| Spinal cord injury (SCI) is one of the central nervous system (CNS) diseases with high mutilation, the treatment of which is a major problem of neuroscience. Recently, many scientists have focused their interest on this field and have acquired important new insights in treating SCI related diseases, while the molecular mechanisms responsible for repair of the injured spinal cord are poorly understood. The repair process of damaged cord involves numerous molecular events, which are seen as changes in gene structure, expression, and activity. Some of these changes are important to the development and progression of the disease in which they directly contribute to the failure of axon regeneration or they improve recovery after traumatic injury of the spinal cord. So far, scientists have paid much attention to explore the changes of gene expression profile within 3 days after spinal cord injury, while the molecular mechanisms responsible for repair process were rarely reported. In the present study, we used PCR-based improved subtractive hybridization for analysis of genes differentially expressed during restoration after spinal cord injury, identifying of target genes and seeking pathways for drug development and therapeutic intervention.Subtractive hybridization is a powerful approach for identifying differentially expressed genes in defined tissues or cell lines. In this study, we applied improved subtractive hybridization, which means that several new techniques were adopted on the basis of classical subtractive hybridization, such as cap-finder reverse transcription method, long-distance PCR, streptavidin magnetic bead-mediated subtraction and spin column chromatography. Based on the spinal cord injury and regeneration model, a cDNA subtractive library was constructed after two rounds of improved subtractive hybridization. The subtractive efficiency of this library was examined by amplifying the housekeeping gene G3PDH and basigin, which is highly expressed in damaged spinal cord, using the non-subtractive cDNA library as a control. To identify the differentially expressed genes, this cDNA subtractive library were ligated to pGEM-T Easy(a) vector, then transformed into E.coli JM109 competent cells. Of the 110 clones randomly pickedout using a-complementary screening method, 93 clones were found to have the recombinant plasmids after examined by EcoR I digestion. Sequenced and analyzed, 51 differentially expressed sequences (not including repeat sequences) were gained. Reverse dot blot was conducted to remove the pseudo-positive clones and finally 40 differentially expressed sequences were gained. Then we design primers for several ESTs and conducted semi-quantative RT-PCR to confirm that they are differentially expressed genes hi damaged spinal cord. The sequences of 40 ESTs were compared with Genbank database in order to get their homology with gene sequences in the database. The outcome was that 32 ESTs show high homology to known sequences and the other 8 represent novel sequences.Among the 32 known ESTs, such genes as synuclein, clusteruu alpha acid glucosidase> vimentin^ reticulon and neurofascin have already been reported to be involved in development of nervous system, survival of neurons, nurite outgrowth or nerve degenerative diseases. Analyzed with SMART software, a Leu zipper structure was found at the end of one novel sequence (number 69). This structure is one of the conservative domains where transcription factors bind their DNA regulatory sequences specifically and it is mainly found in cell cycle regulatory factors, which are closely related with regulation of cell growth and differentiation. Thus we conclude that 69 might be a crucial transcription regulatory factor during spinal cord injury and regeneration. Then we tried to amplify the 5' and 3' full sequence of 69 gene through RACE technique and successfully obtained the 5' full sequence, which layed a base for further research on the role that 69 might play during repair of the contused spinal cord. |
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