本文選題：miR-29b　切入點(diǎn)：體外受精　出處：《吉林大學(xué)》2017年碩士論文

【摘要】：哺乳動(dòng)物早期胚胎發(fā)育又稱植入前胚胎發(fā)育,是指由合子形成開始到與子宮建立聯(lián)系之前的游離階段。在早期胚胎發(fā)育過程中基因表達(dá)除了受DNA甲基化、組蛋白修飾等表觀遺傳修飾的調(diào)控之外,轉(zhuǎn)錄后調(diào)控如micro RNA同樣起到重要的作用。miR-29b(micro RNA-29b)為miR-29家族的一員,有研究表明其通過靶向作用于甲基轉(zhuǎn)移酶基因Dnmts,引起腫瘤細(xì)胞中全基因組DNA甲基化下調(diào)。而在小鼠胚胎中的研究表明,干擾小鼠胚胎中miR-29b的表達(dá)不但導(dǎo)致全基因組DNA甲基化水平異常而且影響多種胚胎發(fā)育相關(guān)基因的表達(dá),包括重要的全能性基因,引起桑椹胚階段發(fā)育阻滯。豬不僅是重要的經(jīng)濟(jì)動(dòng)物,還是重要的實(shí)驗(yàn)動(dòng)物,在轉(zhuǎn)化醫(yī)學(xué)上有重要的應(yīng)用價(jià)值。但是對(duì)于miR-29b的研究在豬中還沒有任何報(bào)道,因此,本實(shí)驗(yàn)選取豬為研究對(duì)象,利用孤雌激活(parthenogenetic activation,PA)及體外受精(in vitro fertilization,IVF)技術(shù)在體外獲得早期胚胎,并使用micro RNA-29b inhibitor進(jìn)行胞質(zhì)注射干擾其表達(dá),來探究miR-29b在豬早期胚胎發(fā)育過程中表達(dá)情況和對(duì)胚胎發(fā)育的影響,從而為miR-29b在豬這一物種中的功能研究提供理論及實(shí)驗(yàn)依據(jù)。本實(shí)驗(yàn)主要結(jié)果如下:1、通過分析精子密度、精子獲能處理、精卵共孵育培養(yǎng)體系以及卵母細(xì)胞成熟激素等影響豬卵母細(xì)胞體外受精及胚胎發(fā)育能力的因素,對(duì)豬卵母細(xì)胞體外受精體系進(jìn)行優(yōu)化,其卵裂率提高至(61.33±0.77)%,囊胚率提高至(28.33±1.08)%。2、分別使用體外受精及孤雌激活技術(shù)在體外獲得各階段的豬早期胚胎,并利用q-PCR及免疫熒光染色技術(shù)檢測(cè)miR-29b及其主要靶基因Dnmts表達(dá)水平的動(dòng)態(tài)變化在兩種胚胎發(fā)育過程中的動(dòng)態(tài)表達(dá)模式。結(jié)果顯示,僅在體外受精胚胎中miR-29b呈時(shí)空特異性表達(dá),其表達(dá)水平在8細(xì)胞期最高,其次是囊胚期,而在4細(xì)胞期表達(dá)水平最低,而Dnmt3a,Dnmt3b表達(dá)水平與miR-29b呈明顯的負(fù)相關(guān)。說明miR-29b的表達(dá)及通過Dnmt3a/3b發(fā)揮作用需要受精過程的激活。3、通過注射miRNA inhibitor到MII期卵母細(xì)胞胞質(zhì)干擾miR-29b表達(dá),檢測(cè)干擾后體外受精胚胎發(fā)育率,并通過q-PCR,免疫熒光染色及甲基化測(cè)序(BSP)技術(shù)檢測(cè)干擾后囊胚階段Dnmts的表達(dá),全基因組及全能性基因Nanog啟動(dòng)子區(qū)域DNA甲基化水平。結(jié)果顯示干擾之后胚胎發(fā)育的囊胚率降低,囊胚總細(xì)胞數(shù)顯著減少,但卵裂率無顯著差異。Dnmt3a,Dnmt3b m RNA表達(dá)水平顯著上調(diào),同時(shí)全基因組甲基化水平及Nanog啟動(dòng)子DNA甲基化水平升高。說明miR-29b的表達(dá)確實(shí)與胚胎發(fā)育相關(guān),其主要通過靶向Dnmt3a,Dnmt3b調(diào)控DNA甲基化水平,進(jìn)而影響囊胚階段的發(fā)育。4、通過q-PCR技術(shù)檢測(cè)干擾后囊胚多能性基因Nanog、Sox2、Oct4以及凋亡基因Bax、Casp3及抗凋亡基因Bcl-xl m RNA表達(dá)水平,并利用免疫熒光染色技術(shù)檢測(cè)干擾后細(xì)胞凋亡數(shù),發(fā)現(xiàn)全能性基因Nanog,Sox2基因表達(dá)水平顯著下調(diào),凋亡基因顯著上調(diào),而抗凋亡基因顯著下調(diào),并且凋亡細(xì)胞數(shù)增多。說明miR-29b在豬早期胚胎的囊胚階段通過調(diào)節(jié)多能性基因和凋亡基因的表達(dá),進(jìn)而影響胚胎的早期發(fā)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

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