| The mechanism of tinnitus is not clearly understood. As we now known, the animal model of tinnitus induced by sodium salicylate is the basical tool of tinnitus research. In this research, we want to provide insights into mechanism of tinnitus by studying the molecular mechanism of sodium salicylate ototoxity through gene chip.The first part Objective To extract high quality total RNA from cochleas of normal rats, the rats treated with long-term sodium salicylate injections or single injection and determine their RNA yield. Method Dissected the temporal bones to collect cochlea ,and the otic capsule was removed . Then the cochlear materials from ten to twelve rats were pooled and homogenized , total RNA was extracted from the homogenized tissues combination TRIzol method with RNeasy method. Finally spectrophotometric analysis was used to determine results of spectrophotometric showed the values of the yield and purity of total RNA and gel electrophoresis was used to test whether there existed RNA degeneration. Result Total RNA extracted from normal cochlea was much more than that from cochlea treated with sodium salicylate. The value of A260/ A280 were 2.05, 2.08 and 2.12 and gel electrophoresis showed no degeneration sign. Conclusion The total RNA extracted combination TRIzol with RNeasy was fit to downstream experiments. And the RNA yield were different among rats different treated, a further research would be carry out to investigate changes of the specific genes by gene expression profile method.The second part Objective Using gene chip technique to analyze the gene expression profile changes of cochleas in rats induced by single high dose salicylate injection and long term injection. Methods. Double-stranded cDNA were synthesized by the reverse transcription of the total RNAs. Biotin-labeled cRNAs were prepared from cDNA by in vitro transcription and further fragmented for target preparation. After an aliquot was analyzed by agarose gel electrophoresis, part of cRNAs were hybridized with test chip, as the results were good, the remaining target preparation were hybridized with the real chip (Rat Genome U-34A, containing over 8800 genes and ESTs). The hybridized chips were scanned and analyzed by Affymetrix Microarray Suite software 5.0. Results As a result, three type of gene expression profiles of cochleas were obtained. When the profile of cochleas treated by single salicylate injection was compared with the profile of normal cochleas, it was found that 42 genes up-regulated and 59 genes down-regulated over two times, and when the profile of cochleas treated by long term salicylate injection was compared with the profile of normal cochleas, it was found that 53 genes up-regulated and42 genes down-regulated over two times. Genes that changed over two times involved in signal transduction, gene transcription regulation, ion channels, synaptic proteins, calcium binding proteins, oxidation reduction, et al. Most noteworthy, evident gene expression changes of Scn8a and other types of ion channels and synaptic proteins genes were found, in which Scn8a is sodium ion channel gene and associated with auditory function. Conclusion This may be contributed to interpret the mechanism of ototoxicity of salicylate, e.g. hearing loss and tinnitus. And may help to understand the broad pharmacological effects of salicylate.The third part Objective To study the mechanism of electrophysiologic changes caused by different type of sodium salicylate injection. Method Three gene expression profiles of different groups of cochlea were obtained. Differentially expressed genes among three groups were analyzed by SOM. The SOM result were analogized to electrophysiologic changes induced by different type of sodium salicylate injection. Genes in clusters of analog result were interpreted by Gene Ontology. Results 6 clusters genes by SOM analysis were obtained, in which cluster 3 and cluster 4 were chose as candidate cluster. There are 46 genes in cluster 3 and 30 genes in cluster 4 employing GO analysis, which involved in cell...
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