【作者】 马兴红；

【导师】 杨增明；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2006， 博士

【摘要】 胚胎着床涉及胚胎和子宫之间一系列复杂的相互作用,许多基因参与其中。尽管已利用寡核苷酸芯片、cDNA芯片、DDRT-PCR等各种不同的技术筛选到一些着床相关基因,但胚胎着床的机制仍不清楚。为了全面了解胚胎着床相关分子和发现新的胚胎着床相关信号通路,我们首次应用基因表达系列分析(SAGE)技术,分析了小鼠早期妊娠第五天非着床位点和着床位点子宫的基因表达谱。我们利用早期妊娠小鼠第五天的子宫分别构建了非着床位点和着床位点标签长10 bp的两个SAGE文库,非着床位点测序的标签数为51306个,对应16964个特异标签,着床位点测序的标签数为53214个,对应16733个特异的标签。为了提高SAGE库中标签对应基因的比例,我们利用同一个SAGE文库的序列文件,构建了标签长11 bp的两个文库,非着床位点的标签总数为48121,对应16677个特异标签,着床位点的标签总数为50227,对应16696个特异的标签。共有1039个标签在非着床位点特异性表达,1252个标签在着床位点特异性表达。根据p值,195个标签在非着床位点显著上调,其中100个标签是单一匹配的,261个标签在着床位点显著上调,其中127个是单一匹配的。为了验证SAGE结果的可信性,我们随机选取14个单一匹配的标签对应的基因(其中7个在着床位点上调,7个下调)进行RT-PCR验证,所有这些基因的RT-PCR结果与SAGE结果中的表达趋势一致。我们利用原位杂交对SAGE结果也进行了验证。与非着床位点相比,1810014L12Rik、Psmb5、Cd63、Npm1、Fads3和Tagln2在着床位点处的腔上皮下基质细胞中高表达。Ddx39在第5天着床位点腔上皮下基质中强表达,在非着床位点及假孕第5天未检测到杂交信号。利用假孕及延迟着床模型发现,Ddx39的表达受正在着床的活性胚泡的诱导。在卵巢切除的小鼠中,Ddx39的表达受雌激素显著上调,孕酮对Ddx39的表达没有明显影响。这表明Ddx39可能在胚胎着床过程中发挥重要作用。总之,我们成功地构建了早期妊娠小鼠第5天子宫的非着床位点和着床位点两个SAGE文库,筛选得到了一批与胚胎着床相关的基因,为进一步研究胚胎着床的分子机理提供了大量有价值的信息。更多还原

【Abstract】 Implantation is a complex interaction between the uterus and blastocyst. A lot of genes are imvolved in embryo-uterine interactions during implantation. Oligonucleotide chips, cDNA microarrays, differential display RT-PCR and other approaches have been used to screen implantation-related molecules. To identify implantation-related genes and novel pathways during implantation, the differential gene expression between inter-implantation site and implantation site in mouse uterus on day 5 of pregnancy was profiled by serial analysis of gene expression (SAGE). The SAGE libraries of 10 bp tags constructed from mouse uteri at inter-implantation site and implantation site were deposited in the GEO repository. The total numbers of tags sequenced were 51,306 tags for inter-implantation library and 53,214 tags for implantation library, corresponding to 16,964 and 16,733 unique tags, respectively. In order to increase the percentage of the tags with single match, we also used the same sequence files of SAGE libraries to construct two SAGE libraries of 11-bp tags. The total numbers of tags in 11-bp libraries were 48,121 for inter-implantation site and 50,227 for implantation site, corresponding to 16,677 and 16,696 unique tags, respectively. There were 1,039 tags specifically expressed at inter-implantation site, and 1,252 tags specifically expressed at implantation site. Based on p-value, there were 195 tags significantly up-regulated at inter-implantation site and 261 tags significantly up-regulated at implantation site, of which 100 genes were single-matched at inter-implantation site and 127 genes single-matched at implantation site, respectively.In order to validate SAGE data, RT-PCR was performed to examine differential expression of 14 randomly selected tags with single match, 7 for significantly up-regulated ones and 7 for down-regulated ones. For all of the genes examined, the trend of relative expression of their transcripts was comparable between SAGE and RT-PCR. SAGE data were further confirmed by in situ hybridization. 1810014L12Rik, Psmb5, Cd63, Npm1, Fads3 and Tagln2 were shown to be highly expressed at implantation site compared to inter-implantation site. Compared to inter-implantation site, Ddx39 was strongly expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy. Ddx39 expression at implantation site was specifically induced by active blastocysts. Additionally, Ddx39 expression was significantly up-regulated by更多还原