Protromics Study Of Steroid Induced Osteonecrosis Of Femoral Head Serum Protein Markers

Objective: avascular necrosis (Osteonecrosis of femoral head, ONFH) is a commonorthopedic clinical disease, treatment difficulties, high morbidity, is one of threemajor problems yet to overcome the medical field. Avascular necrosis in patients withprimary gene mutation, secondary avascular necrosis is divided into traumatic andnon-traumatic avascular necrosis. Pathogenesis of traumatic avascular necrosis clearerfor patients with femoral head trauma resulting in blood flow is interrupted, causingischemia and necrosis of bone cells. Rather traumatic osteonecrosis clinical commonin long-term use of large doses of hormones and alcohol caused [1], its pathogenesisis unclear, in recent years, with the corticosteroid widely used in clinical practice,cases of osteonecrosis also increased significantly. For the pathology of avascularnecrosis is particularly urgent, the exact pathogenesis will find the ideal treatmentplan to lay the foundation. This research project aims for long-term use of large dosesof hormones avascular necrosis patients, serum proteins extracted fromsteroid-induced osteonecrosis (SONFH) in a large sample of patients, according toARCO stage (Ⅰ stage, Ⅱ, Ⅲ or, phase Ⅳ) classification, serum proteinscomprehensive proteomic analysis system. This study will make up fewer cases ofprevious studies, ignoring osteonecrosis causes and pathological staging deficiencies.By analyzing more than proteomics, screening candidate serum protein markers haveexpressed further study the long-term use of large doses of hormones cause thepathogenesis of osteonecrosis of the candidate proteins based on differences in thesemolecules. Protein expression and study of these candidates correlation stagingosteonecrosis of femoral head necrosis as a prognostic and predictive assessment oftherapeutic efficacy of hip osteonecrosis Paul biochemical markers. According to thecorrelation of these candidate proteins with avascular necrosis of the pathogenesis of early diagnosis for the future to find efficient methods to determine the direction ofdrug treatment, while filtering out specific biological markers, as a quick, specificosteonecrosis.Method:1, total protein extracted from human serum: According to inclusion andexclusion criteria, each drawn normal population and in patients with osteonecrosis3mL blood, centrifuged, serum was collected after purification, placed in-80℃refrigerator spare.2, serum proteins, the sample processing: using TCA/acetoneprecipitation process protein samples, the kit is removed and the high abundance ofalbumin globulin to give the final sample.3, two-dimensional gel electrophoresis(2-DE): on the prepared strip has conducted isoelectric focusing (IEF) andpolyacrylamide gel electrophoresis (SDS-PAGE), remove the gel strip, carriedCoomassie blue staining.4, the image analysis: an imaging scan using the scanner,the image analysis using the dye test PDQuest8.0software (Bio-Rad) were analyzed.Through the analysis of comparative analysis of glue, you can find the comparisongroup showed differences in the expression of proteins, these differences in the testprotein spots stained gel protein spots corresponding to the mass spectrometry as anobject.5, time of flight mass spectrometry (MALDI-TOF MS): on target proteinstrypsin digestion, peptide extraction mixture. Take0.3ul peptide extract with anequal volume of α-cyano-4-hydroxy cinnamic acid in acetonitrile mixed solution ofsaturated0.1TFA/50after the point in the mass spectrometer sample plate. Follow theinstructions.6, protein database query: NCBInr using Mascot database retrievalsoftware to retrieve, inputThe results reported.7, Western-Blot analysis ofdifferentially expressed proteins: Select fibrinogen γ chain (FGG) and fibrinogen βchain (FGB), carried out Western-Blot (Western blot), measuring the concentrationof the protein content, test results with2-DE phase verification.Results: separated by2-DE600±20protein spots, significantly differentiallyexpressed proteins73, digging up more than two-fold differential expression of11proteins huge difference, using MALDI-TOF/TOF mass spectrometry to identifythese protein spots, get four kinds of proteins, which are fibrinogen γ chain (FGG),fibrinogen β chain (FGB), ATP synthase, apolipoprotein AI (APOAI). Which necrosisgroup apolipoprotein AI (APOAI) downregulated the remaining three proteins wereup-regulated expression. Through a multi-database query, these proteins are involved in the formation of cytoskeleton involving participation, energy metabolism,redox and other aspects of the signal transduction function, there may be a potentialprotein markers. Select the fibrinogen γ chain (FGG) and fibrinogen β chain (FGB)do Western-Blot, results consistent with the2-DE results.Conclusion:1. Isolated from the serum of patients with osteonecrosis SONFH relatedproteins that help to clarify the pathophysiology of femoral head necrosis caused byhormones, and easily obtained, protein sample processing easier, less invasive,patients easily accepted.2. Using two-dimensional gel electrophoresis (2-DE) toeffectively separate the serum proteome, and access to femoral head necrosis(SONFH) differences in protein expression profiles.3. These were identified usingmass spectrometry MALDI-TOF/TOF protein spots obtained four kinds of proteins,analysis of the biological information, these proteins may be associated with thepathogenesis SONFH, screening for clinical diagnosis biomarker, SONFH moleculartargeted therapy provides a theoretical basis.4. By Western-Blot fibrinogen γ chain(FGG) and fibrinogen β chain (FGB) to validate the expression of consistent resultsand2-DE results prove the accuracy and reliability of the above study.