| With the widespread clinical application of hormone, SANFH has become the most common disease in non-traumatic femoral head necrosis. Currently, the pathogenesis of SANFH remains unclear, many theories can only explain part of the pathogenesis from single molecular or genetic level, not whole, dynamic, multi-level to observe the type, quantity, peak variation of the SANFH related regulatory genes and associated gene protein product.With the post-genome era coming and the rise of proteomics, proteomics can provide new ideas and techniques to study the pathogenesis of SANFH.Objective: (1)To create a rabbit model of early SANFH by given a high doses of dexamethasone and increase the loading of femoral head. (2)To optimize femoral head dimensional gel electrophoresis (2-DE) technology. (3) To create protein expression profiles of early SANFH(3w, 6w, 9w, 12w after the injection of hormones)and normal femoral head, identify the difference proteins between early SANFH and normal femoral head by image analysis and mass spectrometry and observe their changing patterns. (4) To verify the expression of Annexin A1 by Western blot. Methods: (1)The 48 rabbits were randomly divided into the model groups and control group. Rabbits in the model group were injected dexamethasone (10mg/kg) and forced to hold upright position 1 hour everyday, while rabbits in the control group were injected the same amount of saline. Animals were sacrificed at 3w,6w,9w,12w after injection, using optical microscope and transmission electron microscopy to observe the histopathological changes and measuring the Caspase-3 activity in osteocyte.(2)Femoral head from normal rabbits were used for samples preparation, optimizating the technique by improving the 2-DE process in some steps, including sample preparation, isoelectric focusing procedures, IPG strip selection and gel staining.(3) Preparing samples of early SANFH(3w, 6w, 9w, 12w)and normal femoral head, using MALDI-TOF-MS to identify the difference protein after creating 2-DE images. (4) Using Western Blot to detect Annexin A1 expression levels in each group.Results: (1) Histopathological changes could be observed under the light microscope at 3 weeks after administration, including fat cell hypertrophy and relatively decreasing of mesenchymal stem cells, the percentage of fat cell area is higher (P<0.05). The percentage of empty osteocyte lacunae in the model group was significantly higher than in the control group at 6 weeks after administration (P<0.05). Trabecular bone becomes thinner even collapse in model group at 9 weeks after administration. Under the electron microscope, morphological changes in bone cells were happened. At 6 weeks after administration, osteocyte Caspase-3 activity in the model group was higher than the control group (P<0.05). (2)Ideal images can be obtain after optimizing the 2-DE technique.(3) 20 different proteins were identified,7 proteins up-regulate in the early SANFH,meanwhile 13 proteins down-regulate. (4)Result in Western Blot showing that the expression of Annexin A1 in early SANFH was lower than the control group, the expression trends were consistent with 2-DE.Conclusions: (1) Using high-dose dexamethasone combined with increasing loading of the femoral head can creat a rabbit model of early SANFH. (2) The optimization of the femoral head protein 2-DE technology are important to the further research on SANFH. (3)20 differentially expressed protein were found, they may become the potential biomarks to SANFH. (4)The result is consistent with the 2-DE, the trend of Annexin A1 expression down-regulate in SANFH was verified by Western Blot,Annexin A1 may be one of importance proteins in early SANFH.
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