發(fā)布時(shí)間：2018-09-13 09:45

【摘要】：目的: 應(yīng)用Real-time RT-PCR、原位雜交對microRNAs(miRNAs)芯片篩選出的小鼠胚胎著床前后子宮內(nèi)膜中差異表達(dá)最大的mmu-miR-193進(jìn)行表達(dá)規(guī)律研究;運(yùn)用靶基因預(yù)測數(shù)據(jù)庫檢索mmu-miR-193調(diào)控的靶基因;在體外通過轉(zhuǎn)染實(shí)驗(yàn)對靶基因進(jìn)行驗(yàn)證;通過小鼠子宮角注射mmu-miR-193激動(dòng)劑研究其對胚胎著床的影響。從而探明mmu-miR-193在小鼠胚胎著床過程中的作用和機(jī)制。為研究microRNAs在生殖過程中的作用,以及闡明人類正常生殖過程機(jī)制和診斷、治療生殖疾病打下基礎(chǔ)。 方法: 1 1.標(biāo)本的收集:建立妊娠NIH小鼠動(dòng)物模型,分離孕d4、d6天小鼠的子宮內(nèi)膜組織,分別凍存于-80℃?zhèn)溆谩?2. Real-time RT-PCR檢測芯片篩選出差異表達(dá)最大的mmu-miR-193,根據(jù)mmu-miR-193成熟序列設(shè)計(jì)特異性的逆轉(zhuǎn)錄引物和PCR上、下游引物進(jìn)行real-time RT PCR。 3.原位雜交:原位雜交檢測mmu-miR-193在胚胎著床前后(孕d4、d6)子宮內(nèi)膜中的表達(dá)部位。 4.生物信息學(xué)分析:應(yīng)用miRNAs靶基因預(yù)測數(shù)據(jù)庫網(wǎng)站Pictar、Targetscan、miRGen來檢索mmu-miR-193的預(yù)測靶基因。 5.轉(zhuǎn)染實(shí)驗(yàn):通過轉(zhuǎn)染mmu-miR-193模擬物和抑制劑使其功能增強(qiáng)或者降低,Western blot檢測轉(zhuǎn)染后GRB7蛋白的表達(dá)變化。 6.功能實(shí)驗(yàn):在小鼠孕D2天,通過子宮角注射mmu-miR-193激動(dòng)劑,孕D5天檢測GRB7蛋白表達(dá),孕D7天剖腹計(jì)數(shù)胚胎著床數(shù)。 結(jié)果: 1. Real-time RT PCR結(jié)果顯示mmu-miR-193在小鼠胚胎著床前后(孕D4、D6)子宮內(nèi)膜中表達(dá)差異顯著,孕D6與孕D4比顯著上調(diào)。結(jié)果與芯片結(jié)果趨勢一致。 2.原位雜交顯示mmu-miR-193在小鼠子宮內(nèi)膜基質(zhì)細(xì)胞、腔上皮細(xì)胞和腺上皮細(xì)胞均有表達(dá)。 3.利用生物信息學(xué),通過數(shù)據(jù)庫預(yù)測得到Grb7等為mmu-miR-193所調(diào)控的靶基因。 4.在體外,我們通過轉(zhuǎn)染mmu-miR-193的模擬物和抑制劑證實(shí)了GRB7的表達(dá)受mmu-miR-193調(diào)控。 5.通過子宮角注射mmu-miR-193的激動(dòng)劑,導(dǎo)致了GRB7蛋白表達(dá)水平及胚胎著床數(shù)的明顯下降。 結(jié)論: 1. mmu-miR-193在小鼠胚胎著床前后子宮內(nèi)膜中存在明顯差異表達(dá),胚胎著床后較胚胎著床前顯著升高。 2. mmu-miR-193在小鼠胚胎著床過程中通過調(diào)節(jié)其靶基因Grb7的表達(dá)而調(diào)控小鼠胚胎著床。
[Abstract]:Objective: to study the expression of mmu-miR-193, which was the most differentially expressed in mouse endometrium before and after implantation with microRNAs (miRNAs) microarray by Real-time RT-PCR, in situ hybridization, and to search the target genes regulated by mmu-miR-193 by using target gene prediction database. The target gene was verified by transfection experiment in vitro, and the effect of mmu-miR-193 agonist on embryo implantation was studied by injection of mmu-miR-193 agonist into mouse uterus horn in vitro. The role and mechanism of mmu-miR-193 in mouse embryo implantation were investigated. It provides a basis for studying the role of microRNAs in reproductive process, clarifying the mechanism and diagnosis of human normal reproductive process, and treating reproductive diseases. Methods: 1. Collection of specimens: the animal model of pregnant NIH mice was established and the endometrial tissues of mice on the 4th day of gestation were isolated and frozen at -80 鈩，

本文編號：2240811