Differential Proteomics Study On Bone Tissues Of Femoral Head From The Patients With Non-traumatic Osteonecrosis

Background Osteonecrosis of the femoral head (ONFH) was a pathologic process caused by multi-pathogenic factors that damaged the blood supply of the femoral head, which led to the death of myeloid element, adipocyte and osteocytes. It is one of the common clinic diseases and mainly divided into two groups:traumatic osteonecrosis of femoral head (TONFH) and non-traumatic osteonecrosis of femoral head(NONFH). Some data indicates that ONFH has replaced the hip tuberculosis as the first place in hip diseases. Osteonecrosis of the femoral head have troubled domestic and foreign scholars for a long time. it is also called "the deadless cancer". But now, due to a lack of effective early screening and detection methods, it is rarely detected and intervented in early time. So most patients have a feel of hip'pain and limitation of hip'motion for a long time, then, think about the possibility of osteonecrosis of the femoral head. At this time, these patients had to be operated with THA because of the collapse and defomation of femoral head and osteoarthritis. It is the clinical and laboratory experts'task to make clear its pathogenesis, search valuable biomarkers for early screening and treatment.The pathologic process of traumatic osteonecrosis of femoral head is already clear, but the etiology and pathogenesis of non-traumatic osteonecrosis of femoral head are still unclear. Currently, hormone and alcohol are thought to be the most causes of non-traumatic osteonecrosis. Researchers have put out more than 10 theories up to now, they all involve in many kinds of enzymes, cytokines, gene, protein and so on, which are all closely related with proteins. Proteins are the performer and participant in course of disease occurrence and development, and also the signal of disease process. Previous researchs about protein only aimed at one or a few proteins in life activity, therefore, large-scale, omni-directional protein study is necessary.The proteome can entirely research the expression, composition and activity discipline of protein. It include three main parts:Expression Proteomics,Structural Proteomics and Functional Proteomics. Expression Proteomics refers to identify all the protein produced by specified cell, tissue or organ. Structural Proteomics refer to determine the first level and 3D structure of all-above metioned proteins. Functional Proteomics illuminate the physiological function of the protein and the dynamic change of the expression level in its different periods. Proteomics research can realize the docking and confirm with the genome, directly reveal the pathological mechanism of occurrence and development of the human major disease.Proteomics research on most orthopaedic disease is still in the initial stage. In recent years, proteomics research about osteonecrosis of femoral head is mainly on serology and experimental animal tissue. Some research has found that a few high expression proteins are closely related with osteonecrosis. But problems are:serum protein component is not stable, easily influenced by other factors, repeatability of experimental data is poor; and the data from animal experiment is different.Moreover, these proteomics research mostly based on conventional two-dimensional gel electrophoresis (2-DE) technology which has certain limitation: (1) the sample weight restrictions on that low abundance protein (<1 ng) hard to detection; (2) the results of electrophoresis-need dyeing and the results of dyeing combined with different proteins are different; (3) this technology is no-automatic processing, time-consuming, cannot undertake high-throughput analysis.Extracting proteins from bone tissue generally have two methods:chemical method and physical method. Several experiments are taken with chemical methods, although this method can isolate a variety of bone protein and low abundance protein can be effectively identified, but it needs about a week' time, and easily led to the protein denaturation,so it is not suited for Western blot study. In 2009, WangLe used hammering grinding method to extract proteins from bone tissue, easily and do not destroy the structure and physical and chemical properties of proteins, worthy of using for reference.At present there are many new proteomics analysis methods, among which the difference in-gel electrophoresis (DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. Because of these advantages, DIGE has become the most popular research means on proteomics.Objective In order to find different proteins, explore its pathogenesis, provide theory'basis for screening markers and targeted therapy. Based on above background, we designed this experiment. We collected femoral head from the patients with non-traumatic osteonecrosis of the femoral head and femoral neck' fracture, used liquid nitrogen grinding to extract total proteins from bone tissue, separated and identified protein with DIGE and MALDI-TOF/TOF, then made a bioinformatic analysis,Methods Our research is divided into four parts. The first part, collecting bone tissue sample. The second part, establishing a electrophoregram of DIGE. The third part, obvious difference protein identification and bioinformatics analysis. The fourth part, confirming expression level of some protein through Western blot test.Results 1600±50 protein points were successfully separated, and 274 points were obviously different. We dug 50 points which expressed differently more than two times with EttanTM Spot picker automatic workstations, identified with MALDI TOF/TOF mass spectrometer. Finally,9 different proteins were identified, which are apolipoprotein Al (APOA1), heat shock proteinβ-1 (HSPβ-1), ATP synthaseβsubunit (ATP5β), fibrinogenγchain (FGG), fibrinogenβchain (FGB), serum albumin (ALB), sulfur oxygen protein peroxide oxidoreductase (PRDX2), transferrin (TF) and actinl (ACTG1) respectively. They are all up-regulation except fibrinogen. Through a variety of database, these proteins involved in lipids metabolism, energy metabolism, cytoskeleton, signal transduction, redox etc. Results of Western blot showed the expressions of APOA1 and ATP5βwere consistent with DIGE.Conclusions:1. Extracting total protein of bone tissue by liquid nitrogen grinding can meet the require of subsequent proteomics research and proteins have little degradation.2. DIGE can separate total proteins of bone tissue and find different proteins efficiently.3. By identifying different proteins with MALDI-TOF/TOF mass spectrometry, we obtained 9 proteins. By bioinformatics analysis, these proteins are all related to occurrence and development of osteonecrosis. So our results provide theoretical basis for screening diagnosis biomarkers and molecular target therapy for ONFH.4. Through Western blot, we proved the reliability of our research.Innovations:to our knowledge, there are no reports on proteomic analysis of bone tissue from patients of ONFH with using DIGE and MALDI-TOF/TOF. In our study, we directly use bone tissue from patients of ONFH for research, data is more objective; use physical method of liquid nitrogen grinding to extract protein to reduce protein degradation; use DIGE to find different proteins,the result is more repetitive and sensitive.