小鼠精原细胞分化的蛋白质组学研究

Proteomics Study on Spermatogonia Differentiation in Mice

对小鼠睾丸中未分化(Undifferentiated)和分化中(Differentiating)的两类精原细胞进行定量蛋白质组研究,探讨两类精原细胞蛋白质组的表达差异,探索与精原细胞分化相关的蛋白质。利用Thy1和c-Kit两个特异抗体结合的磁珠分选技术,分别将生后7 d雄性小鼠睾丸中的Thy1+细胞和c-Kit+细胞分别作为未分化和分化中的精原细胞分选出来,其中Thy1阳性细胞3组,c-Kit阳性细胞4组,分别代表了未分化精原细胞和分化中的精原细胞。采用高效液相色谱串联质谱方法(LC-MS/MS)分析两类细胞蛋白表达差异。并且对两类细胞的差异蛋白进行基因本体(Gene ontology,GO)功能注释、KEGG代谢通路和聚类分析。质谱分析共鉴定了3228种蛋白,其中有256种蛋白在两类细胞中表达差异。其中,差异蛋白的富集分析发现,在生物过程方面,注释结果显示差异蛋白主要在代谢过程(Primary metabolic process),细胞代谢过程(Cellular metabolic process),分子代谢过程(Macromolecule metabolic process)和氮化合物代谢过程(Nitrogen compound metabolic process)中富集;在细胞组分方面,蛋白主要富集在细胞(Cell part)、细胞内组分(Intracellular part)和细胞器(Intracellular organelle)中;在分子功能方面,鉴定到的蛋白主要参与蛋白结合(Protein binding)、核苷结合(Nucleotide binding)、水解酶活性(Hydrolase activity)和核酸结合过程(Nucleic acid binding)。基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路注释结果显示:88个蛋白质在KEGG通路分析数据库中有功能注释,共参与了18条代谢通路,其中,最主要的代谢通路是剪接体(Spliceosome)和泛素介导的蛋白水解作用(Ubiquitin mediated proteolysis)。获得小鼠睾丸中未分化和分化中的两类精原细胞蛋白质组表达谱,揭示精原细胞分化的蛋白质组的组成、筛选出差异蛋白。

This work aims to study the quantitative proteomics of undifferentiated spermatogonia and differentiating spermatogonia in mice testis,to explore the difference of proteome expression between the two types of spermatogonia,and to explore the proteins related to spermatogonial differentiation.Thy1+cells and c-Kit^+cells in testis of 7-days male mice were separated as undifferentiated and differentiating spermatogonia by magnetic bead sorting technique combined with Thy1 and c-Kit specific antibodies.Three groups of Thy1 positive cells and four groups of c-Kit positive cells represented undifferentiated spermatogonia and differentiating spermatogonia respectively.High performance liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to analyze the difference of protein expression between the two types of cells.GO function annotation,KEGG metabolic pathway and cluster analysis were performed on the differentially expressed proteins of the two types of cells.A total of 3228 proteins were identified by mass spectrometry,of which 256 were differentially expressed in two types of cells.Among them,enrichment analysis of differential proteins discovered that in biological processes,annotations showed that differential proteins were mainly enriched in primary metabolic process,cellular metabolic process,macromolecule metabolic process and nitrogen compound metabolic process.In terms of cell components,proteins were mainly concentrated in cell parts,intracellular parts and organelles.In terms of molecular functions,the identified proteins were mainly involved in protein binding,nucleotide binding and nucleic acid binding.KEGG annotation results showed that 88 proteins were functionally annotated in KEGG database and involved in 13 metabolic pathways.The main metabolic pathways were spliceosome and ubiquitin mediated proteolysis.The proteome expression profiles of undifferentiated and differentiating spermatogonia in mouse testis are obtained.The proteome composition of undifferentiated and differentiating spermatogonia is revealed and differential proteins are screened out.