| Objective We identified an efficient protocol for extracting protein from rat spinal cord for 2-DE,among phenol, TCA-A and lysis. To explore the special proteins that were significant regulated after acute spinal cord injury in rat. Understanding the subcellular localization and function of these proteins, may help explore complex molecular cascades underlying the progressive pathologic changes in spinal cord injury.Material and method For each normal rat, a 1cm spinal cord segment(0.5cm proximal and 0.5cm distal to T10) was collected for subsequent analyses. Three protein extraction protocols were tested and evaluate using Bradford, SDS-PAGE and 2-DE. A severe injury was conducted by dropping a 10 g rod 25 mm onto T10 by IMPACTOR MODEL-â…¡. Depending on the different sacrificed time, 9 adult wistar rats were randomly divided into 3 groups: control group(sacrificed without spinal cord injury), 3h group(3h after spinal cord injury) and 8h group(8h after spinal cord injury). 2-DE was used to separete the protein collected from the three groups. Siginificant regulated proteins were identified by PDQuest. The interested protein spots were excised and detected by MALDI-TOF/TOF. We searched NCBI and Uni Prot KB for the subcellular localization and function of these proteins. Then, STRING software was used to analysis the signal path with these proteins. At last, we verified the one down-regulated protein GFAP(glial fibrillary acidic protein) by Western blot.Result 1. Compare with the other two protocol, TCA-A provided more clear band in SDS-PAGE and more protein spots in 2-DE. So we adopted TCA-A in the later study. 2. Compared with control group, we found 30 significant regulated proteins(11up-regulated and 19 down-regulated) in experimental group, including 9 metabolic proteins, 6 oxidative stress proteins, 5 neurofibrillar proteins, 3 skeleton proteins, 3 ATP enzymes, 1signal transduction protein and 3 others. 3. Western blot verified the result of GFAP. GFAP was significant decreased in 3h group and started to rise again in 8h group.Conclusion The study was designed to screening the significant regulated proteins after with proteomics technique. Exploring the potential special mole molecular maker for spinal cord injury, might provide the reliable foundation of treatment for spinal cord injury. |
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