Analysis Of Gene Expression And Biological Process In Proximal And Distal Nerve Segments Post Sciatic Nerve Transection

Objectives:In modern society, there are many peripheral nerve injuries in accident andmanufacturing works. Not as the central nervous system which could not regenerate,the injured peripheral nerves have the ability to regenerate by their own miraculously.And this kind of spontaneous regeneration process is controlled by various moleculargenetic levels of peripheral nerve cells. In earlier studies, people prefer to the researchof morphology. A lot of genes which have the adjustment ability of regeneration afterperipheral nerve injury were found in nearly study stage with the development of genetechnology. System knowledge and comparative study of gene expression changesand the related biological processes during peripheral nerve injury and regeneration,however, is still lacking.This study aimed to obtain system knowledge of gene expression changesfollowing the model of rat sciatic nerve transection injury and discover the possiblebiological processes via the contrast of differentiation of the proximal and distalsegments. We wish to have a deeply integrated insight into the molecular mechanismsregulating peripheral nerve regeneration from much broader perspectives on the basisof early research.Materials and Methods:150adult male Sprague-Dawley (SD) rats were randomly divided into6groupsaccording to the different time of sciatic nerve transection. The rats were executed at0day,4day,7day,14day,21day,28day respectively, and the proximal and distalsegments of0.5cm sciatic nerve were taken for biological analysis. The total RNAwere extracted by Trizol, the gene chips for expression were prepared, repeated threetimes at each time points, using the related software to chip numerical standardized analysis. We compared the data of different time points with blank control group (0day) for a statistical analysis through the T test, and choosed genes which were p <0.05, the fold change>2as have significant differences in statistical analysis to abiological function analysis.We applied the functin of Gene Ontology (GO) analysis of DAVID softwareonline, made the analysis of total11biology expression of biological processes whichare response to stimulus, inflammantory response, immune response, cell proliferation,cell migration, cell apoptosis, axon guidance and axonogenesis, myelination,extracellular matrix, signal transduction and protein kinase activity, were reflected bythe genes of proximal and distal segments. We observed expression trends inbiological process of the total of11biological processes about the variations of genes,analyzed and summarized the distinct of proximal and distal segments.In order to verify the experimental accuracy of the gene chips, we selected fourknown genes have a regulating effects on peripheral nerve, respectively, by used realtime fluorescence quantitative analysis, Western Blot protein analysis andhistochemistry immunofluorescence staining from three aspects of the molecules,protein, and morphology respectively.Research Results:The gene chips analysis showed that the most genes had the expression trend ofpositive regulation at7day, and the genes had a stable expression trend of negativeregulation at4day later, the two types maintained the balance after14day, the genesof positive regulation were dominant in the overall number in the proximal segmentsof sciatic nerve post transection. The genes had the top expression trend of positiveregulation at7day, and the numbers of genes with expression trend of negativeregulation were increased with time points, the two types maintained the balance intotal numbers in the distal segments of sciatic nerve post transection.In biological process, the11biological characteristics: inflammantory response,immune response, cell proliferation, cell migration, cell apoptosis, axon guidance andaxonogenesis, myelination, extracellular matrix, signal transduction and protein kinase activity which were associated with peripheral nerve regeneration, had thespecific changes in the proximal and distal segments in every time points. All thesewere had the accordance to peripheral nerve regeneration process we had knoun.Research Conclusions:1、In the model of rat with sciatic nerve transection, the genes of positive regulationwere dominant in the proximal segments, and the genes numbers of positive andnegative regulation were equally in the distal segments. There had the mostpositive expression in the same time point(7day), but a opposite expression trendafter the point in both proximal and distal segments however.2、The11biological characteristics which were inflammantory response, immuneresponse, cell proliferation, cell migration, cell apoptosis, axon guidance andaxonogenesis, myelination, extracellular matrix, signal transduction and proteinkinase activity which were related to peripheral nerve regeneration had thespecific changes in the proximal and distal segments in every time points inbiological process. The maximum of different genes and the biggest difference ofpositive and negative regulation appeared mostly in7day. They were connectedto each other and consisted a regulating and controlling networks that haddynamic development.3、The results of gene chips were true and correct according to the comfirmation ofreal-time fluorescence quantitative analysis, Western Blot protein analysis andhistochemistry immunofluorescence staining.