryos at the preimplantation stages.

表目錄………………………………………………………………3
圖目錄………………………………………………………………4
附錄目錄……………………………………………………………5
重要名詞中英文對照及縮寫表……………………………………6
中文摘要……………………………………………………………7
英文摘要……………………………………………………………8
第一章 文獻回顧………….………………………………………9
第一節 哺乳類動物著床前期胚胎發育的過程………………10
第二節 影響哺乳動物著床前期胚胎發育之因子………………12
第三節 哺乳類動物胚胎著床之機制……………………………15
第四節 囊胚期胚胎型態之評估…………………………………22
第五節 血癌抑制因子之生化特性及生理功能…………………25
第六節 反意寡核苷酸抑制基因表現之原理及應用……………30
第二章 研究動機…………………………………………………35
第三章 材料與方法………………………………………………36
第一節 實驗材料…………………………………………………37
第二節 實驗方法…………………………………………………41
第四章 結果………………………………………………………55
第一節 以小鼠囊胚型態分級評估其著床率……………………56
第二節 血癌抑制因子之反意寡核苷酸對著床前期鼠胚發育及著床的影 響…………………59
第五章 討論…………………………………… …………………66
第一節 以小鼠囊胚型態分級評估其著床率……………………67
第二節 血癌抑制因子之反意寡核苷酸對著床前期鼠胚發育及著床的影響…………………71
第六章 未來研究方向…………………………… ………………80
參考文獻………………………………………… …………………81
表…………………………………………………………………… 117
圖…………………………………………………………………… 124
附錄………………………………………………………………… 134

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