| With the post genomic era jumping on the stage of human development, thetranscriptome of high-throughput technology has become the main force of studyinglife science research after proteomics and metabolomics technology. In recent years,with the social progress and development, the degree of the burden which was takento the society and patients by spinal cord ischemia-reperfusion injury(SCIRI) as acommon complication in the field of Neuroscience is more obvious, so the clinicalmedicine pays more and more attention to the invaliding. However, because of thecomplicated mechanisms of injury and repair, the damage is still the emphasis anddifficulty in clinical research and treatment. Through the results of early protemic itis founded that spinal cord ischemia-reperfusion injury had undergone an importantregulatory changes in time interval of24-48h, so the existing traditional andcommon research methods are not sufficient to react all of essence of the dynamicchanges. The emergence of transcriptome which is a high-throughput detectionmeans makes up for this deficiency and broadens the research ideas to this damagevery well. So in this thesis,the characteristics of different timing changes after spinalcord ischemia-reperfusion injury were took as the breakthrough point, based on theprevious research foundation the transcriptome analysis method was adopt topreliminary analysis and study multiple genes, pathways of change and theirrelationship involved in different sequential dynamic evolution of damage to hopethat the characteristic and law of dynamic changes in the whole damage processcould be showed fully and systemly from the source. All of them play a greatpushing effect for revealing the action mechanism of damage from gene level, whichhas the important practical significance. 【Objective】Reveal the dynamic change characteristics of the occurrence and thedevelopment of spinal cord ischemia-reperfusion injury at the transcriptional levelsystemly, which laid a theoretical foundation of screening disease-related key genesand making clear the mechanisms of damage in the future.【Method】Set up the model of spinal cord ischemia-reperfusion injury in SD rats of cleangrade divided into sham operation control, ischemia, reperfusion24h and reperfusion48h a total of4groups. Systemly evaluated behavior of model animals in differenttiming of damage and observed the pathological change characteristics of theinjuried spinal cord tissue. Took the lumbar spinal cord of injury segment as theresearch object and applicated of whole genome expression profile chip and miRNAmicroarray technology, screen out different expression of mRNA in damage of eachsequence through twopacket analysis method which then were analyzed of biologicalfunction and signaling pathway based on GO, KEGG database. At the same timecombined with multi packet and Serial Test Cluster (STC) analysis methods to getdynamic expression patterns of different mRNA, miRNA in the process of evolutionof the damage which were based to integrated analysis of specific correlationbetween mRNAand miRNA.【Results】The model group behavior scores display rat would appear different degreeoffunctional damage in awake,but along with the reperfusion time series to extendthe function situation would gradually improve; The scores of thecontrol,ischemia,reperfusion24h and reperfusion48h group were6.000±0.000,2.000±0.000,2.722±0.251and3.556±0.272respectively, and the scores betweeneach two groups had satistical significance (P<0.05). Pathological HE stainingshowed that started from ischemic period of invaliding, the pathological changes ofspinal cord tissue appeared showing cell shape changes and the interstitial edema,with the process of reperfusion began, the degree of cell damage gradually aggravated along with appearance of tissue hemorrhage, and status of interstitialedema came to peaks in reperfusion24h then eased in reperfusion48h.Through Two-Class Dif analysis,3groups of genes which had significantdifference were obtained in different time series when the screening criteria wasP<0.05, Fold Change≥2.0, FDR<0.05. The up-regulated genes were3032,528,3348in the timing of ischemia, reperfusion24h and reperfusion48h, while thedown-regulated genes were2442,183,2222. GO and Pathway analysis of thesegenes, the significant GO in the up-regulated expression were238,420,190itemswhile in down-regulated expression were1298,79,1003items. The significant GOwere analyzed in enrichment,the biosynthesis, positive biological process regulationand positive cell processes regulating were the common GO items for each stage;The significant pathway of each time group in up-regulated genes were16,30,15strips and in down-regulated genes were62,10,38strips. The most significantchange pathways in ischemia group were Ribosome and ECM-receptor interactionpathway, in reperfusion24h group was Apoptosis pathway and in reperfusion48hgroup was ECM-receptor interaction pathway.Through Multi-Class Dif analysis,8242different expression genes and172different expression miRNA were obtained based on time series of spinal cordischemia-reperfusion injury evolution when the screening criteria was P<0.05,FDR<0.05. Then through STC analysis,16significant differences in gene dynamicexpression patterns and10significant difference in miRNA dynamic expressionpatterns were obtained when the screening criteria was P<0.05/80. Among them,N0.28, N0.11and N0.40, N0.25and N0.27models of different genes and No.35,No.46, N0.19, N0.45models of different miRNA had important significance ofbiology.Based on the regulated relationship between miRNA and the correspondingmRNA and combined with all different genes in16significant expression patternsand the corresponding target gene of all different miRNA in10significantexpression patterns, the negative correlation analyzed to get4miRNA which were miR-22-3p, miR-743b-3p, miR-201-5p and miR-144-5p and2698different targetgenes which had negative correlations with the4miRNA.The common target geneswhich were negatively regulated by the4miRNA were23genes, such as Tmem69,Cxcl10and so on, all of them were used to construct negative correlation networkregulation of miRNAand mRNA.【Conclusion】1. It is comfired that the rat model of spinal cord ischemia-reperfusion injury hadbeen established successfully combined with behavioral score and pathologresults.2. It is founded that gene groups which related with the biosynthesis, positiveregulation of biological process, the positive cell processes regulating genefunction and Ribosome, ECM-receptor interaction, Apoptosis signal transductionpathways play a leading role in the change of spinal cord ischemia-reperfusioninjury through the application of the whole genome expression profilemicroarray and miRNAexpression microarray detection technology on the injurysection of spinal cord tissue.3. In the damage evolution, different expression of gene and miRNA existed somespecific significant dynamic expression patterns which had a biologicalsignificance.Among them, differential gene dynamic expression patterns ofN0.11and N0.40contains gene groups which could distinguish between normaland disease, and these genes were regulated by the miRNA which were includedin dynamic expression of No.46,19,45.4. Biological information analysis comfired that the group of different expressiongenes involving damage evolution were mainly regulated by4types of miRNAwhcih were respectively rno-miR-22-3p, rno-miR-743b-3p, rno-miR-201-5pand rno-miR-144-5p.All these characteristics of regularity in the dynamic evolution of injury mightbe the concrete manifestation of the new mechanism, it was also the above fourkinds of miRNA involved in the damage of the whole process and play an important role in the regulation. All of these laid the theoretical foundation of tamp for thethorough analysis, research and verification of transcriptome in spinal cordischemia-reperfusion injury in the future.【Innovation Point】The transcriptome of technology was innovatively used in the study of spinalcord ischemia-reperfusion injury, it respectively showed the changes of mRNA andmiRNA in every damage timing and evolution of damage, which provided anoriginal important theoretical basis for pathology control mechanism of spinal cordischemia-reperfusion injury and guiding the clinical treatments.This research was supported by the National Natural Science Foundation ofChina, Grant No.:81350013. |
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