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目的应用蛋白质组学实验方法对在异体异位生长发育的新生小鼠睾丸组织进行差异蛋白分析,从而对睾丸组织体外移植模型用于生精细胞发育研究的可行性提供进一步的理论依据。方法以新生小鼠睾丸组织为供体,免疫缺陷小鼠为受体进行组织移植,移植8周后收集移植物组织,采用双向凝胶电泳(2-DE)分离移植物组织总蛋白,同时分离8周正常小鼠睾丸组织总蛋白作对比分析,运用Im ageM aster 2D E lite 5.0图像分析软件识别移植物组织与正常组织差异表达的蛋白质点,应用基质辅助电离解析飞行时间质谱(MALD I-TOF-MS)获取肽质量指纹图谱(PMF),检索数据库鉴定差异表达的蛋白质点。结果正常睾丸组织和移植物组织双向电泳图谱的平均蛋白质点数分别为(1 326±15)个和(1 561±20)个,3次重复实验的蛋白质点位置重复性较好。通过比较两者的双向凝胶电泳图谱,得到表达差异量较大的蛋白质点21个。选取6个仅在正常睾丸组织中有表达的蛋白质点进行质谱和生物信息学分析,鉴定出的6种蛋白质,除血红蛋白α1链(Hbα-α1)外,其他5种蛋白均为在小鼠睾丸中有较高表达的蛋白质,包括A激酶锚定蛋白结合的精子蛋白(ASP)、磷脂过氧化氢物谷胱甘肽过氧化物酶(GPX4)、精子蛋白(Sp17)、天门冬酰胺酶样蛋白(ASRGL1)和过氧化物氧化还原因子4(Prdx4)。结论在本实验中,从异体异位生长发育成熟的新生小鼠睾丸组织中检测到5种在睾丸中特异性高表达蛋白质的缺失,这些蛋白质主要在精子运动、精子顶体形成以及精子的受精等功能中发挥作用。
OBJECTIVE: To apply differential proteome analysis of testicular tissue of allogeneic ectopic growth and development in proteomics experiment and provide further theoretical basis for the feasibility of in vitro testicular germ cell transplantation. Methods The newborn mouse testis was used as the donor and the immunodeficient mouse was used as the recipient for the tissue transplantation. After 8 weeks of transplantation, the graft tissue was harvested and the total tissue protein of the graft was separated by two-dimensional gel electrophoresis (2-DE) 8 weeks normal mice testis tissue protein for comparative analysis, the use of Im ageM aster 2D E lite 5.0 image analysis software to identify differentially expressed between the graft tissue and normal tissue protein spots, using matrix-assisted ionization time of flight mass spectrometry (MALD I-TOF -MS) to obtain peptide mass fingerprinting (PMF), and search the database to identify differentially expressed protein spots. Results The average number of protein spots in the two-dimensional gel electrophoresis of normal testes and grafts were (1 326 ± 15) and (1 561 ± 20), respectively. The reproducibility of protein spots in three replicates was good. By comparing the two-dimensional gel electrophoresis pattern, 21 protein spots with large amount of expression difference were obtained. Six of the six proteins identified by normal and testicular tissue were selected for mass spectrometry and bioinformatics analysis. All the five proteins except for hemoglobin α1 (Hbα-α1) Among these proteins, there are higher expressed proteins including A-kinase-anchored sperm protein (ASP), phospholipid hydroperoxide glutathione peroxidase (GPX4), sperm protein (Sp17), asparaginase- Protein (ASRGL1) and peroxide redox factor 4 (Prdx4). Conclusions In this study, five kinds of testicular proteins with high expression were detected in testicular tissue of allogeneic ectopic newborn mice. These proteins mainly involved in sperm motility, acrosome formation and spermatogenesis And other functions play a role.