【摘要】：目的:1、探討雌激素受體α(estrogen receptor alpha,ERα)及其磷酸化激活形式p ERα-S118在小鼠1-細(xì)胞胚和2-細(xì)胞胚中的階段特異性表達(dá)與小鼠合子型基因激活的關(guān)系。2、探討ERα特異性抑制劑MPP處理對(duì)小鼠植入前胚發(fā)育的階段特異性影響,及其對(duì)合子型基因激活的作用機(jī)制。3、探討ERα通過(guò)micro RNA途徑影響小鼠合子型基因激活的機(jī)制,并研究其對(duì)干細(xì)胞關(guān)鍵因子Oct4和Sox2的調(diào)控作用。方法:1、采用輸卵管沖洗的方式,于hCG注射后15 h~54 h每隔3 h收集小鼠植入前胚,利用細(xì)胞免疫熒光技術(shù)檢測(cè)ERα和p ERα-S118的表達(dá)定位。2、收集昆明小鼠1-細(xì)胞胚、2-細(xì)胞胚和4-細(xì)胞胚,以空白KSOM培養(yǎng)液(單純型優(yōu)化的胚胎培養(yǎng)基)為對(duì)照組,以KSOM中添加MPP為實(shí)驗(yàn)組;將各時(shí)間點(diǎn)收集的植入前胚培養(yǎng)3 h后再移入新KSOM培養(yǎng)液中繼續(xù)培養(yǎng)至囊胚階段,并記錄囊胚數(shù)。于hCG注射后21 h收集小鼠1-細(xì)胞胚,置于MPP中培養(yǎng),于hCG后27 h,提取總RNA,RT-q PCR檢測(cè)合子型標(biāo)志基因e IF1A和Mu ERV-L的表達(dá)變化;采用細(xì)胞免疫熒光技術(shù)檢測(cè)經(jīng)21~27 h MPP處理后,組蛋白甲基化(H3K4me3和H3K27me3)水平的改變。于hCG注射后21 h收集小鼠1-細(xì)胞胚,分別置于KSOM+BrdU和KSOM+MPP+BrdU培養(yǎng)液中培養(yǎng),于hCG后27 h收集樣品,采用抗BrdU抗體免疫染色實(shí)驗(yàn)觀察DNA復(fù)制情況;于hCG注射后27h收集小鼠1-細(xì)胞胚,經(jīng)不同濃度MPP處理后,于hCG后42 h固定細(xì)胞,用免疫熒光技術(shù)觀察2-細(xì)胞胚核像的變化;并且用BrdU摻入法檢測(cè)DNA復(fù)制是否受到影響;采用免疫熒光技術(shù)檢測(cè)S期啟動(dòng)相關(guān)因子c-MYC和E2F1的表達(dá)變化。3、于hCG注射后27 h收集小鼠1-細(xì)胞胚,建立27~45 h MPP處理(20μmol/L)實(shí)驗(yàn)組和KSOM對(duì)照組模型;提取總RNA,經(jīng)Megaplex逆轉(zhuǎn)錄以及預(yù)擴(kuò)增后,進(jìn)行Taq Man定量PCR芯片檢測(cè),收集數(shù)據(jù);通過(guò)交集分析,篩選出差異表達(dá)的micro RNA與已知小鼠精子micro RNA表達(dá)譜的一致部分;然后通過(guò)micro RNA靶基因注釋、信號(hào)通路研究等手段,進(jìn)一步分析ERα通過(guò)micro RNA途徑影響小鼠植入前胚發(fā)育的可能機(jī)制;采用細(xì)胞免疫熒光技術(shù)驗(yàn)證靶基因表達(dá)產(chǎn)物(Oct4和Sox2)的變化。結(jié)果:1、ERα在小鼠植入前胚中的核內(nèi)定位首次出現(xiàn)于S期中期,于第一次有絲分裂期核內(nèi)定位消失,重新出現(xiàn)于2-細(xì)胞胚早期(第一次有絲分裂的末期),并保持核內(nèi)定位和胞質(zhì)定位至2-細(xì)胞胚分裂期。p ERα-S118在小鼠1-細(xì)胞胚的早期可見(jiàn)細(xì)胞核表面的定位,并且在1-細(xì)胞胚和2-細(xì)胞胚有絲分裂前期和前中期出現(xiàn)核內(nèi)定位增強(qiáng),中期出現(xiàn)胞質(zhì)表達(dá)水平升高,于后期迅速降低;p ERα-S118在2-細(xì)胞核內(nèi)的定位首次出現(xiàn)于S期啟動(dòng)時(shí)。2、MPP處理對(duì)小鼠植入前胚發(fā)育具有顯著的抑制作用,其階段特異性影響與小鼠ZGA發(fā)生的時(shí)間一致。經(jīng)21~27 h MPP處理,小鼠合子型基因e IF1A和Mu ERV-L m RNA表達(dá)下調(diào);1-細(xì)胞胚雄原核H3K27me3表達(dá)水平降低。MPP處理對(duì)小鼠的1-細(xì)胞胚和2-細(xì)胞胚的S期進(jìn)程都有顯著的抑制作用。c-MYC和E2F1在小鼠2-細(xì)胞胚內(nèi)的表達(dá)不受MPP處理的影響。3、Micro RNA芯片結(jié)果顯示,表達(dá)上調(diào)的micro RNA數(shù)目(126個(gè))多于表達(dá)下調(diào)的micro RNA(64個(gè))。受MPP處理影響的37個(gè)精源性micro RNA顯著參與“應(yīng)激反應(yīng)、大分子物質(zhì)代謝、基因表達(dá)和轉(zhuǎn)錄后調(diào)控、基因表觀遺傳學(xué)調(diào)控、負(fù)反饋調(diào)控(大分子物質(zhì))代謝過(guò)程、抑制基因表達(dá)、抑制m RNA翻譯”等多種分子功能。精源性micro RNA靶基因集在“DNA依賴性轉(zhuǎn)錄調(diào)控”中有顯著的富集;而且其中的小規(guī)模合子型靶基因更顯著地富集于多種生物學(xué)過(guò)程。以“Akt1 Foxo3 Stat3 ERα Oct4”為主軸的調(diào)控網(wǎng)絡(luò)參與了小鼠小規(guī)模合子型基因激活的DNA依賴轉(zhuǎn)錄調(diào)控。經(jīng)MPP處理后,小鼠2-細(xì)胞胚OCT4表達(dá)水平升高,SOX2表達(dá)下調(diào);經(jīng)Akt抑制劑API-2處理后,p ERα-S118表達(dá)上調(diào)。結(jié)論:1.ERα可通過(guò)影響小鼠早期植入前胚的S期進(jìn)程、精源性染色質(zhì)的組蛋白甲基化修飾、精源性micro RNA表達(dá)等途徑,參與小鼠小規(guī)模合子型基因的DNA依賴性轉(zhuǎn)錄。2.ERα在小鼠合子型基因激活中的作用機(jī)制還包括對(duì)干性維持關(guān)鍵基因Oct4和Sox2的調(diào)控。3.而且,ERα在小鼠合子型基因激活中的作用受到Akt的磷酸化激活調(diào)控,而ERα又可以通過(guò)mi R-125a-5p和mi R-125a-3p途徑,影響Akt的功能,形成反饋調(diào)控網(wǎng)絡(luò)。
[Abstract]:Objective: 1. To investigate the relationship between the specific expression of estrogen receptor alpha (ER) and its phosphorylation-activated form (p ER)-S118 in mouse 1-cell embryo and 2-cell embryo and the activation of zygotic gene in mice. Objective: To investigate the specific effect of ER proton-specific inhibitor (MPP) on the stage-specific development of mouse preimplantation embryos and its effect on the activation of zygotes-type genes. The key factors of stem cell factor Oct4 and Sox2 were studied and studied. Methods: 1. The pre-implantation embryos were collected from 15 h ~ 54 h after hCG injection. The expression and localization of ER and p ER-S118 were detected by immunofluorescence technique. The 1-cell embryos, 2-cell embryos and 4-cell embryos of Kunming mice were collected. The blank KSOM culture medium was used as the control group, and MPP was added to KSOM as experimental group. The pre-implantation embryos collected at each time point were cultured for 3 h, then moved into the new KSOM culture medium and then cultured to the blastocyst stage, and the blastocyst count was recorded. 1-cell embryos of mice were collected for 21 h after hCG injection, cultured in MPP, extracted for 27h after hCG, total RNA was extracted, RT-q PCR was used to detect the expression changes of zygotic marker genes e IF1A and Mu ERV-L, and after 21-27h MPP treatment was detected by immunofluorescence technique, Changes in Histone Methylation (H3K4me3 and H3K27me3) levels. The 1-cell embryos of mice were collected at 21h after hCG injection, respectively placed in KSOM + BrdU and KSOM + MPP + BrdU culture solution, samples were collected at 27h after hCG, DNA replication was observed with anti-BrdU antibody immunostaining experiment, 1-cell embryos were collected after hCG injection, and treated with MPP treatment at different concentrations, The expression of c-MYC and E2F1 in S phase was detected by immunofluorescence technique, and the expression of c-MYC and E2F1 was detected by immunofluorescence technique. The 1-cell embryos of mice were collected after hCG injection for 27 h, and 27-45h MPP treatment (20 umol/ L) test group and KSOM control group model were established; total RNA was extracted, and after Meaplex reverse transcription and pre-amplification, a TaqMan quantitative PCR chip detection and data collection were carried out; and by intersection analysis, The results showed that the microRNAs of differentially expressed microRNAs were consistent with those of the known mouse sperm micro RNA expression profiles, and then the possible mechanism of ER gene expression was further analyzed by micro RNA target gene annotation, signal path research and so on, and the possible mechanism of the mouse embryo development was further analyzed by micro RNA pathway. The changes of target gene expression products (Oct4 and Sox2) were verified by cell immunofluorescence. Results: 1. The nuclear localization of ER IUD in the embryos of mice was first observed in the mid-stage of S phase, disappeared in the nucleus of the first mitotic phase, and reappeared in the early stage of 2-cell embryo (the end of the first mitosis). and keeping the nuclear localization and cytoplasmic localization to the 2-cell embryo division stage. In the early stage of mitosis of 1-cell embryo and 2-cell embryo, nuclear localization was enhanced in the early stage and metaphase of 1-cell embryo and 2-cell embryo, the level of cytoplasmic expression increased in the medium term, and decreased rapidly in later stage. At the start of S phase, p ER 044-S118 appeared in S phase for the first time. MPP treatment had a significant inhibitory effect on the development of mouse embryos before implantation, and its stage-specific effects were consistent with the time of ZGA in mice. After 21-27h MPP treatment, mouse zygotic gene e IF1A and Mu ERV-L mRNA were down-regulated; 1-cell embryo male pronucleus H3K27me3 expression level decreased. MPP treatment had significant inhibitory effect on the S phase processes of 1-cell and 2-cell embryos in mice. The expression of c-MYC and E2F1 in mouse 2-cell embryos was not affected by MPP treatment. 37 sperm source micro RNAs affected by MPP treatment were significantly involved "Stress response, macromolecular substance metabolism, gene expression and post-transcriptional regulation, gene epigenetic regulation, negative feedback regulation (macromolecular substance) metabolism, inhibition of gene expression, and inhibition of m RNA translation" and the like. The fine-source micro RNA target gene set has significant enrichment in the 鈥淒NA-dependent transcriptional regulation鈥，

本文編號(hào)：2312335