| [Objective]This dissertation, on the basis of Ischemia/reperfusion spinal injury and energy metabolism, applying the methods of fluorescence labeling Proteome and blood-serum metabonomics. observes I/R spinal injuryand the changing characteristics of proteinum and enzyme concerned with energy metabolism, designing to use omics level to analyze the characteristics of dynamic variation in proteomics and metabolism atlas in the process of I/R spinal injury; meanwhile, the dissertation makes an attempt to expound that there is a relativity between delayed reversible ischemic neurological deficit caused by acute I/R spinal injury and energy metabolism, which provides a significant gist in finding its damage mechanisms and new drug target.[Methods]Experimental I/R rabbit spinal cord damage model, pathological morphology observation and ethology observation are established. Animals are divided into 3 groups (n = 6) using random sampling:sham operating group (SHAM). merely ischemia 30min group (I30min). ischemia 30min reperfusion 24h group (I30minR24h), assessed by functional score (Tarlov score) and pathomorphology observation for them. Using two-dimensional gel electrophoresis (2D-DIGE) to make quantitative analysis; firstly. applying CyDye fluorescent marker. two-dimensional electrophoresis separated proteins, and scanning the fluorescence images, and using DeCyder analysis software to make image analysis is exerted. therefore it acquires information of difference protein. After extracting rabbit serum by means of gas chromatography-Time Of Flight Mass Spectrometer(GC-TOF-MS). Masslynx4.0 data processing system:through using Nist 02 database we could analyze the name and change of the peaks of wave of the small molecules product. all GC/TOF-MS dates of rabbits have obtained corresponding load figure and score figure by ingredient analysis:Through the space 2-D figure we would observe the continuous variation pattern of metabolic small molecule product of three groups.[Result]18 successful models of rabbits were established. Compared with the SHAM group, scores of the hind legs inâ… 30min andâ… 30minR24h groups were 2.6 and 4.6 respectively. After ischemia-reperfusion, pathological morphology shows nuclei swelling, nuclear membranes obvious, which mostly are located at the center of the cell. The shape of the neurons changed from the initial extension to deformation gradually with less neurite, until the nuclei shifting and hyperchromatic appears the phenomenon of nuclear fragmentation. Along with the prolongation of time, we can observe that the nuclei of the neurons are bigger partly, and intranuclear heterochromatin slightly increases and the lipofuscin of the cytoplasm mounts up with rough endoplasmic reticulum and mitochondria visible. The myelin shelf of the surrounded myelinated nerve fibers are mildly separated, with nuclear and cytoplasmic matrix mild cavitated and decrease of the organelles. The Neuropil is swelled and cavitated, with the reduction of the neurofilament, the compact mitochondria, and the thinning myelin of the nerve fibers, etc. By means of the fluorescent marker and two-dimensional electrophoresis image analysis, and with two-fold differences for threshold,49 spots are detected to screening out the differentialy expressed proteins. We identified 21 unique proteins within 49 spots that differentially changed by mass spectrometry (MALDI-TOF/TOF MS) technique. The result:2 proteins are up-regulated in the I/R tissue(neurofilament protein M,alpha-tubulin), while 19 proteins are down-regulated in the I/R tissue. These proteins which contains Glyceraldehyde-3-phosphate dehydrogenase(GAPDH), Beta-enolase (EN03), Succinate dehydrogenase(SDHA), Glycogen phosphorylase (PYGM) and Pyruvate kinase(PKM2) are down-regulated apparently in the I/R tissue significantly(p<0.05). Through the analysis of metabolic GC/TOF-MS we have identified 51 small molecules (including 1 as internal marked material). The main metabolites are divided into four categories:amino acid, carbohydrate, lipid and others. The result shows:there exist obvious category differences in the scoring charts between groups. Experimental results show that metabolic changes of the small molecules peaked at 24h time point, and then tended to return to a normal metabolic course.[Conclusion] 1. The results have confirmed the existence of the spinal cord reperfusion injury. Ischemica and oxygen deficit can injury the spinal cord, however concerning the restore blood circulation and reperfusion, the pathological changes did not lessen but increased, which indicates that after the I/R injury of the spinal cord there is a secondary injury. The research results have indicated that with the delayed time of I/R and severe pathological changes, significant protein group and metabolomics have taken place; it was discovered that 24h after reperfusion is the most important point for small molecule metabolites active. As a result, the pathological extent of I/R spinal cord injury in rabbit has compliance with phamacogenomics, namely, protein group and metabonomics are also changed with the prolonged time of I/R, which contains significant answering relationship from the aspect of time. The dissertation presents that this stage may be the most important time zone of the reversible adjustment for ischemia-reperfusion injury of spinal cord.2.The five related key enzymes and regulatory enzymes(GAPDH, EN03, SDHA. PYGM, and PKM2) involved in its energy metabolism by means of screening and identifying play an important adjustable role in the developing process of spinal cord I/R. which may be its important pathology foundation. The five enzymes in the process of spinal cord I/R injury shows an apparent down regulation tendency; especially Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and Succinate dehydrogenase(SDHA) acting as the key enzymes which participate in glucolysis and citric acid cycle under inhibition have shown down-regulated expression:The changes indicate that the energy and glucose have a dysbolism after spinal cord ischemic. and it runs through the whole process of early ischemic spinal cord injury and reperfusion injury. It suggests that regulation of metabolism changes of the five enzymes may play an important role in spinal cord injury and regeneration.3. Acute I/R spinal injury metabolism, with the prolonging time of reperfusion, makes a change from the original anaerobic metabolism to aerobic metabolism as the main metabolism. It presents that acute I/R spinal injury in the early process, severe energy metabolism dysbolism takes place in intramedullary spinal cord, especially dysglycemia.[Contribution and innovation]1. By innovative means of combining metabolomics with proteomics, we have the High Flux Screening analysis and mamutual authentication to the pathological process of Acute I/R SCI and its Rehabilitation, especially the integration of two kinds of High Flux Experimental Techniques provides an olfactory pathways and technology platform for us, which finally makes us obtain a large number of data. But until now no reports on the research in I/R SCI by combining two kind of technology have been observed.2. We initially put forward the Compliance with Time between reperfusion of rabbit I/R SCI pathological severity and Phamacogenomics. Namely, the variation of metabolomics and proteomics turn up as the change of time of I/R. The research results elucidate that the change of omics corresponds to the histomorphological changes of spinal cord, which suggests pivotal time-window of special change of omics. What is the most important key time of reversibility regulation is 24h after Ischemia reperfusion Injury.3. The Key Enzyme of energy metabolism is identified and Screened:GAPDH,ENO3,SDHA,PYGM and PKM2. these enzyme plays a vital important role in the development of I/R. which shows that the disturbance of energy metabolism is the important pathology base in I/R of spinal cord. It presents that acute I/R spinal injury metabolism, with the prolonging time of reperfusion, makes a change from the original anaerobic metabolism to aerobic metabolism as the main metabolism. And the dissertation considers that acute I/R spinal injury in the early process, severe energy metabolism dysbolism takes place in intramedullary spinal cord, especially dysglycemia. The research results will provide a fresh evidence for new mechanism on I/R of spinal cord. |
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