| Spinal cord injury (SCI) is an unexpected, catastrophic event that leads to varying degrees of paralysis and sensory loss below the level of the lesion. The consequence of which often persists for the life of the patient and influence in diverse ways not only the patient, but also family members and society at large. Thus, it is essential to investigate the regeneration after SCI. A flock of progresses have been made in this field. However, the molecular mechanism of regeneration after SCI is still not clear.In the past decades of years, a lot of methods were developed to explore the molecular mechanism of regeneration after SCI, such as in situ hybridization, RT-PCR, Western Blotting and immunohistochemistry. Some SCI-induced gene and protein expression changes have been identified with these methods. However, these techniques are quite slow and laborious thereby enabling only a few genes to be investigated at a time. Recently, several multiplex techniques capable of monitoring global changes in mRNA and protein abundance have been developed, such as microarray analysis, differential display PCR, total gene expression analysis (TOGA), serial analysis of gene expression (SAGE), rapid analysis of gene expression (RAGE), massively parallel signature sequencing (MPSS), restriction-mediated differential display (RMDD) and proteomic technique. Microarray analysis and related techniques is a powerful new tool to quickly examine thousands of genes simultaneously and has enabled us to profile the changes of global genes expression after SCI.Adult littermate male Wistar rats, weighting 200~250g, were divided randomly into injured group and control group. The injured group rats were subjected to a completelytransected injury at thoracic spinal cord (T8-T9). The control group rats received only a laminectomy. The animals, allowed to survive for another 4.5 days, were killed. The spinal cords of rats from same group were dissected out and pooled for isolation of total RNA. The total RNA was converted to cDNA probe by reverse transcription. Two kinds of fluorescent labeled probes were hybridized to the cDNA microarray. In the present study, we identified 65 genes that showed a greater than 2-fold increase hi SCI tissue. 79 genes were down-regulated greater than 50% in SCI tissue. The 65 up-regulated genes consist of 21 known genes, 30 known ESTs and 14 unknown genes. The 79 down-regulated genes comprise 20 known gene, 42 known ESTs and 17 unknown genes.The 5 up-regulated known gene, tissue inhibitor of metalloproteinase 1, transgelin, vimentin, Fc gamma receptor, cathepsin S, and 3 down-regulated known genes, stearyl-CoA desaturase, coagulation factor II, endosulfin alpha, were further confirmed with reverse transcription polymerase chain reaction (RT-PCR). The data indicated that cDNA microarray is an efficient and useful tool to identify genes changed after SCI. These differential expression genes may play a role in the response to tissue damage or repair following SCI.At the same time, we utilized Q-TOF to identify 20 protein related to SCI which failed to be identified by MALDI-TOF. The same animal model was adopted in the experiment. The animals, survived for another 5 days, were killed. Soluble protein and membrane protein was extracted from the spinal cord, respectively. We separated protein with two-dimensional gel electrophoresis and cut 20 differential protein according to previous image analysis. The amino acid sequences of part polypeptides of 15 protein were obtained by Q-TOF. Out of 15 protein, 2 protein was identified as unknown protein by Blast analysis. We are cloning mRNA encoding unknown protein.From above experiments, we screened some genes related to SCI, and identified 2 unknown protein on the previous basis. These data enable us to further understand the molecular mechanism of regeneration after SCI. |
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