| As china entering into an aged society, spinal degenerative disease has increasedyear by year. Surgical of decompression and stabilization is the only way to solvesevere spinal cord compression disease and restore the spinal cord function. But insome cases of severe spinal cord compression, the spinal function has been graduallydeteriorated after transient recover. This phenomenon been called “Secondaryparalysis”. After been oppressed for a long time, spinal cord was in chronic ischemiaand hypoxia state. And decompression is a kind of instant changes to chroniccompression. Spinal cord blood perfusion increased rapidly compared withpreoperation, beats of the spinal cord was visible after surgery, At present, spinal cordischemia reperfusion injury(SCIRI) has been known as main cause of “Secondaryparalysis”.The course of the SCIRI is simple, but the mechanism of SCIRI iscomplicated. I/R damage to the spinal cord is a kind of stress injury. Tens ofthousands different kinds protein exist in cell, which serving different function. Oneof them able to withstand adverse stress damage, we called them “Stress relatedproteins”. Stress related proteins involving multiple protein family, contains hundredskinds of proteins, most of them are important members of molecular chaperone familyin the cell. Studying space time variety rule of stress related proteins expression afterSCIRI is vital for the prevention and treatment of SCIRI. Using differential proteomics technology can analyze the relative abundance of protein expressiondifferent cell or tissue at different time. So the stress related protein which expressionchange is obvious could be selected by this technique. studying the expression rule ofthe target protein will reveal its role in the SCIRI.【Objective】Through the SCIRI animal models, to obtain the two timesupregulated or downregulated protein by the method of comparative proteomicstechnology. Picking up the stress related protein, to study the temporal and spatialvariation of the protein expression, and discuss the function of stress related protein.【method】Using New Zealand white rabbits as the experimental animals, SCIRImodel is set up by Zivin method. According occlusion and reperfusion time ofabdominal aorta, the experimental animals were divided into control group(abdominal aorta exposing only, without occlusion), ischemia for30minutes andreperfusion group0hours,6hours,12hours,24hours,48hours, a total of six groupfor research. Bilateral hind legs function of experimental animals was evaluatedthrough the method of modification Tarlov score after reperfusion6,12,24and48hours. And spinal cord tissue of each experimental animals groups were examined forhistological study under light microscope and electron microscope.Using fluorescentdifference two-dimensional gel electrophoresis and mass spectrometry identification,bioinformatics analysis, obtained the proteins which expressed obvious differentially.The types, quantity and the expression changes of the protein were studied. Stress related proteins were selected, and using western blot to confirm mass spectrometry.Using immunohistochemical studied spatial and temporal change of stress relatedprotein expression in spinal cord. Neurological function, histology,immunohistochemistry and western blot, and differential proteomics were integratedto analysis the role of stress related protein in SCIRI.【results】 A total of21differences protein were identified, according to theexpression changes24hours after reperfusion, protein was divided into up regulatedprotein and down regulated protein. There were18protein in down regulated proteinmodels,including3stress related proteins, HSC70, PDIA3and STIP1. Metabolicenzymes: succinodehydrogenase,enolase3,glycogen phosphorylase B,Glyceraldehyde-3-phosphate dehydrogenase, Mitochondrial glycerol-3-phosphatedehydrogenase2, ubiquitin-activating enzyme E1, Pyruvate kinase isozymes M1/M2,Glycogen Phosp-horylase B; Nerve fiber protein: glial fibrillary acidic; cytoskeletalprotein: Spna2protein, FSCN1protein, dynamin1;others: N-ethylmaleimide-sensitive factor, ATP synthase F1complex, alpha subunit precursor, serum albuminprecursor; Up regulated proteins including neurofilament protein M, neurofilament-Land alpha-tubulin. The translation motion phenomenon of5proteins was seen in Gelelectrophoresis figure. Tarlov scores showed the function of hind limbs ofexperimental animals gradually improved after reperfusion, the function reach the bestlevel of early phase at24hours after reperfusion, and then the function was slightly down at48hours after reperfusion. HE stain showed the spinal cord tissue damageincreased gradually as prolonged of reperfusion time.The lesion was most severe at12hours after reperfusion which showed dense cavity formation in the gray matter.The extent of cavity of gray matter was mitigated gradually from24hours to48hoursafter reperfusion. Neurons in the spinal cord were gradually reduced as thereperfusion time last.The mortality rate of motorneurons was higher thaninterneurons.,Near all kinds of organelles damage were found by electron microscopyobservation after30minutes ischemia performance, and lesion was graduallyaggravated with the time last. The pathological changes was same basically at24hours and48hours after reperfusion. HSC70, STIP1and PDIA3immunoblot grayscale analysis revealed that the expression of three proteins change were consistentwith DIGE analysis. The expression of the three protein were downregulatedobviously after24hours of reperfusion. we found that Induced-inhibited-induced wasthese protein expression regularity curve in SCIRI. Immunohistochemistry showedthat immune response was seen in neuron cytoplasm. As reperfusion time goes on, theintensity of immune response was enhanced. and reach to the peak level during6hours to12hours after reperfusion. At24hours after reperfusion the intensity of theimmune response turned to be alleviate, and enhanced once again at48hours afterreperfusion. Immune response of HSC70was observed at membrane of glial cells andschwann cells, and with prolongation of reperfusion time the nucleus was gradually appear strong immune reaction, the change characteristic of immune response wassimilar with that in neuronal cytoplasm. The intensity of immune response in motorneuron cytoplasm was relatively weaker than that of interneurons, and immuneresponse appeared in interneurons was earlier than that in motor neurons.【conclusion】1,21proteins involving a variety of functions were identified bythe method of comparative proteomics technology, The function of some proteinsmay be achieved by glycosylation or phosphorylation;2Spinal cord histology andfunction studies showed they were inconsistent, The function of spinal cord was notcompletely depend on the number of neurons or the damage degrees of spinal cord,the neurons with normal morphology may be have an abnormal neurologicalcondition;3. When spinal cord tissue suffered severe injury, edema within gray matter,expression of most protein might be shut down, and the energy was supplied priorused to adjust the water and salt metabolism. when spinal cord edema was relieved,the expression of the protein would be up regulated, and the protein continue to play arole of nerve protective effect;4Vulnerability of motor neurons in the SCIRI might beassociated with lack of stress related protein expression.5The strong SCIRI toleranceof glial cells and schwann may be related to HSC70transfer into the nucleus.【Innovative Pionts】1In this research,ischemia and reperfusion injury wasconsidered as a kind of stress injury, we studied SCIRI from the aspects of stressinjury;2we selected the target protein by the method of comparative proteomics technology which is very efficient and accurate;3we differentiate spinal cordischemic injury from reperfusion injury, put forward an idea of two-phase damage ofSCIRI4we integrated immunohistochemistry and immunoblot method to study thetarget protein, which increase accuracy of the result in this paper. The temporal andspatial variation of proteins expression was precise elaborated;5By combininghistology and behavioral research, we systems analysed SCIRI characteristics and therole of stress related protein. |
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