發(fā)布時(shí)間：2018-03-13 14:08

  本文選題：小鼠　切入點(diǎn)：胚胎　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：DNA去甲基化參與哺乳動(dòng)物的諸多生命過(guò)程,在早期胚胎發(fā)育和原始生殖細(xì)胞生成過(guò)程中發(fā)揮重要作用。在過(guò)去幾十年的研究中有關(guān)DNA主動(dòng)去甲基化的機(jī)制引發(fā)了很多討論和關(guān)注,但很多觀點(diǎn)仍存在爭(zhēng)議。Tet蛋白以及它的氧化產(chǎn)物5hmC的發(fā)現(xiàn),為DNA主動(dòng)去甲基化機(jī)制的研究開辟了全新的視野。5hmC是重要的表觀遺傳標(biāo)記,它主要作為一種中間產(chǎn)物,參與DNA去甲基化過(guò)程。本論文研究了Tet蛋白在小鼠早期胚胎DNA去甲基化過(guò)程中的作用,為深入了解Tet蛋白的功能提供了理論基礎(chǔ),并且可進(jìn)一步了解小鼠早期胚胎發(fā)育過(guò)程中DNA主動(dòng)去甲基化所依賴的機(jī)制。本研究首先系統(tǒng)驗(yàn)證了小鼠早期胚胎發(fā)育過(guò)程中5mC和5hmC在體內(nèi)受精胚胎和孤雌激活胚胎中的定位及動(dòng)態(tài)變化趨勢(shì),發(fā)現(xiàn)：受精卵中5mC和5hmC呈不對(duì)稱分布,從PN1到PN5時(shí)期雌原核5mC水平緩和下降,而5hmC水平緩和上升,雄原核5mC水平劇烈下降,而5hmC水平劇烈上升。孤雌激活胚胎在原核期發(fā)育階段僅有少量的DNA 5hmC發(fā)生,但在2-細(xì)胞期出現(xiàn)劇烈的DNA 5hmC重編程,說(shuō)明父本基因組大量的5hmC生成主要發(fā)生于1-細(xì)胞時(shí)期,而母本基因組大量的5hmC生成主要發(fā)生于2-細(xì)胞時(shí)期。進(jìn)一步檢測(cè)發(fā)現(xiàn)2-細(xì)胞時(shí)期Stella含量會(huì)顯著下降,這可能減弱了其對(duì)母本基因組的保護(hù)作用從而有利于Tet蛋白發(fā)揮其羥甲基化的作用。從2-細(xì)胞期到囊胚期發(fā)育過(guò)程中,孤雌激活胚胎和體內(nèi)受精胚胎在5mC、5hmC以及Tet家族基因表達(dá)上呈現(xiàn)類似的動(dòng)態(tài)變化模式,但孤雌激活胚胎的5hmC水平和Tet基因表達(dá)水平普遍低于體內(nèi)受精胚胎,顯示出二者在DNA甲基化重編程過(guò)程中的差異。通過(guò)進(jìn)一步的研究發(fā)現(xiàn),無(wú)論是孤雌激活胚胎還是體內(nèi)受精胚胎,2一細(xì)胞晚期的5hmC水平顯著高于早期,說(shuō)明在2-細(xì)胞胚胎發(fā)育過(guò)程中有大量的5hmC的生成。為了研究這一現(xiàn)象與Tet蛋白的相關(guān)性,通過(guò)RNAi實(shí)驗(yàn)下調(diào)Tet基因的表達(dá)發(fā)現(xiàn)：孤雌激活2-細(xì)胞胚胎和受精2-細(xì)胞胚胎DNA 5hmC的生成均受Tet基因的調(diào)控,在孤雌激活胚胎中Tetl的作用更強(qiáng),而在體內(nèi)受精胚胎中Tetl和Tet3的作用更強(qiáng)。說(shuō)明Tet基因不僅介導(dǎo)1-細(xì)胞胚胎DNA 5hmC的生成,亦可介導(dǎo)2-細(xì)胞胚胎DNA 5hmC的生成。同時(shí)RNAi實(shí)驗(yàn)還發(fā)現(xiàn),Tet基因的下調(diào)會(huì)顯著影響胚胎發(fā)育的囊胚率,說(shuō)明Tet家族在胚胎發(fā)育過(guò)程中發(fā)揮比較重要的作用。最后本實(shí)驗(yàn)利用體細(xì)胞核移植技術(shù),將小鼠胎兒成纖維細(xì)胞轉(zhuǎn)入小鼠2-細(xì)胞胚胎細(xì)胞中,發(fā)現(xiàn)小鼠胎兒成纖維細(xì)胞的5hmC水平顯著提高,說(shuō)明小鼠2-細(xì)胞胚胎細(xì)胞質(zhì)仍然具有調(diào)控DNA5hmC重編程的能力。綜上所述,父本基因組大量的5hmC生成主要發(fā)生于1-細(xì)胞時(shí)期,母本基因組大量的5hmC生成主要發(fā)生于2-細(xì)胞時(shí)期。Tet基因不僅參與1-細(xì)胞時(shí)期5hmC的生成,也參與2-細(xì)胞時(shí)期5hmC的生成。2-細(xì)胞胚胎細(xì)胞質(zhì)具有調(diào)控5hmC重編程的能力。本研究結(jié)果為了解Tet蛋白家族功能及哺乳動(dòng)物早期胚胎DNA去甲基化機(jī)制提供了新的信息。
[Abstract]:Many life processes DNA demethylation in mammals, play an important role in early embryonic development and primordial germ cell formation. In the past several decades of research on the mechanism of DNA initiative to methylation caused a lot of discussion and attention, but many ideas are still controversial.Tet protein and found its oxidation product 5hmC the study of DNA active demethylation mechanism opens up a new field of.5hmC are important epigenetic marks, it is mainly used as an intermediate product, participate in DNA demethylation process. This paper research the Tet protein demethylation function in the process of early mouse embryo DNA, provides a theoretical basis for the understanding the function of Tet protein, and to further understand the mechanism of DNA active demethylation on mouse early embryos. In this study, the system verifies the early mouse embryo 5mC and 5hmC during the development of embryo in vivo and parthenogenetic activation and trends in embryos found that asymmetric distribution of 5mC and 5hmC were fertilized eggs, from PN1 to PN5 during the period of the female pronucleus 5mC level decreased and 5hmC level ease, ease up, the male pronucleus 5mC level in severe decline, and the level of 5hmC rises sharply. The parthenogenetic embryos had only a small amount of DNA 5hmC in prokaryotic developmental stage, but in 2- cell cycle DNA 5hmC reprogramming dramatically, indicating that 5hmC generates a large number of paternal genome occurred mainly in the 1- cell stage and 5hmC generated a large number of maternal genome occurred mainly in the 2- cell stage further. Detection of 2- cells during the Stella content significantly decreased, which may weaken the protective effect of the maternal genome to Tet protein play its polymethylolated role. From the 2- cell stage to blastocyst development. In the process of embryo and in vivo fertilized embryos in 5mC parthenogenetic activation, expression of 5hmC and Tet genes showed similar patterns of dynamic changes, but the parthenogenetic activation level of 5hmC and Tet gene expression levels are generally lower than embryos fertilized embryos in vivo, showed two differences in DNA methylation reprogramming through further process. The study found that both embryos and parthenogenetic activation in vivo fertilized embryos, 2 cells in late 5hmC level was significantly higher than that in early generation of a large number of 5hmC in 2- cells during embryonic development. In order to study the correlation between this phenomenon and Tet protein, the down-regulation of Tet gene expression by RNAi experiments that generate 2- cell embryo and fertilization embryo 2- cells DNA 5hmC parthenogenetic activation are regulated by Tet gene, in stronger embryo Tetl parthenogenetic activation, and Tetl in the embryo in vivo and Tet3 for more That generation of Tet gene is not only mediated by 1- cell embryos DNA 5hmC, can generate 2- cells mediated by DNA 5hmC RNAi. At the same time, embryo experiments also found that down-regulation of Tet gene may influence embryo development rate of blastocyst, indicating that Tet family play a more important role in the embryonic development process. At the end of the experiment using the technology somatic cell nuclear transplantation, mouse fetal fibroblast cells into mouse 2- cells in embryonic cells, found that mice fetal fibroblast cells significantly increased the level of 5hmC, indicating that 2- cells of Mice Embryonic cytoplasm still has the regulation of DNA5hmC reprogramming. In summary, 5hmC generates a large number of paternal genome occurs mainly in 1- cells, 5hmC genome of creation a large number of mainly generated in.Tet 2- cells in 1- cell 5hmC gene is not only involved in the period of the generation of.2- cells from embryonic 5hmC 2- cells are also involved in the fine period Cytoplasm has the ability to regulate 5hmC reprogramming. The results provide new information for understanding the function of Tet protein family and the mechanism of DNA demethylation in early mammalian embryos.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q954.4
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本文編號(hào)：1606710