胎鼠与成鼠脊髓蛋白质组图谱的构建及其差异分析

Proteome map establishment and difference analysis of embryonic and adult rat spinal cord

目的初步建立并优化用于脊髓组织蛋白质组分析的双向电泳技术。应用双向电泳技术建立胎鼠与成鼠脊髓蛋白质组图谱,并对两者的差异进行比较,从整体水平上探究成鼠脊髓丧失再生能力的蛋白质基础。方法以大鼠脊髓为研究对象,应用以固相pH梯度等电聚焦为第一向、均一水平十二烷基硫酸钠-聚丙烯酰胺凝胶电泳为第二向的双向电泳体系,对样品制备、电泳参数、染色等技术环节中影响双向电泳分辨率的关键因素进行一系列的优化。采用ImageMaster 2D Elite图像分析软件对获得的胎鼠与成鼠脊髓蛋白质组2-DE图谱进行图像分析,比较两者在蛋白质组表达上的差异。结果在此优化的实验条件下,获得了质量较好的脊髓组织蛋白质组双向电泳图谱。胎鼠脊髓蛋白质组图谱经图像分析后,可检测到838个蛋白点,成鼠脊髓蛋白质组图谱可检测到847个蛋白点。对胎鼠与成鼠脊髓蛋白质组匹配差示图谱的分析发现,两者间存在813个差示蛋白。结论通过对各种条件的优化,初步建立了脊髓组织2-DE技术。对胎鼠与成鼠脊髓蛋白质组的比较发现,两者存在明显差异。胎鼠与成鼠脊髓蛋白质组差示图谱的建立,为进一步筛选、鉴定与脊髓再生能力丧失有关的蛋白,并探究其作用机制打下了基础。

Objective To establish and optimize the two-dimensional electrophoresis（2-DE） technique for spinal cord tissue proteome analysis. To establish proteome maps of embryonic and adult rat spinal cord by 2-DE, to compare them with each other and to explore why adult rat spinal cord loses the regeneration ability. Methods First-dimensional isoelectric focusing（IEF） was carried out by using immobilized pH gradient（IPG） gel and second- dimensional electrophoresis was performed by using homogeneous Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis（SDS-PAGE） with rat spinal cord tissue as object. A series of important factors, which affect the resolution, such as sample preparation, electrophoresis parameters, visualization and so on, were optimized. The proteome maps of embryonic and adult rat spinal cord were obtained by 2-DE, respectively. Proteome maps were digitized with the ImageScanner. The images analysis was performed with ImageMaster 2D Elite software. Results In the optimized experiment condition, the 2-DE proteome maps of rat spinal tissue were achieved with satisfactory quality. In the analyses, 838 protein spots were distinguishable in proteome map of embryonic rat spinal cord and 847 protein spots were distinguishable in proteome map of adult rat spinal cord. In the analysis of match differentially displayed map between embryonic and adult rat spinal cord, 813 differentially displayed proteins were found. Conclusion With optimization of each condition, 2-DE technique for spinal cord tissue has been preliminarily established. There are significant differences between proteome of embryonic and adult rat spinal cord. Establishment of the match differentially displayed map between embryonic and adult rat spinal cord provides basis for selecting and identifying the proteins that relates to the loss of the regeneration ability of adult spinal cord, and exploring the roles of those proteins.