本文選題：Qpct + Aqp1　； 參考：《哈爾濱工業(yè)大學(xué)》2016年博士論文

【摘要】：Qpct(glutaminyl-peptide cyclotransferase)及Aqp1(Aquaporin-1)基因分別位于小鼠的17號(hào)和6號(hào)染色體。Qpct基因編碼的蛋白名稱為谷氨酰基環(huán)化酶,其在β淀粉樣蛋白的形成中發(fā)揮重要作用,且該淀粉樣蛋白的過(guò)度積累是阿爾茲海默病的誘因,進(jìn)一步研究發(fā)現(xiàn)Qpct基因?qū)σ恍┌┌Y產(chǎn)生抑制作用。Aqp1基因編碼的蛋白是水通道蛋白家族成員之一,在生物體中負(fù)責(zé)水分的運(yùn)輸,因此在生物的生長(zhǎng)過(guò)程中發(fā)揮重要的作用。鑒于Qpct及Aqp1在小鼠中的印記狀態(tài)及胚胎發(fā)育中的功能尚不清楚,因此,本文通過(guò)SNP位點(diǎn)測(cè)序法對(duì)Qpct及Aqp1在小鼠胚胎及胎盤發(fā)育中的印記狀態(tài)進(jìn)行鑒定;通過(guò)了解Qpct及Aqp1基因在胚胎及胎盤發(fā)育中的表達(dá)模式及調(diào)控機(jī)制,揭示兩者在胚胎及胎盤發(fā)育中的潛在功能;并通過(guò)Aqp1敲除鼠模型研究該印記基因?qū)ε咛ゼ疤ケP發(fā)育中的產(chǎn)生的影響,旨在揭示Aqp1在小鼠胚胎發(fā)育中的重要作用。本研究首先對(duì)生物信息學(xué)篩選出的兩個(gè)基因Qpct及Aqp1進(jìn)行了印記鑒定。利用基因上存在的SNP位點(diǎn)對(duì)E15.5小鼠胚內(nèi)及胚外組織的c DNA進(jìn)行測(cè)序,結(jié)果表明Qpct及Aqp1只有在小鼠胎盤中的SNP位點(diǎn)處測(cè)序峰是單峰,且都只母本表達(dá),而在胚胎的各組織中SNP處均為雙等位表達(dá)。由此可見(jiàn),小鼠的Qpct及Aqp1基因均是只在胎盤中特異性母本表達(dá)、父本印記的印記基因。其次,本研究分析了小鼠Qpct基因的時(shí)空表達(dá)譜及其印記的調(diào)控機(jī)制。通過(guò)實(shí)時(shí)定量PCR、原位雜交(切片或全胚胎)及免疫組化的實(shí)驗(yàn)方法對(duì)印記基因Qpct在胚胎及胎盤中的表達(dá)特點(diǎn)進(jìn)行分析。定量結(jié)果表明,Qpct基因在胚胎發(fā)育的囊胚期表達(dá)較高,在E8.5-E15.5胚胎發(fā)育中后期Qpct基因表達(dá)逐漸升高,E12.5-E18.5胎盤發(fā)育中后期Qpct基因的表達(dá)表現(xiàn)為先升高后降低的趨勢(shì),且E15.5為表達(dá)峰值時(shí)期。原位雜交數(shù)據(jù)表明Qpct基因主要在中期胚胎的腦、神經(jīng)管、聽(tīng)泡中高表達(dá),后期胚胎中該基因廣泛表達(dá),且在腦、肺、肝中表達(dá)最高。胎盤中后期(E12.5-E18.5)免疫組化結(jié)果表明,Qpct基因主要在蛻膜層及迷路層高表達(dá)。研究發(fā)現(xiàn),Qpct基因的啟動(dòng)子區(qū)上存在一個(gè)Cp G島,對(duì)該Cp G島進(jìn)行甲基化分析及Ch IP實(shí)驗(yàn),證實(shí)該Cp G島不調(diào)控基因的印記,但可以調(diào)控其在胚胎內(nèi)主要臟器中的表達(dá),而組蛋白H3K4me3可以調(diào)控該基因的母本表達(dá)、父本印記。最后,本文對(duì)Aqp1的時(shí)空表達(dá)譜、表達(dá)調(diào)控機(jī)制及功能進(jìn)行了研究。通過(guò)實(shí)時(shí)定量PCR結(jié)果表明,Aqp1在胚胎發(fā)育中后期(E8.5-E15.5)表達(dá)逐漸升高,E15.5時(shí)表達(dá)最高,且主要在肺、肝中表達(dá)較高,在腦及腎臟中表達(dá)較低。E15.5胎盤免疫組化結(jié)果表明,Aqp1基因主要在胎盤的蛻膜層及迷路層高表達(dá)。Aqp1基因從目前的生物信息庫(kù)數(shù)據(jù)中未見(jiàn)Cp G島的存在,但通過(guò)分析發(fā)現(xiàn)該基因的啟動(dòng)子區(qū)(region A)及第一外顯子(region B)有較大密度的CG位點(diǎn)存在。甲基化分析結(jié)果表明,region A和region B的甲基化調(diào)控了該基因在胚胎及胎盤中的表達(dá)。利用Aqp1敲除鼠模型,對(duì)Aqp1基因在胚胎及胎盤發(fā)育中的功能進(jìn)行探索。實(shí)驗(yàn)結(jié)果表明,Aqp1-/+(母本缺失)及Aqp1-/-(雙缺失)小鼠胎盤變大,同時(shí)胎盤與胚胎的重量明顯增加,胎盤中間層及迷路層均出現(xiàn)明顯擴(kuò)增,且迷路區(qū)的血管數(shù)目及分支明顯增多。證實(shí)在小鼠孕期,Aqp1基因主要通過(guò)調(diào)節(jié)胎盤供給給胚胎的營(yíng)養(yǎng)物質(zhì)對(duì)胚胎的發(fā)育產(chǎn)生抑制作用。綜上所述,本文鑒定出兩個(gè)胎盤特異性的印記基因Qpct及Aqp1,兩者在小鼠胚胎及胎盤的發(fā)育中廣泛表達(dá)。Qpct基因的母本表達(dá)、父本印記受到了組蛋白H3K4me3的調(diào)控。Aqp1的啟動(dòng)子區(qū)(region A)及第一外顯子(region B)共同調(diào)控該基因在胚胎及胎盤中的表達(dá),同時(shí)發(fā)現(xiàn)Aqp1基因?qū)π∈笤衅谂咛ゼ疤ケP的生長(zhǎng)發(fā)育具有重要的抑制作用。
[Abstract]:The Qpct (glutaminyl-peptide cyclotransferase) and Aqp1 (Aquaporin-1) genes encoded by the.Qpct gene of chromosome 17 and 6 in mice are glutamyl cyclase, which play an important role in the formation of beta amyloid, and the excessive accumulation of the amyloid is the cause of Alzheimer's disease. It is found that the protein encoded by the Qpct gene, which inhibits the.Aqp1 gene, is one of the members of the aquaporin family, responsible for the transport of water in organisms, and therefore plays an important role in the growth of organisms. In view of the imprint status of Qpct and Aqp1 in mice and the function of embryo development, it is not clear. The imprinting state of Qpct and Aqp1 in mouse embryo and placenta development was identified by SNP sequencing method. The potential function of Qpct and Aqp1 gene in the development of embryo and placenta was revealed by understanding the expression pattern and regulation mechanism of Qpct and Aqp1 gene in the development of embryo and placenta; and the imprinting gene was studied by the Aqp1 knockout mouse model. The effects of the embryogenesis and placenta development are aimed at revealing the important role of Aqp1 in the development of mouse embryos. First, two genes Qpct and Aqp1 screened by bioinformatics were imprinted and identified. The C DNA in the embryo and outer embryo of E15.5 mice was sequenced using the SNP locus existing in the gene, and the results showed Qpct and Aq. P1 only in the SNP site of the mouse placenta is a single peak, and all only mother parents are expressed, but both in the SNP of the embryo are double alleles. Thus, the Qpct and Aqp1 genes in the mice are all the specific maternal and paternal imprinting genes in the placenta. Secondly, this study analyzed the time and space of the mouse Qpct gene. The characteristics of the expression of the imprinted gene Qpct in the embryo and placenta were analyzed by real time quantitative PCR, in situ hybridization (slice or whole embryo) and immunohistochemistry. The quantitative results showed that the expression of Qpct gene was higher in the blastocyst stage of embryonic development, and the Qpct based in the late stage of the development of E8.5-E15.5 embryos. As the expression increased gradually, the expression of Qpct gene expression in the middle and late stages of E12.5-E18.5 placenta was first increased and then decreased, and E15.5 was the peak expression period. In situ hybridization data showed that the Qpct gene was highly expressed in the brain, neural tube and auditory vesicles of the metaphase embryos, and the gene was expressed extensively in the later embryos, and the most expressed in the later embryos, and the most expressed in the brain, lung and liver. The results of E12.5-E18.5 immunohistochemical staining showed that the Qpct gene was mainly expressed in the decidua and labyrinth layers. It was found that there was a Cp G island in the promoter region of the Qpct gene. The methylation analysis of the Cp G island and the IP experiment of Ch showed that the Cp G Island did not regulate the gene's imprint, but it could regulate its main viscera in the embryo. Expression in the organ, and histone H3K4me3 can regulate the maternal expression of the gene and the paternal imprint. Finally, this paper studies the temporal and spatial expression spectrum of Aqp1, the regulation mechanism and function of the expression. The results of real-time quantitative PCR show that the expression of Aqp1 in the middle and late embryonic development (E8.5-E15.5) increases gradually, the expression of E15.5 is highest, and mainly in the lung, The expression of the liver in the brain and kidney is higher. The results of low.E15.5 placental immunohistochemical expression in the brain and kidney show that the Aqp1 gene is mainly expressed in the decidual and labyrinth layer of the placenta and the.Aqp1 gene is not found in the Cp G Island, but the promoter region of the gene (region A) and the first exon (region B) are found by analysis. The presence of large density CG loci. Methylation analysis shows that the methylation of region A and region B regulates the expression of the gene in the embryo and placenta. Using Aqp1 knockout mouse model, the function of the Aqp1 gene in the development of embryo and placenta is explored. The experimental results show that Aqp1-/+ (mother missing) and Aqp1-/- (double deletion) mouse fetus are found. At the same time the weight of placenta and embryo increased obviously, the placental interlayer and the labyrinth layer were enlarged obviously, and the number and branch of the blood vessels in the labyrinth area increased obviously. It was confirmed that the Aqp1 gene was mainly controlled by the nutrition substance supplied by the placenta to the embryo during pregnancy. Two placental specific imprinting genes, Qpct and Aqp1, are widely expressed in the development of mouse embryos and placenta, and the paternal imprint is regulated by the promoter region of.Aqp1 (region A) and the first exon (region B) regulated by histone H3K4me3, and the expression of the gene in the embryo and placenta is simultaneously regulated. The Aqp1 gene has an important inhibitory effect on the growth and development of mouse embryos and placenta during pregnancy.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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