| Objective:Using proteomic iTRAQ technology for osteoarthritis,knee osteoporosis and the two comorbidities model of New Zealand white rabbit knee joint cartilage differences in protein quantitative analysis,then looking for osteoporosis and the material basis of comorbidity of ost-eoarthritis,determine the specific protein markers.From the aspect of proteomics of osteoporosis and osteoarthritis is the material basis of comorbid specific protein markers.Method:Select 35 only 5 months white female rabbits in New Zealand,who were randomly divided into four groups in which A(OP and OA model group,n = 10)and B(OP model group,n = 10),C(OA model group,n = 10),D(blank control group,n = 5).Let build OA and OP model in group A,build the OP model in group B,and build the OA model in group C respectively.After the success of the building for each model validation,each group extractsright leg knee articular cartilage.After marking them,put cartilage specimens in-80?fridge cryopreservation timely.Then get protein samp-es after each group of articular cartilage specimens respectively in liquid nitrogen grinding + TCA/acetone precipitation+SDT cracking liquid boiling water bath + centrifugal technology such as operations.Then gradually to the protein sample by BCA protein electrophoresis and quan-titatively analysis method,and USES the Filter aided proteome preparat-ion(FASP)method to get peptides after enzyme solution,100?g peptides were taken from the samples,according to the AB SCIEX company iTRAQ kit instructions marking.Marker peptides are mixture sample,after a strong cation exchange chromatography column(SCX)classification into 8 points,then desalination freeze-dried respectively.After grading samples of peptides for 1 hour and gradient Q-exactive analysis respectively,that a total of eight LC-MS/MS analysis.The original mass spectrometry data using analysis software Proteome Discoverer for retrieval,by FDR<0.01 standard in protein screening identified differences in groups of protei-ns.Then in the difference in the protein A/B and the A/C expression is significantly higher or lower,and at the same time there was no significa-nt difference in the B/C standard screening of osteoporosis and osteoart-hritis comorbidity of specific proteins.And study the protein features and functions to conclud that the protein markers of comorbidity of both.Results:In the OA + OP and OP group analysis of the total screen:123 different proteins of which 65 difference proteins will elevate,and the rest will reduce.In the OA + OP and OA group analysis of the total screen:103 different proteins,including a significant rise in 62 different proteins,the remaining 41 different protein is significantly reduced.In the OP and OA group analysis,screen out:89 different proteins,inclu-ding 45 different proteins were significantly elevated,and the rest of the 44 difference protein is significantly reduced.From the different proteins of the three groups,get out a total of 12 specific protein:alpha 2-HS glycoprotein,Creatine kinase M-type,Fibrinogen beta chain,Fibrin-ogen gamma chain,Serum albumin,Vitamin D binding protein,N-acylethanol-amine acid amidase,Tubulin polymerization-promoting protein family mem-ber 2,Asporin protein and myelin protein zero,Uncharacterized protein(G1TQR3),Uncharacterized protein(U3KLT3).The above 12 specific protei-ns are in significant rise in the amount of protein expression in the OP and OA comorbidities model group.Conclusions:This experiment found a total of 12 specific proteins in the osteoporosis and knee osteoarthritis model rabbit,including Alpha-2-HS-glycoprotein,Creatine kinase M-type,Fibrinogen beta chain,Fibrino-gen gamma chain Serum albumin,Vitamin D-binding protein,Asporin protein and myelin protein zero.Their respective features are closely related to the osteoporosis and osteoarthitis comorbidity.And they may become to the specific protein markers for both disease,and can be used as the material basis of comorbidity for further mechanisms of disease research. |
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