miR-29b的表达及其对猪早期胚胎发育影响的研究

发布时间：2018-03-31 19:09

本文选题：miR-29b　切入点：体外受精　出处：《吉林大学》2017年硕士论文

【摘要】：哺乳动物早期胚胎发育又称植入前胚胎发育,是指由合子形成开始到与子宫建立联系之前的游离阶段。在早期胚胎发育过程中基因表达除了受DNA甲基化、组蛋白修饰等表观遗传修饰的调控之外,转录后调控如micro RNA同样起到重要的作用。miR-29b(micro RNA-29b)为miR-29家族的一员,有研究表明其通过靶向作用于甲基转移酶基因Dnmts,引起肿瘤细胞中全基因组DNA甲基化下调。而在小鼠胚胎中的研究表明,干扰小鼠胚胎中miR-29b的表达不但导致全基因组DNA甲基化水平异常而且影响多种胚胎发育相关基因的表达,包括重要的全能性基因,引起桑椹胚阶段发育阻滞。猪不仅是重要的经济动物,还是重要的实验动物,在转化医学上有重要的应用价值。但是对于miR-29b的研究在猪中还没有任何报道,因此,本实验选取猪为研究对象,利用孤雌激活(parthenogenetic activation,PA)及体外受精(in vitro fertilization,IVF)技术在体外获得早期胚胎,并使用micro RNA-29b inhibitor进行胞质注射干扰其表达,来探究miR-29b在猪早期胚胎发育过程中表达情况和对胚胎发育的影响,从而为miR-29b在猪这一物种中的功能研究提供理论及实验依据。本实验主要结果如下:1、通过分析精子密度、精子获能处理、精卵共孵育培养体系以及卵母细胞成熟激素等影响猪卵母细胞体外受精及胚胎发育能力的因素,对猪卵母细胞体外受精体系进行优化,其卵裂率提高至(61.33±0.77)%,囊胚率提高至(28.33±1.08)%。2、分别使用体外受精及孤雌激活技术在体外获得各阶段的猪早期胚胎,并利用q-PCR及免疫荧光染色技术检测miR-29b及其主要靶基因Dnmts表达水平的动态变化在两种胚胎发育过程中的动态表达模式。结果显示,仅在体外受精胚胎中miR-29b呈时空特异性表达,其表达水平在8细胞期最高,其次是囊胚期,而在4细胞期表达水平最低,而Dnmt3a,Dnmt3b表达水平与miR-29b呈明显的负相关。说明miR-29b的表达及通过Dnmt3a/3b发挥作用需要受精过程的激活。3、通过注射miRNA inhibitor到MII期卵母细胞胞质干扰miR-29b表达,检测干扰后体外受精胚胎发育率,并通过q-PCR,免疫荧光染色及甲基化测序(BSP)技术检测干扰后囊胚阶段Dnmts的表达,全基因组及全能性基因Nanog启动子区域DNA甲基化水平。结果显示干扰之后胚胎发育的囊胚率降低,囊胚总细胞数显著减少,但卵裂率无显著差异。Dnmt3a,Dnmt3b m RNA表达水平显著上调,同时全基因组甲基化水平及Nanog启动子DNA甲基化水平升高。说明miR-29b的表达确实与胚胎发育相关,其主要通过靶向Dnmt3a,Dnmt3b调控DNA甲基化水平,进而影响囊胚阶段的发育。4、通过q-PCR技术检测干扰后囊胚多能性基因Nanog、Sox2、Oct4以及凋亡基因Bax、Casp3及抗凋亡基因Bcl-xl m RNA表达水平,并利用免疫荧光染色技术检测干扰后细胞凋亡数,发现全能性基因Nanog,Sox2基因表达水平显著下调,凋亡基因显著上调,而抗凋亡基因显著下调,并且凋亡细胞数增多。说明miR-29b在猪早期胚胎的囊胚阶段通过调节多能性基因和凋亡基因的表达,进而影响胚胎的早期发育。
[Abstract]:Early embryo development also called preimplantation embryo development, by means of zygote formation to free stage prior to establish contact with the uterus. In early embryonic development process in addition to gene expression by DNA methylation, histone modifications beyond the regulation of epigenetic modification, post transcriptional regulation such as micro RNA also play a role in.MiR-29b important (micro RNA-29b) is a member of the miR-29 family, the research has shown that by targeting methyltransferase gene Dnmts, caused by DNA methylation in tumor cells. The down-regulation of research in mouse embryo showed that expression of miR-29b interference in mouse embryos not only leads to the genome DNA methylation level and the influence of various abnormal genes related to embryonic development including the expression of pluripotency genes important cause, morula stage development block. The pig is not only an important economic animal, or heavy The experimental animal, has important application value in translational medicine. But for the research of miR-29b has not reported any, therefore in the pig, this experiment selects the pig as the research object, using parthenogenetic activation (parthenogenetic activation, PA) and in vitro fertilization (in vitro fertilization IVF) technology to obtain early embryos in vitro, and were microinjected into the cytoplasm of the expression of RNA-29b inhibitor using micro interference, to explore the expression of miR-29b in the process of pig early embryonic development and the influence on embryonic development, so as to provide theoretical and experimental basis for the study of miR-29b function in pigs in this species. The main results are as follows: 1, through the analysis of sperm density, sperm capacitation, factors affecting fertilization and embryo developmental competence of porcine oocytes in vitro sperm egg incubation culture system and oocyte maturation hormone, on porcine oocytes in vitro Fine system optimization, the cleavage rate increased to (61.33 + 0.77)%, the blastocyst rate increased to (28.33 + 1.08)%.2, were used in in vitro fertilization and parthenogenetic activation of porcine embryos obtained at various stages in vitro, dynamic changes and the use of q-PCR and immunofluorescence staining technique to detect the expression level of miR-29b and its main the dynamic expression of Dnmts target genes during embryonic development of the two models. The results showed that only miR-29b in in vitro fertilization embryo in a specific expression pattern, its expression level is the highest in the 8 cell stage, followed by the blastocyst stage, and in the 4 cell stage was the lowest, and Dnmt3a, a significant negative correlation the expression level of Dnmt3b and miR-29b. The expression of miR-29b and Dnmt3a/3b play a role in activation of.3 through the process of fertilization, through the injection of miRNA inhibitor to the MII phase of oocyte cytoplasmic expression of miR-29b interference, interference detection after in vitro fertilization embryo The rate of fetal development, and by q-PCR, immunofluorescence staining and methylation sequencing (BSP) expression of blastocyst stage Dnmts detection after interference, and genomic totipotency of the promoter region of Nanog gene DNA methylation level. Results show that the interference after the embryonic development of blastocyst rate, total cell number of blastocyst was significantly reduced, but the rate of cleavage no significant differences in.Dnmt3a, Dnmt3b m RNA was up-regulated, while the methylation level of Nanog promoter and the whole genome DNA methylation level increased. The expression of miR-29b is associated with embryonic development, mainly through targeting Dnmt3a, Dnmt3b regulation of DNA methylation level, thereby affecting the development of.4 blastocyst stage, through the detection of interference q-PCR technology after blastocyst pluripotency genes Nanog, Sox2, Oct4 and apoptosis gene Bax, Casp3 and anti apoptosis gene Bcl-xl m expression levels of RNA and immunofluorescence staining technique for the detection of interference After the number of apoptotic cells, found pluripotency genes Nanog, Sox2 gene expression levels were significantly reduced, significantly upregulated genes, and anti apoptotic genes were significantly reduced, and the number of apoptotic cells increased. MiR-29b through the regulation of pluripotency genes and apoptosis gene expression at the blastocyst stage of early embryo of pig, and then affect the development of mouse embryos.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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