| The model of middle cerebral artery (MCA) occlusion used in this study has been well characterized in terms of infarct pathophysiology and development of infarct. Neuropathologic changes occurred in MCA occlusion animal model replicate them after a stroke in human. This pathophysiology was known to be associated with the alteration of gene expression. Since many genes with complex responses are differentially expressed in cerebral ischemia, the knowledge of highly selective gene expression at various time points can provide a convenient shortcut for implicating a therapeutic target in cerebral ischemia. Differential gene expressions in cerebral ischemia were detected by differential display,and DNA microarray . However, little is known about which JAK/STAT-related genes are regulated in adult male rat after transient focal cerebral ischemia.In order to identify genes regulated by transient focal cerebral ischemia, we used membrane-based focused microarray technology. The microarray technique is very powerful in that it allows one to screen over a hundred genes at one time. This screening technique will allow us to further focus our research question based on the changes in JAK/STAT related gene expression patterns in adult male rat after transient focal cerebral ischemia. The expression levels of several genes were validated using Real-time PCR techniques.Gene expression profiles in rat hippocampus after transient focal cerebral ischemiaUsing superarray Q Series Signal Transduction PathwayFinder Gene Array, contain 96 marker genes associated with 18 signal transduction pathways (Mitogenic Pathway,Wnt Pathway, Hedgehog Pathway, TGF-b Pathway, Survival Pathway, p53 PathwayStress Pathway, NF-κB Pathway, NFAT Pathway, CREB Pathway, Jak-Stat Pathway, Estrogen Pathway,Androgen Pathway,Calcium and Protein Kinase C Pathway,Phospholipase C Pathway,Insulin Pathway,LDL Pathway,Retinoic Acid Pathway.). Through a simple side-by-side hybridization, using RNA samples and the arrays and reagents provided,determine whether any of the 18 signal transduction pathways may be involved in adult male rat hippocampus after transient focal cerebral ischemia. The results showed NF-kB Pathway and Jak-Stat Pathway were invoiced in this experimental system. Using superarray Q series Jak/STAT signaling pathway gene array, a total of 96 genes were screened in adult male rat hippocampus after transient focal cerebral ischemia. The results showed that 27 genes were at least 2-fold up-regulated by ischemia treatment, and 18 genes were at least 2-fold down-regulated by ischemia treatment compared to the controls.A subset of genes detected by gene array was tested by quantitative Real-Time PCR. Of the thirty-four genes found to be regulated by ischemia treatment, we selected eight genes that were up-regulated (STAT2, STAT5a, STAT5b, STAT6, SOCS4, PIAS2, PIAS3, PIAS4) following transient focal cerebral ischemia. Confirming the microarray data, we found that STAT2, STAT5a, STAT5b, STAT6, SOCS4, PIAS2, PIAS3, PIAS4 mRNA were up-regulated by ischemia treatment.Gene expression profiles in Band 3 cell line during cell hypoxia/reoxygenationUsing superarray Q series Jak/STAT signaling pathway gene array, a total of 96 genes were screened in adult male rat hippocampus after hypoxia/reoxygenation treatment. The results showed that 42 genes were at least 2-fold up-regulated by hypoxia/reoxygenation treatment, and 6 genes were at least 2-fold down-regulated by hypoxia/reoxygenation treatment compared to the controls.A subset of genes detected by gene array was tested by quantitative Real-Time PCR. Differential gene expression was consistent between the two independent methods of analysis, and the level of expression of all selected genes changed in the same direction as the changes measured by the microarrays, although there were quantitative differences between the results of gene array analysis and quantitative Real-Time PCR. Of the forty-two genes found to be regulated by hypoxia/reoxygenation treatment, we selected eight genes that were up-regulated (STAT2, STAT5a, STAT5b, STAT6, SOCS4, PIAS2,PIAS3, PIAS4) following transient focal cerebral ischemia. Confirming the microarray data, we found that STAT2, STAT5a, STAT5b, STAT6, SOCS4, PIAS2, PIAS3, PIAS4 mRNA were up-regulated by hypoxia/reoxygenation treatment.In conclusion, the obtained data suggest that the change of JAK/STAT pathway related genes is induced by the ischemia in brain and hypoxia/reoxygenation in cell line probably due to the compensation for damage. |
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