牛囊胚ICM转录组分析及体外培养
发布时间：2018-03-11 21:25
本文选题：差异基因 切入点：多能性标记 出处：《甘肃农业大学》2015年硕士论文 论文类型：学位论文
【摘要】：牛胚胎干细胞(embryonic stem cells,ES)在畜牧业中具有重要的应用价值。但是目前,公认的牛ES细胞系仍未成功建立,其主要原因是牛ES细胞多能性维持机理尚不清楚。随着第二代测序技术(如Illumina,454和SOLID)的快速发展,RNA-Seq已成为研究基因表达和转录组分析新的重要手段。本试验利用RNA-seq筛选牛囊胚ICM和TE的差异表达基因并体外培养牛囊胚ICM克隆,旨在探究牛ES细胞多能性维持机制以及牛类ES细胞的多能性标记基因与表面标记,为优化牛类ES细胞培养条件和相关研究提供依据。试验一:基于RNA-Seq技术的牛囊胚ICM与TE差异表达基因筛选本研究利用免疫磁珠分选和新一代高通量测序技术RNA-Seq,筛选牛囊胚ICM和TE之间的差异表达基因,并经qRT-PCR验证,进一步探索与维持牛胚胎干细胞多能性相关的特异基因、转录因子与细胞信号通路。试验中包括ICM和TE两个组,每个组包含三个重复,采用DESeq2分析测序数据,结果筛选出207个组间显著差异表达基因(Padj≤0.05并且|log2Ratio|≥1),其中159个基因在ICM中上调表达,48个基因在ICM中下调表达。筛选出14条显著富集于生物学过程的GO功能条目(P-value≤0.05),以及12条显著富集的Pathway(P-value≤0.05)。这些通路的功能涉及细胞命运的控制、细胞分化以及细胞多能性的维持和自我更新。试验二:牛囊胚ICM克隆的体外培养利用2i/LIF培养液,通过全胚接种及机械传代法分离与培养牛囊胚ICM;用免疫荧光染色法检测其多能性标记基因与表面标记;并通过qRT-PCR检测免疫磁珠法分选获得的牛囊胚ICM和TE的多能性标记基因的差异表达。试验成功分离出了牛囊胚ICM克隆,并体外培养至第10代。结果表明,ICM克隆表面标记SSEA1、SSEA4和TRA-1-60染色为阳性,且多能性标记基因OCT4、SOX2和NANOG在其中均有表达;同时q RT-PCR结果显示SOX2在牛囊胚ICM和TE中的表达差异最为显著(P㩳0.01)。综上所述,2i/LIF培养液有助于牛囊胚ICM的培养;SOX2很有可能成为牛ICM克隆的候选多能性标记基因。
[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，
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