囊胚内细胞团和滋养外胚层基因表达研究
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摘要
目的研究人胚胎植入前囊胚内细胞团和滋养外胚层细胞基因的表达。方法收集辅助生殖来源人受精后6 d Gardner评分5AA囊胚,显微镜下微吸管机械分离内细胞团和滋养外胚层细胞,单细胞测序检测,选取筛选的差异表达基因并进行无监督层次聚类分析和Gene Ontology(GO)功能分类分析。结果对3枚受精后6 d的5AA囊胚内细胞团14个单细胞和滋养外胚层19个单细胞测序,结果显示内细胞团1 283个基因表达上调,其G O生物学功能主要是参与DNA依赖的转录、生物种间相互作用、抗原加工和表达、免疫反应、信号转导、氧化还原、细胞分化、抗凋亡、神经系统发育、细胞黏附等,参与丝裂原活化蛋白激酶(MAPKs)信号通路、肌动蛋白细胞骨架、黏附、轴引导、Jak-STAT、Wnt信号通路。滋养外胚层1 073个基因表达上调,其GO生物学功能主要是参与转录、DNA依赖的转录、细胞周期、蛋白分解代谢、蛋白氨基酸磷酸化、蛋白质运输、细胞分裂、有丝分裂、泛素依赖性蛋白分解、细胞内蛋白质转运等;参与泛素介导蛋白水解、鞘脂代谢、缬氨酸,亮氨酸和异亮氨酸降解、果糖和甘露糖代谢、氨基磷酸酯代谢、类固醇的生物合成、抗原处理和表达等信号通路。结论从空间维度揭示囊胚内细胞团和滋养外胚层的基因表达,显示两者相互协调,精细调节囊胚发育和胚胎植入过程,进一步数据分析有望寻找到调控胚胎植入的内源性关键特异分子。
Objective To investigate the global gene expression of inner cells mass and trophectoderm of the human primplantational blastocyst. Methods We used blastocysts which was 6 d after fertilization, and Gardner score 5AA. Microcapsules were used to separate the inner cells mass and trophectoderm. Single cell sequencing was performed. And the gene expression was analyzed by bioinformatics software. The unidentified hierarchical clustering analysis and Gene Ontology(GO) functional classification were used to analyze the differentially expressed genes.Results The 11 single cells from inner cells mass and 16 single cells from trophectoderm in 5AA preimplantational blastocysts were sequenced. We found there were 1 283 genes up-regulated in inner cell mass compared with trophectoderm. The GO biological function was mainly involved in DNA-dependent transcription resistance, signal transduction, cell differentiation, anti-apoptosis, nervous system development, cell adhesion, etc. These genes involved in MAPKs signaling pathway, actin cytoskeleton, adhesion,axis guidance, Jak-STAT, Wnt signaling pathway.We found there were 1 073 genes with up-regulated expression in trophectoderm compared with inner cell mass. Its GO biological function was mainly involved in transcription, DNA dependent transcription, cell cycle, protein catabolism, protein amino acid phosphorylation, protein transport, cell division, mitosis, ubiquitin-dependent protein decomposition, intracellular protein transport, etc. Those genes involved in ubiquitin-mediated proteolysis,sphingolipid metabolism, valine, leucine and isoleucine degradation, fructose and mannose metabolism, aminophosphonate metabolism, steroid biosynthesis, antigen processing and expression signal pathways. Conclusion The global geneexpression of inner cells mass and trophectoderm of the human preimplantational blastocyst is revealed from the spatial dimension, indicating that they are coordinated with each other, and the process of embryo implantation are finely regulated. Further data analysis is expected to find the endogenous specific molecules for regulating embryo implantation.
Objective To investigate the global gene expression of inner cells mass and trophectoderm of the human primplantational blastocyst. Methods We used blastocysts which was 6 d after fertilization, and Gardner score 5AA. Microcapsules were used to separate the inner cells mass and trophectoderm. Single cell sequencing was performed. And the gene expression was analyzed by bioinformatics software. The unidentified hierarchical clustering analysis and Gene Ontology(GO) functional classification were used to analyze the differentially expressed genes.Results The 11 single cells from inner cells mass and 16 single cells from trophectoderm in 5AA preimplantational blastocysts were sequenced. We found there were 1 283 genes up-regulated in inner cell mass compared with trophectoderm. The GO biological function was mainly involved in DNA-dependent transcription resistance, signal transduction, cell differentiation, anti-apoptosis, nervous system development, cell adhesion, etc. These genes involved in MAPKs signaling pathway, actin cytoskeleton, adhesion,axis guidance, Jak-STAT, Wnt signaling pathway.We found there were 1 073 genes with up-regulated expression in trophectoderm compared with inner cell mass. Its GO biological function was mainly involved in transcription, DNA dependent transcription, cell cycle, protein catabolism, protein amino acid phosphorylation, protein transport, cell division, mitosis, ubiquitin-dependent protein decomposition, intracellular protein transport, etc. Those genes involved in ubiquitin-mediated proteolysis,sphingolipid metabolism, valine, leucine and isoleucine degradation, fructose and mannose metabolism, aminophosphonate metabolism, steroid biosynthesis, antigen processing and expression signal pathways. Conclusion The global geneexpression of inner cells mass and trophectoderm of the human preimplantational blastocyst is revealed from the spatial dimension, indicating that they are coordinated with each other, and the process of embryo implantation are finely regulated. Further data analysis is expected to find the endogenous specific molecules for regulating embryo implantation.
引文
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[21]Reijo Pera RA,De Jonge C,Bossert N,et al.Gene expression profiles of human inner cell mass cells and embryonic stem cells[J].Differentiation,2009,78(1):18-23.DOI:10.1016/j.diff.2009.03.004.
[22]Fujii T,Moriyasu S,Hirayama H,et al.Aberrant expression patterns of genes involved in segregation of inner cell mass and trophectoderm lineages in bovine embryos derived from somatic cell nuclear transfer[J].Cell Reprogram,2010,12(5):617-625.DOI:10.1089/cell.2010.0017.
[23]Driver AM,Pe?agaricano F,Huang W,et al.RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo-and in vitro-derived bovine blastocysts[J].BMC Genomics,2012,13:118-136.DOI:10.1186/1471-2164-13-118.
[24]Giritharan G,Delle Piane L,Donjacour A,et al.In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm[J].Reprod Sci,2012,19(3):243-252.DOI:10.1177/1933719111428522.
[2]Levin D,Hasson J,Cohen A,et al.The effect of endometrial injury on implantation and clinical pregnancy rates[J].Gynecol Endocrinol,2017:1-4.DOI:10.1080/09513590.2017.1318369.
[3]Wang ET,Kathiresan ASQ,Bresee C,et al.Abnormal implantation after fresh and frozen in vitro fertilization cycles[J].Fertil Steril,2017,107(5):1153-1158.DOI:10.1016/j.fertnstert.2017.03.012.
[4]Tang F,Barbacioru C,Nordman E,et al.RNA-seq analysis to capture the transcriptome landscape of a single cell[J].Nat Protoc,2010,5:516-535.DOI:10.1038/nprot.2009.236.
[5]Tang F,Barbacioru C,Wang YZ,et al.m RNA-Seq whole-transcriptome analysis of a single cell[J].Nat Methods,2009,6(5):377-382.DOI:10.1038/nmeth.1315.
[6]Li H,Durbin R.Fast and accurate short read alignment with BurrowsWheeler transform[J].Bioinformatics,2009,25(14):1754-1760.DOI:10.1093/bioinformatics/btp324.
[7]Huang da W,Sherman BT,Lempicki RA.Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J].Nat Protoc,2008,4(1):44-57.DOI:10.1038/nprot.2008.211.
[8]Bevilacqua E,Hoshida MS,Amarante-Paffaro A,et al.Trophoblast phagocytic program:roles in different placental systems[J].Int J Dev Biol,2010,54(2-3):495-505.DOI:10.1387/ijdb.082761eb.
[9]Nakashima A,Aoki A,Kusabiraki T,et al.Role of autophagy in oocytogenesis,embryogenesis,implantation,and pathophysiology of pre-eclampsia[J].J Obstet Gynaecol Res,2017,43(4):633-643.DOI:10.1111/jog.13292.
[10]Adjaye J,Huntriss J,Herwig R,et al.Primary differentiation in the human blastocyst:comparative molecular portraits of inner cell mass and trophectoderm cells[J].Stem Cells,2005,23(10):1514-1525.DOI:10.1634/stemcells.2005-0113.
[11]Hosseini SM,Dufort I,Caballero J,et al.Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts[J].BMC Dev Biol,2015,15:49.DOI:10.1186/s12861-015-0096-3.
[12]Rossant J,Tam PPL.Blastocyst lineage formation,early embryonic asymmetries and axis patterning in the mouse[J].Development,2009,136(5):701-713.DOI:10.1242/dev.017178.
[13]Zhao XM,Cui LS,Hao HS,et al.Transcriptome analyses of inner cell mass and trophectoderm cells isolated by magnetic-activated cell sorting from bovine blastocysts using single cell RNA-seq[J].Reprod Domest Anim,2016,51(5):726-735.DOI:10.1111/rda.12737.
[14]Giritharan G,Delle Piane L,Donjacour A,et al.In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm[J].Reprod Sci,2012,19(3):243-252.DOI:10.1177/1933719111428522.
[15]Treff NR,Northrop LE,Kasabwala K,et al.Single nucleotide polymorphism microarray-based concurrent screening of 24-chromosome aneuploidy and unbalanced translocations in preimplantation human embryos[J].Fertil Steril,95(5):1606-1612.DOI:10.1016/j.fertnstert.2010.11.004.
[16]Yan L,Yang M,Guo H,et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells[J].Nat Struct Mol Biol,2013,20(9):1131-1139.DOI:10.1038/nsmb.2660.
[17]Berg DK,Smith CS,Pearton DJ,et al.Trophectoderm lineage determination in cattle[J].Dev Cell,2011,20:244-255.DOI:10.1016/j.devcel.2011.01.003.
[18]Ozawa M,Hansen PJ.A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting[J].Fertil Steril,2011,95(2):799-802.DOI:10.1016/j.fertnstert.2010.10.006.
[19]Zhang J,Chiodini R,Badr A,et al.The impact of next-generation sequencing on genomics[J].J Genet Genom,2011,38(3):95-109.DOI:10.1016/j.jgg.2011.02.003.
[20]Ozawa M,Sakatani M,Hankowski KE,et al.Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells[J].Theriogenology,2012,78(6):1243-1251.DOI:10.1016/j.theriogenology.2012.05.020.
[21]Reijo Pera RA,De Jonge C,Bossert N,et al.Gene expression profiles of human inner cell mass cells and embryonic stem cells[J].Differentiation,2009,78(1):18-23.DOI:10.1016/j.diff.2009.03.004.
[22]Fujii T,Moriyasu S,Hirayama H,et al.Aberrant expression patterns of genes involved in segregation of inner cell mass and trophectoderm lineages in bovine embryos derived from somatic cell nuclear transfer[J].Cell Reprogram,2010,12(5):617-625.DOI:10.1089/cell.2010.0017.
[23]Driver AM,Pe?agaricano F,Huang W,et al.RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo-and in vitro-derived bovine blastocysts[J].BMC Genomics,2012,13:118-136.DOI:10.1186/1471-2164-13-118.
[24]Giritharan G,Delle Piane L,Donjacour A,et al.In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm[J].Reprod Sci,2012,19(3):243-252.DOI:10.1177/1933719111428522.