SD大鼠视网膜蛋白质组双向电泳图谱建立和初步分析

Establishment and preliminary analysis of the two-dimensional gel electrophoresis map of neural retinal proteome in rats

目的建立优化SD大鼠神经视网膜双向电泳（two-dimensional gel electrophoresis，2-DE）图像技术方法，并对正常视网膜蛋白质组图像进行初步分析。方法雄性SD大鼠，断颈处死取眼球。在解剖显微镜下分离神经视网膜组织，液氮速冻，提取总蛋白质组份，-70℃保存。部分视网膜标本立即固定后做光镜或电镜分析。应用2-DE技术，对提取的蛋白质进行分离。在一维等点聚焦、垂直SDS凝胶电泳、银染（考染）等关键技术上进行优化和重复实验，建立分辨率高和重复性良好的2-DE图像；联合图像扫描和图像分析软件，对正常SD大鼠神经视网膜组织蛋白质组图像进行初步分析。结果成功获得了分辨率和重复性良好的视网膜蛋白质2-DE图谱。每张电泳图谱可获得约3000个左右可辨认的蛋白质斑点。其中大部分蛋白质点分布在pH4～8范围内，其相对分子质量主要集中在25～100区域。结论2-DE技术可用于对神经视网膜蛋白质组份的分离和鉴定。本实验建立的技术为进一步进行视网膜蛋白质组学研究奠定了基础。

Objective To establish and optimize the two-dimensional gel electrophoresis （2-DE） map of normal neural retinal proteome in Sprague- Dawley （SD） rats and to make preliminary analysis of the map. Methods Male SD rats were sacrificed by decapitation. The eyes were enucleated and the neural retina layers were carefully peeled off with the aid of dissecting microscope and immediately frozen in liquid nitrogen. Some samples were stored in fixative solution for further examination by light microscopy or by electron microscopy. The total proteins extracted from rat neural retinas were separated by 2-DE. Repeated experiment was conducted to optimize the experimental conditions for the isoelectric focusing （IEF）, SDS polyacrylamide gel electrophoresis （SDS-PAGE） separations, silver staining （ or coomassie-bluestaining） and other 2-DE techniques, The gel images were acquired by scanner and 2-DE analysis soft ware. Results Well resolved, reproducible 2-DE profiles of rat neural retinal proteome were obtained. From 2-DE analysis,approximately 3 000 protein spots were observed in each pattern,most of which were distributed in the Mr range from 25 to 100 with pH 4 - 8. Conclusion The technique of 2-DE can be used to separate and identify the proteins of rat neural retina. The results of the present study provided the way for further research on retinal proteomics.