Proteomics And Pathology Study In Multiple Sclerosis

ObjectiveThe aim of this study was to explore the molecular mechanism of multiple sclerosis,to find the biomarker in CSF and serum and to observe the pathological expression in spinal cord of EAE(animal model of multiple sclerosis).The role played by the biomarker protein was investigated in the development of multiple sclerosis and in the rebuilding of tissue.It would provide more experimental data for therapy research.Methods1.CSF samples from 8 patients of tension type headache were enriched and desalted through ultra filtration.The abundance proteins were depleted by the ProteoExtract Albumin/IgG Removal Kit.The concentration of protein was quantified with Bradford method.After the preparation of samples,proteins were separated in the SDS gels stained by silver nitrate and Coomassie brilliant blue respectively with two-dimensional gel electrophoresis.The mappings of gels were analyzed by ImageMaster 2D Platinum 5.0 software to locate the differential protein spots.Among which,the interested protein spots were chosen with the compare to SWISS-2DPAGE protein spots database,cut off from the gels, digested,and analyzed with MALDI-TOF-MS.With the results,we searched the protein information in MASCOT software and NCBInr database related to PMF and identified the interested proteins.2.According the proteomics technology system,a comparative proteomic analysis of CSF proteins was employed to 6 patients with multiple sclerosis(MS) and compared to 6 controls in order to identify the interested proteins and explore the molecular mechanism of multiple sclerosis.3.Serum samples were harvested from 2 health volunteers.The abundance proteins were depleted by the ProteoExtract Albumin/IgG Removal Kit.Samples were desalted and enriched through ultra filtration.The concentration of protein was quantified with Bradford method.After the preparation of samples,proteins were separated in the SDS gels stained by silver nitrate and Coomassie brilliant blue respectively with two-dimensional gel electrophoresis.The mappings of gels were analyzed by ImageMaster 2D Platinum 5.0 software to locate the differential protein spots.Among which,the interested protein spots were chosen with the compare to SWISS-2DPAGE protein spots database,cut off from the gels, digested,and analyzed with MALDI-TOF-MS.With the results,we searched the protein information in MASCOT software and NCBInr databases related to PMF and identified the interested proteins.4.According the proteomics technology system,a comparative proteomic analysis of serum proteins was employed to 8 patients with MS and compared to 8 controls in order to identify the interested proteins and explore the molecular mechanism of multiple sclerosis.5.The animal model was established in Wistar rat by immunization with complete Freud adjuvant and GPSCH.The clinical symptoms were evaluated in scales.The pathological change was observed with the aid of light microscopy.6.Brain and spinal cord were dissected,embedded in paraffin,and sliced. The expression and distribution of Apo E was detected with the "two steps" immunohistochemistry method.The expression and distribution of GFAP was detected with the SP immunochemistry method.Results1.316 protein spots were detected in gel stained by silver nitrate. Repeatability evaluation of it showed that protein spots match ratio was 93.2%. Compare with the pI and MW scale of 30 match spots chosen in random,there was not significant difference in statistics between two gels.201 protein spots were detected in gel stained by Coomassie brilliant blue.From the above result, the repeatability of this proteomics technological system was excellent.2.The gels containing MS CSF revealed 38 spots that were different to the control gels.Ten of the differential spots were chose to be identified with PMF technology.Seven of those were proteins present in normal human CSF.They were haptoglobin,clusterin,leucine-rich alpha-2-glycoprotein,retinol binding protein,thansthyretin,and albumin(isoform CRA_n and isoform CRA_j.)The three exceptions were ATP synthase,general transcription factor IIH and tetranectin.3.233 protein spots were detected in gel stained by silver nitrate. Repeatability evaluation of it showed that protein spots match ratio was 94.6%. Compare with the pI and MW scale of 30 match spots chosen in random,there was not significant difference in statistics between two gels.168 protein spots were detected in gel stained by Coomassie brilliant blue.From the above result, the repeatability of this proteomics technological system was excellent.4.The gels containing MS serum revealed 23 spots that were different to the control gels.Ten of the differential spots were chose to be identified with PMF technology.They were apolipoprotein E,serine proteinase inhibitor,albumin chain A,Alpha-1-acid glycoprotein 1,Alpha-1 microglycoprotein,clusterin, haptoglobin,leucine-rich alpha-2-glycoprotein,retinol binding protein,and thansthyretin.5.The experimental group developed the typical symptoms of EAE on (14.1±2.6)days after immunization with the incidence of 100%and showed a chronic monophasic course.The infiltration of inflammation cells in cerebrum, cerebellum,brainstem and myeloid tissue and the demyelination of the white matter were observed through HE staining and Luxol fast blue staining respectively under an optical microscope.The typical pathological changes mainly distributed in white matter and junction area of grey matter and white matter.No similar pathological change was found in the control.6.Apo E immunoreactivity was markedly increased in perivascular cuffings infiltrated by inflammation cells and slightly increases in grey matter compared with the control.Apo E immunostaining cells was more than the control in white matter.There was DAB staining in negative control.Most Apo E immunostaining cells were also GFAP immunostaining.This eliminated those cells were probable astrocytes.ConclusionIt suggested that the mechanism of MS includes the damage of energy metabolic pathway,inflammation,reconstruction of tissue.The expreesion of Apo E in EAE might reflect that astrocyte involved the reconstruction mechanism of MS.