Qpct及Aqp1在小鼠胎盘的印记鉴定及表达调控机制研究

发布时间：2018-07-05 15:12

本文选题：Qpct + Aqp1　；参考：《哈尔滨工业大学》2016年博士论文

【摘要】：Qpct(glutaminyl-peptide cyclotransferase)及Aqp1(Aquaporin-1)基因分别位于小鼠的17号和6号染色体。Qpct基因编码的蛋白名称为谷氨酰基环化酶,其在β淀粉样蛋白的形成中发挥重要作用,且该淀粉样蛋白的过度积累是阿尔兹海默病的诱因,进一步研究发现Qpct基因对一些癌症产生抑制作用。Aqp1基因编码的蛋白是水通道蛋白家族成员之一,在生物体中负责水分的运输,因此在生物的生长过程中发挥重要的作用。鉴于Qpct及Aqp1在小鼠中的印记状态及胚胎发育中的功能尚不清楚,因此,本文通过SNP位点测序法对Qpct及Aqp1在小鼠胚胎及胎盘发育中的印记状态进行鉴定;通过了解Qpct及Aqp1基因在胚胎及胎盘发育中的表达模式及调控机制,揭示两者在胚胎及胎盘发育中的潜在功能;并通过Aqp1敲除鼠模型研究该印记基因对胚胎及胎盘发育中的产生的影响,旨在揭示Aqp1在小鼠胚胎发育中的重要作用。本研究首先对生物信息学筛选出的两个基因Qpct及Aqp1进行了印记鉴定。利用基因上存在的SNP位点对E15.5小鼠胚内及胚外组织的c DNA进行测序,结果表明Qpct及Aqp1只有在小鼠胎盘中的SNP位点处测序峰是单峰,且都只母本表达,而在胚胎的各组织中SNP处均为双等位表达。由此可见,小鼠的Qpct及Aqp1基因均是只在胎盘中特异性母本表达、父本印记的印记基因。其次,本研究分析了小鼠Qpct基因的时空表达谱及其印记的调控机制。通过实时定量PCR、原位杂交(切片或全胚胎)及免疫组化的实验方法对印记基因Qpct在胚胎及胎盘中的表达特点进行分析。定量结果表明,Qpct基因在胚胎发育的囊胚期表达较高,在E8.5-E15.5胚胎发育中后期Qpct基因表达逐渐升高,E12.5-E18.5胎盘发育中后期Qpct基因的表达表现为先升高后降低的趋势,且E15.5为表达峰值时期。原位杂交数据表明Qpct基因主要在中期胚胎的脑、神经管、听泡中高表达,后期胚胎中该基因广泛表达,且在脑、肺、肝中表达最高。胎盘中后期(E12.5-E18.5)免疫组化结果表明,Qpct基因主要在蜕膜层及迷路层高表达。研究发现,Qpct基因的启动子区上存在一个Cp G岛,对该Cp G岛进行甲基化分析及Ch IP实验,证实该Cp G岛不调控基因的印记,但可以调控其在胚胎内主要脏器中的表达,而组蛋白H3K4me3可以调控该基因的母本表达、父本印记。最后,本文对Aqp1的时空表达谱、表达调控机制及功能进行了研究。通过实时定量PCR结果表明,Aqp1在胚胎发育中后期(E8.5-E15.5)表达逐渐升高,E15.5时表达最高,且主要在肺、肝中表达较高,在脑及肾脏中表达较低。E15.5胎盘免疫组化结果表明,Aqp1基因主要在胎盘的蜕膜层及迷路层高表达。Aqp1基因从目前的生物信息库数据中未见Cp G岛的存在,但通过分析发现该基因的启动子区(region A)及第一外显子(region B)有较大密度的CG位点存在。甲基化分析结果表明,region A和region B的甲基化调控了该基因在胚胎及胎盘中的表达。利用Aqp1敲除鼠模型,对Aqp1基因在胚胎及胎盘发育中的功能进行探索。实验结果表明,Aqp1-/+(母本缺失)及Aqp1-/-(双缺失)小鼠胎盘变大,同时胎盘与胚胎的重量明显增加,胎盘中间层及迷路层均出现明显扩增,且迷路区的血管数目及分支明显增多。证实在小鼠孕期,Aqp1基因主要通过调节胎盘供给给胚胎的营养物质对胚胎的发育产生抑制作用。综上所述,本文鉴定出两个胎盘特异性的印记基因Qpct及Aqp1,两者在小鼠胚胎及胎盘的发育中广泛表达。Qpct基因的母本表达、父本印记受到了组蛋白H3K4me3的调控。Aqp1的启动子区(region A)及第一外显子(region B)共同调控该基因在胚胎及胎盘中的表达,同时发现Aqp1基因对小鼠孕期胚胎及胎盘的生长发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.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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