發(fā)布時(shí)間：2018-03-11 21:25

  本文選題：差異基因　切入點(diǎn)：多能性標(biāo)記　出處：《甘肅農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：牛胚胎干細(xì)胞(embryonic stem cells,ES)在畜牧業(yè)中具有重要的應(yīng)用價(jià)值。但是目前,公認(rèn)的牛ES細(xì)胞系仍未成功建立,其主要原因是牛ES細(xì)胞多能性維持機(jī)理尚不清楚。隨著第二代測(cè)序技術(shù)(如Illumina,454和SOLID)的快速發(fā)展,RNA-Seq已成為研究基因表達(dá)和轉(zhuǎn)錄組分析新的重要手段。本試驗(yàn)利用RNA-seq篩選牛囊胚ICM和TE的差異表達(dá)基因并體外培養(yǎng)牛囊胚ICM克隆,旨在探究牛ES細(xì)胞多能性維持機(jī)制以及牛類ES細(xì)胞的多能性標(biāo)記基因與表面標(biāo)記,為優(yōu)化牛類ES細(xì)胞培養(yǎng)條件和相關(guān)研究提供依據(jù)。試驗(yàn)一:基于RNA-Seq技術(shù)的牛囊胚ICM與TE差異表達(dá)基因篩選本研究利用免疫磁珠分選和新一代高通量測(cè)序技術(shù)RNA-Seq,篩選牛囊胚ICM和TE之間的差異表達(dá)基因,并經(jīng)qRT-PCR驗(yàn)證,進(jìn)一步探索與維持牛胚胎干細(xì)胞多能性相關(guān)的特異基因、轉(zhuǎn)錄因子與細(xì)胞信號(hào)通路。試驗(yàn)中包括ICM和TE兩個(gè)組,每個(gè)組包含三個(gè)重復(fù),采用DESeq2分析測(cè)序數(shù)據(jù),結(jié)果篩選出207個(gè)組間顯著差異表達(dá)基因(Padj≤0.05并且|log2Ratio|≥1),其中159個(gè)基因在ICM中上調(diào)表達(dá),48個(gè)基因在ICM中下調(diào)表達(dá)。篩選出14條顯著富集于生物學(xué)過(guò)程的GO功能條目(P-value≤0.05),以及12條顯著富集的Pathway(P-value≤0.05)。這些通路的功能涉及細(xì)胞命運(yùn)的控制、細(xì)胞分化以及細(xì)胞多能性的維持和自我更新。試驗(yàn)二:牛囊胚ICM克隆的體外培養(yǎng)利用2i/LIF培養(yǎng)液,通過(guò)全胚接種及機(jī)械傳代法分離與培養(yǎng)牛囊胚ICM;用免疫熒光染色法檢測(cè)其多能性標(biāo)記基因與表面標(biāo)記;并通過(guò)qRT-PCR檢測(cè)免疫磁珠法分選獲得的牛囊胚ICM和TE的多能性標(biāo)記基因的差異表達(dá)。試驗(yàn)成功分離出了牛囊胚ICM克隆,并體外培養(yǎng)至第10代。結(jié)果表明,ICM克隆表面標(biāo)記SSEA1、SSEA4和TRA-1-60染色為陽(yáng)性,且多能性標(biāo)記基因OCT4、SOX2和NANOG在其中均有表達(dá);同時(shí)q RT-PCR結(jié)果顯示SOX2在牛囊胚ICM和TE中的表達(dá)差異最為顯著(P�d0.01)。綜上所述,2i/LIF培養(yǎng)液有助于牛囊胚ICM的培養(yǎng);SOX2很有可能成為牛ICM克隆的候選多能性標(biāo)記基因。
[Abstract]:Bovine embryonic stem cells have important application value in animal husbandry. However, the established bovine es cell line has not been successfully established. With the rapid development of the second generation sequencing techniques (such as Illumina454 and SOLID), RNA-Seq has become a new and important method to study gene expression and transcriptome analysis. The differentially expressed genes of bovine blastocyst ICM and te were screened and the ICM clones of bovine blastocysts were cultured in vitro. The aim of this study was to explore the maintenance mechanism of bovine es cell pluripotency, and the pluripotent marker genes and surface markers of bovine es cells. Experiment 1: screening differentially expressed genes between ICM and te in bovine blastocysts based on RNA-Seq technique. This study used immunomagnetic bead sorting and a new generation of high-throughput sequencing technology RNA-Seq. screening. The differentially expressed genes between ICM and te in bovine blastocysts were selected. The specific genes, transcription factors and cell signaling pathways related to maintaining the pluripotency of bovine embryonic stem cells were further explored by qRT-PCR. The experiment included two groups, ICM and te, each group containing three repeats. DESeq2 was used to analyze the sequencing data. Results two hundred and seven distinct differentially expressed genes (Padj 鈮，

本文編號(hào)：1599957