基于单细胞测序的卵丘颗粒细胞中CCND1和CCND2对牦牛卵母细胞发育影响的研究

发布时间：2018-05-02 04:11

本文选题：牦牛 + 单细胞测序　；参考：《西南民族大学》2017年硕士论文

【摘要】：牦牛是我国青藏高原特有的牛种,是当地牧民主要的生活生产资料,但其繁殖和生产性能低下,阻碍着高原地区畜牧业的发展。卵母细胞的体外成熟培养是体外受精、胚胎移植等繁殖技术的基础环节。与体内成熟相比,体外成熟的卵母细胞进行受精后所获得胚胎的发育能力较低,胚胎移植后也面临着妊娠率低、易流产、活产率低等问题。大量研究证明卵母细胞周围的颗粒细胞在卵母细胞的成熟及发育中起着重要的角色,该类细胞可以通过旁分泌途径及缝隙连接为卵母细胞直接提供营养、代谢小分子、第二信使等,间接影响卵母细胞的减数分裂、胞质成熟、受精乃至后续胚胎的发育潜能。因此本研究以牦牛颗粒细胞为研究对象,挖掘了GⅤ期、MⅠ期、MⅡ期的转录组信息,分析其生物学信息,并对细胞周期蛋白D1和D2(CCND1和CCND2)基因与卵母细胞发育关联性,结果如下:1、利用地衣红染色方法从核形态上确定了卵母细胞成熟培养0 h、12 h、22 h时分别获得了GⅤ期、MⅠ期、MⅡ期的卵母细胞。2、对三个时期卵母细胞周围的颗粒细胞转录组测序结果分析显示,GⅤ期、MⅠ期、MⅡ期分别有7540、8491、7520个转录本;其中GⅤ期与MⅠ期差异表达基因有318个上调,304个下调;MⅠ期与MⅡ期差异表达基因有141个上调,334个下调;GⅤ期与MⅡ期差异表达基因191个上调,88个下调;qPCR验证结果与转录组数据结果一致,表明转录组测序数据可靠。3、对CCND1和CCND2进行免疫组化定位、免疫荧光定性、实时荧光定量和免疫印迹分析结果显示:CCND1和CCND2蛋白在颗粒细胞中的免疫组化均呈阳性,且GⅤ期颗粒细胞中的mRNA和蛋白的表达水平显著高于MⅠ期和MⅡ期。4、与卵母细胞成熟和发育的关联性研究结果显示CCND1和CCND2与牦牛卵母细胞体外成熟率和受精后的囊胚率呈正相关。综上,本研究通过单细胞转录组研究首次获得了牦牛卵母细胞成熟过程中其颗粒细胞的基因表达谱。与牦牛卵母细胞成熟和发育能力的关联性分析表明CCND1和CCND2与卵母细胞发育潜能成正相关。由此推测CCND1和CCND2可作为卵母细胞质量和发育潜能评估的生物标记,为筛选优质卵母细胞、改善体外成熟体系以及牦牛卵母细胞发育机制研究奠定基础。
[Abstract]:Yak is a special breed of cattle in the Qinghai Tibet Plateau of China. It is the main production data of local herdsmen, but its reproduction and production performance is low, which hinders the development of animal husbandry in the plateau. In vitro maturation and culture of oocyte is the basic link of in vitro fertilization, embryo transfer and other reproductive techniques. The embryonic development ability of the cells after fertilization is low, and the embryo transfer is also faced with low pregnancy rate, easy abortion and low survival rate. A large number of studies have shown that the granulosa cells around oocytes play an important role in the maturation and development of oocytes, and this kind of cells can be connected to the oocytes through paracrine pathways and gaps. Cells directly provide nutrition, metabolic small molecules, second messenger and so on, which indirectly affect meiosis, maturation of the oocytes, fertilization and the developmental potential of subsequent embryos. Therefore, this study took yak granular cells as the research object, excavated the transcriptional information of G V, M I, M II, and analyzed the biological information and the cell cycle eggs. The relationship between white D1 and D2 (CCND1 and CCND2) gene and oocyte development was as follows: 1, using the red coloring method to determine the maturation and culture of oocyte from 0 h, 12 h, and 22 h to obtain G V, M I, M II stage oocyte.2, and analyze the sequencing results of the granular cell transcriptional group around three stages of oocyte. It showed that there were 754084917520 transcripts in G V, M I and M II, of which 318 up-regulated genes were up and 304 down-regulated in phase G V and M I, 141 up-regulated and 334 down regulated in phase I and M II stage, 191 up-regulated and 88 down-regulation in G V and M II stage, and qPCR verification results and transcriptional data The results showed that the sequencing data of the transcriptional group were reliable.3, CCND1 and CCND2 were immunofluorescent and immunofluorescent, and the results of real time fluorescence quantitative and Western blot analysis showed that the immunofluorescence of CCND1 and CCND2 protein in granular cells were positive, and the expression level of mRNA and protein in G V granular fine cells was significantly higher than that of M phase I stage. The correlation between M II.4 and the maturation and development of oocyte showed that CCND1 and CCND2 were positively correlated with the rate of in vitro maturation of yak oocytes and the rate of blastocyst after fertilization. In this study, the gene expression profiles of the granulosa cells in Yak oocyte maturation were first obtained by single cell transcriptome. The correlation analysis of the maturation and developmental ability of the parent cells showed that CCND1 and CCND2 were positively related to the developmental potential of oocyte. Thus, it was deduced that CCND1 and CCND2 could be used as biomarkers for evaluating the quality and developmental potential of oocyte, which lay the foundation for the screening of high quality oocyte, the improvement of the mature system in vitro and the development mechanism of yak oocyte. Foundation.

【学位授予单位】：西南民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823.85

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