8-细胞早期胚胎和8-细胞紧密化胚胎基因表达差异的初步研究  

Difference of gene expression between 8-cell early embryos and 8-cell compacted embryos in mice

作　　者：李汶[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,长沙410078

出　　处：《中南大学学报（医学版）》2007年第2期252-258,共7页Journal of Central South University ：Medical Science

基　　金：国家重点基础研究发展规划项目(G1999055901)~~

摘　　要：目的:研究哺乳动物胚胎的紧密化及紧密化前后基因表达谱的变化,寻找紧密化过程可能的基因调控机制。方法:超排、收集昆明白小鼠8细胞早期胚胎626枚和8细胞紧密化胚胎437枚,联合采用SMART-PCR(switching mechanism at5′end of the RNA transcript-PCR)和抑制消减杂交(suppression subtractive hybridization,SSH)技术,以8细胞紧密化胚胎cDNA作为检测子,以8细胞早期胚胎cDNA作为驱动子,研究8细胞早期胚胎及8细胞紧密化胚胎的基因表达差异。结果:共获得478个阳性克隆;进一步分析384个克隆,共获得83条ESTs(expressed sequence tags)。其中,有51个ESTs(61%)分别与36个已知基因高度同源,27个ESTs(33%)分别与7个假想基因明显同源,另有5个(6%)ESTs未发现与已知基因同源,为新的ESTs片段。紧密化过程中表达上调的基因中细胞骨架蛋白基因占4.8%,酶活性蛋白基因占9.6%,转录及翻译调节因子基因占12%,结合蛋白、蛋白间相互作用蛋白/运输蛋白基因占13.3%,核糖体结构蛋白基因占14.5%,多能性相关基因占27.7%,功能未知基因占18%;将17个与假想基因同源的ESTs和新的ESTs片段提交Gene Bank,被接受并给予了新序列编号。结论:联合SMART-PCR和SSH技术是一种研究少量标本之间基因表达差异的有效方法;在哺乳动物胚胎紧密化过程中,维持细胞多能性相关基因表达明显活跃,这可能与维持胚胎细胞在分化环境中保持其全能性相关;所获得的17个新的EST片段均可能为与紧密化密切相关的新基因的表达片段。Objective To investigate the compaction to search for the control mechanism mice） and 8-cell compacted embryos （437 mice differences of gene expression before and after the of compaction. Methods ） were collected from Kunm 8-cell embryos （ 626 ingbai mice, respectively. The cDNA of early 8-cell embryos was used as a tester, and the cDNA of 8-cell compacted embryos as a driver. Differences of gene expression were investigated between early 8-cell embryos and 8-cell compacted embryos using combining SMART （switching mechanism at 5＇ end of the RNA transcript ）-PCR and suppression subtractive hybridization （SSH）. Results Four hundreds and seventy eight positive clones were obtained, of which 384 clones with a range of 200 - 1000 bp and low redundancy were selected for further analysis. Eighty-three ESTs （expressed sequence tags ） of the genes expressed differently were gained between early 8-cell embryos and 8-cell compacted embryos. Bioinformatic analysis showed that among the screened 83 putative positive ESTs, 51 ESTs matched 36 known genes which were up- regulated , 27 ESTs matched 7 hypothetical genes, and 5 ESTs were new. Genes, during the compaction, included cytoskeleton （4.8 % ） ; enzymes （9.6%） ; transcriptional factor/regulatory factor （12%）; binding protein, protein-protein interactions and transport （ 13. 3 % ） ; structural constituent of ribosome （ 14. 5 % ） ; pluripotency associated gene （ 27.7 % ）, and molecular function unknown （ 18 % ）. Novel ESTs and 17 ESTs which matched the hypothetical genes were put into GenBank with accession numbers. Conclusion Combining SMARTPCR and suppression subtractive hybridization is an efficient method to investigate the gene expression difference in a few samples. During the compaction, the genes maintaining cell pluripotentiality express actively, which may be related to maintaining the embryonic cell totipotence in the differentation environment. All the 17 ESTs might be novel genes related to the compaction in the

关 键 词：植入前 紧密化 抑制消减杂交 差异表达 小鼠 

分 类 号：R321.2[医药卫生—人体解剖和组织胚胎学]