發(fā)布時間：2018-03-11 21:25

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

【摘要】：牛胚胎干細胞(embryonic stem cells,ES)在畜牧業(yè)中具有重要的應(yīng)用價值。但是目前,公認的牛ES細胞系仍未成功建立,其主要原因是牛ES細胞多能性維持機理尚不清楚。隨著第二代測序技術(shù)(如Illumina,454和SOLID)的快速發(fā)展,RNA-Seq已成為研究基因表達和轉(zhuǎn)錄組分析新的重要手段。本試驗利用RNA-seq篩選牛囊胚ICM和TE的差異表達基因并體外培養(yǎng)牛囊胚ICM克隆,旨在探究牛ES細胞多能性維持機制以及牛類ES細胞的多能性標記基因與表面標記,為優(yōu)化牛類ES細胞培養(yǎng)條件和相關(guān)研究提供依據(jù)。試驗一:基于RNA-Seq技術(shù)的牛囊胚ICM與TE差異表達基因篩選本研究利用免疫磁珠分選和新一代高通量測序技術(shù)RNA-Seq,篩選牛囊胚ICM和TE之間的差異表達基因,并經(jīng)qRT-PCR驗證,進一步探索與維持牛胚胎干細胞多能性相關(guān)的特異基因、轉(zhuǎn)錄因子與細胞信號通路。試驗中包括ICM和TE兩個組,每個組包含三個重復,采用DESeq2分析測序數(shù)據(jù),結(jié)果篩選出207個組間顯著差異表達基因(Padj≤0.05并且|log2Ratio|≥1),其中159個基因在ICM中上調(diào)表達,48個基因在ICM中下調(diào)表達。篩選出14條顯著富集于生物學過程的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胞命運的控制、細胞分化以及細胞多能性的維持和自我更新。試驗二:牛囊胚ICM克隆的體外培養(yǎng)利用2i/LIF培養(yǎng)液,通過全胚接種及機械傳代法分離與培養(yǎng)牛囊胚ICM;用免疫熒光染色法檢測其多能性標記基因與表面標記;并通過qRT-PCR檢測免疫磁珠法分選獲得的牛囊胚ICM和TE的多能性標記基因的差異表達。試驗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克隆表面標記SSEA1、SSEA4和TRA-1-60染色為陽性,且多能性標記基因OCT4、SOX2和NANOG在其中均有表達;同時q RT-PCR結(jié)果顯示SOX2在牛囊胚ICM和TE中的表達差異最為顯著(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：1599957