Proteomics Of Foreign Nerve Regeneration After Somatic-autonomic Nerve Anastomosis

Part I Establishenment of Two-dimensional Gel Electrophoresis forProteomics of Rat Spinal Nerve TissueObjective: To investigate the application of two-dimensional gel electrophoresis in normal rat spinal nerve proteome studying artificial somatic-autonomic reflex pathway.Methods: Protein of normal rat L4 ventral root was extracted by powerful solubility lysis solution(Urea 9M, CHAPS4%,Tris 40 mM, DTT 65mM, PMSF 2mM, EDTA 5mM, Phamalyte 0.5%) and separated by combination of the immobilized pH gradient (IPG) isoelectric focusing electrophoresis and vertical SDS-PAGE electrophoresis. Silver-staining and CBB-staining was used to detect and quantitate proteins of different gels.Results: The proteins of normal rat spinal nerves separated by 2DGE(first dimension 18cm pH3-10 NL strips, second dimension 12.5% SDS-PAGE gel in the isodalt system had 900±30 spots, and the match rate of protein spots was 80 % of different gels.Conclusion: Lysis solution with high chaotropes system adopted and experimental condition control of the first and the second dimension electrophoresis, are the key to obtain the satisfactory 2DGE maps of normal rat spinal nerves.Part II Comparative Proteomics Analysis between Foreign NerveRegeneration after Somatic-autonomic Nerve Anastomosis and Purely Somatic Nerve Regeneration in RatObjective: To compare the global protein expressions following somatic-autonomic nerve anastomosis with somatic-somatic nerve anastomosis in rat.Methods: After establishment of rat model of regenerated foreign nerve (FNR) and purely somatic nerve (SNR) by 90 days, optimized 2DGE technology was used to separate the global proteins of FNR and SNR. The pattern of proteins expression of two groups was determined by ImageMaster analysis software, and the selected proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry.Results: Results showed good match of 2DGE maps of two groups obtained from the 3 repeated SDS-PAGE, with 918±20 spots detected for FNR and 937±30 spots for SNR, and a average matching rate of 82% and 75%, respectively. The pattern of 2DGE maps of two groups was very similar, with most of proteins fixed in the relatively identical places. It is different in expression for 33 spots in 2DGE maps, of which 17 spots were up-regulated in FNR while 16 spots up-regulated in SNR. Three selected spots were identified as Glucose regulated protein 58, Prohibitin and Tumor protein with MALDI-TOF-MS and MASCOT database.Conclusion: The characteristic of regenerated foreign nerve fibers may be generally identical to somatic nerve fibers in molecular level. There were differences in protein expression quantitation between FNR and SNR, possibly owing to the different target organ they innervated.Part IIIProteomics Analysis during Foreign Nerve Regeneration after Somatic-autonomic Nerve AnastomosisObjective: Using proteomics to explore the temporal expression profiles of proteins in rat during foreign nerve regeneration after somatic-autonomic anastomosis.Methods: Extracts of foreign nerves collected at 7, 14 and 28 days after somatic-autonomic anastomosis were analyzed by two-dimensional gel eletrophoresis and quantitative image analysis. Mass spectrometric analysis was applied to identify the different proteins in expression.Results: Of the ～900 protein spots resolved on each gel, 25 showed significant regulation for at least one time point. These proteins were grouped into two expression profiles of down-regulation and four of up regulation using cluster analysis. Mass spectrometric analysis identified 24 proteins pertaining to several functional classes, including acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal organization and in lipid metabolism.Conclusion: Molecular process underlying foreign nerve regeneration after somatic-autonomic anastomosis is complex and temporal. Glial and inflammatory determinants may play important roles in the process.