Establishment and differential protein identification of two-dimensional gel electrophoresis for proteomics in the spinal cord of morphine-tolerant rats / 中南大学学报(医学版)

ABSTRACT

To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.