| Guillain-Barrésyndrome (GBS) is an acute or subacute autoimmune disease whose pathologic change is peripheral nervous inflammatory demyelination or axonal degeneration, as well as clinical character is flaccid paralysis. The cause and pathogenesis of GBS is still uncertain. In recent years, with the development of neuropathology and electrophysiology, GBS has been divided into acute inflammatory demyelinating polyneuropathy(AIDP),acute motor axonal neuropathy (AMAN),acute motor and sensory axonal neuropathy (AMSAN),Miller-Fisher syndrome(MFS) and so on. AMAN is a more serious clinical subset compared with AIDP, and the character of pathology and electrophysiology is axonal degeneration and decreasing of motor wave amplitude respectively.Proteomics is one of the signs of the post-genome era. Proteomics offers scientists the possibility of identifying disease-associated protein markers to assist in diagnosis or prognosis and to select potential targets for specific drug therapy. At present, there are a few studies about proteomics analysis of cerebrospinal fluid of patients with GBS, while there is no study on the comparative proteomics of AMAN cell model up to now.To investigate molecular mechanisms of AMAN, proteomics was applicated to evaluate the effect of serum from AMAN patients on spinal motor neurons of rat in this study.Methods:1. To obtain serum: Choosing the patients with GBS of acute period who fulfilled the criteria of GBS made by Asbury, then patients with AMAN were diagnosed according to the criteria of Cornblath. The blood of vein was centrifuged to obtain serum. The serum of healthy people was offered by Blood Center of Hebei Province. All serum was preserved in refrigerator of -80°C.2. Cell culture: Spinal ventral tissue was isolated from embryonic rats and digested into dissociated cell suspention for culture in vitro. When spinal motor neurons were cultured for six days, neurons were exposed to serum of patients with AMAN or healthy people, then collected after 2 hours.3. Protein extraction: Neuronal protein was extracted by lysate(9 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.5% CA and a cocktail of protease inhibitors), and the ultrasonic method.4. 2-Dimensional gel electrophoresis: The proteins were run immobilized pH gradient (IPG) isoelectric focusing electrophoresis as the first dimension, and then run vertical SDS-PAGE as the second dimension. The maps were visualized by silver staining or colloidal coomassive blue.5. Gel image analysis: The maps were analyzed with ImageMaster 2D Elite software. Taking the 2-D running gel of control group as reference gel, the 2-D running gel of neurons after AMAN serum exposure was compared with it, for finding the different protein spots. According to the parameter such as position,size,shape and so on, software could match the same proteins from different maps automatically, and consider the unmatched protein spots and more than 50% up-regulated or down-regulated protein spots as the different spots.6. In-gel proteins enzymolysis: After cutted the proteins from gel, 100μl 25mM NH4HCO3/50% acetonitrile were added in and vibrated to remove the dye. The granules of gel were freezed drying by freeze dryer, then digested with trypsin.7. Mass spectrometry identification: The proteins of interest were identified by MALDI-TOF/TOF tandem mass spectrometry.Reasults:Neuronal soma and neurite didn't change obviously after treated by nomal serum for 2 hours, while, after treated by AMAN serum for 2 hours, the halo of a part of soma became weakened, as well as neurite became thin and shortened. More than 1500 spots on the gels stained by silver and 600 spots on the gels stained by Coomassie blue were observed, respectively, when the gels were analyzed by ImageMaster 2D Elite software . We found 21 differently expressed proteins and identified them by MALDI-TOF/TOF tandem mass spectrometry as heterogeneous nuclear ribonucleoprotein H,tubulin,actin Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase,proteasome subunit alpha type 1,phosphatidyletha-nolamine-binding protein 1,14-3-3 protein epsilon,peptidyl-prolyl cis-trans isomerase A,prohibitin,endoplasmic reticulum protein ERp29 precursor,peroxiredoxin-6,60 kDa heat shock protein,F-actin-capping proteins,hypoxanthine-guanine phosphoribosyltransferase,Ras-related protein Rab-7a,triosephosphate isomerase,the receptor for activated protein kinase C 1,glyceraldehyde-3-phosphate dehydrogenase,voltage-dependent anion-selective channel protein 1,stress-70 protein,heat shock cognate 71 kDa protein.Conclusion:We got a number of related-proteins of AMAN including cytoskeleton proteins, apoptosis related proteins, antioxidants proteins and metabolic enzyme. Some of the proteins are quite useful for discovering the molecular mechanisms of AMAN, and some of the proteins could be identified as disease-related protein markers to assist in diagnosis and be selected as potential targets for specific drug therapy. |
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