| Bone metabolism and formation disorder mostly long-term chronic inflammation caused by infection or other factors such as the direct interference. In China, every year there are about 50000000 cases of patients with fracture, fracture nonunion which incidence rate is about 5%-10%, mainly because of fracture after internal fixation or open fracture secondary to infection or other factors cause persistent inflammation. The healing process of fracture, the process is the necrotic bone fragments dissolved plus bone matrix formation and mineralization of large. Its main process includes dissolving bone of osteoclasts and osteoblasts. The role of the two antagonistic to each other constraints, dynamic balance is reached a certain. In general, the inflammatory reaction in traumatic fracture or bone can stimulate the body to start the physiological function, resistance and the exclusion of damage factors, bone tissue to repair damage, promote the healing of fracture. But the long-term chronic inflammatory response to the infection or other factors caused by the bone formation, dissolution and even suffer further destruction of bone. In recent years, more and more scientists began to study the relationship between inflammatory factor and osteoblast and osteoclast cells, including related research inflammation of chronic persistent can accelerate the bone resorption process has become more mature. However, on the other hand, chronic persistent on osteoblast metabolism especially on osteoblast differentiation related research has not yet mature, its interaction with specific mechanisms are still not clear. Osteoblast differentiation is involved in many cellular signal transduction mechanism of the existing channels, talk to each other between certain pathways （cross talk）, mutual influence, in response to inflammatory stimuli the adjusting process of more sophisticated, even on one or more paths are difficult to clearly into bone cells in the inflammatory stimuli and differentiation. In biomedical research in the future, to intervene and control inflammation from multiple inflammation development stage, multiple cell signaling pathways target is the development direction of the study of bone regeneration. Through the deep research on the inflammatory effects on osteoblast cell differentiation and related intervention mechanism, controllable target and identify the specificity in the inflammatory pathway under the control of inflammation to multi-level, multi targets, to promote the osteogenic differentiation, and realize the development of new drugs anti-inflammatory, contributed to the bone.Proteomics is the study of the maturity of the technology leads to the arrival of the post genome era, the biological function of protein is more direct execution, become a research hotspot in the field of life science. Proteomics is defined as:to the organism, tissue or cell all protein expression level, function and their interactions between high flux screening and quantitative and qualitative analysis, is more reliable than previous diagnosis and treatment by a single gene, protein to study disease, more accurately reflect the body state.Using traditional biochemical techniques, such as based on the analysis of two-dimensional gel electrophoresis mass spectrometry, one by one to study large amounts of protein, screening and detection of workload is huge and difficult to complete, not to mention the discovery of new from pathogenesis.ITRAQ technology is a kind of quantitative proteomics using new technology, has an outstanding advantage in comparative proteomics.Through the comparison of the body changes in bone differentiation proteins into healthy and inflammatory conditions, can obtain a large number of candidate marker protein. However, the large number of candidate marker protein and the development of the disease process and relationship specific detection of more explicit validation is a big difficult problem in current study of biomarkers of work. Multiple reaction monitoring mass spectrometry based （Multiple Reaction Monitoring, MRM） biomarkers for technical verification and testing, can through the high sensitivity mass spectrometry selected target protein. The technology is not required for synthesis of specific antibody, RT-PCR and Western-blot than the needs of antibody detection technology, it has more advantage. Research on verification technology using different levels of verification of iTRAQ technology, NBD polypeptide into protein group improved TNF-inhibition of osteogenic differentiation analysis based on the identification result is reliable, can clear the feasibility of further research, and also can be a more comprehensive understanding of the inflammatory stimulation mechanism of bone cell differentiation into functional properties and differences in protein, for subsequent protein expression the establishment of basic verification, so it is very necessary for functional verification.This study includes three parts:Part One:NBD peptide TNF-inhibited and improve establishment of cell differentiation of bone cellsmodel verificationThe prototype of C2C12 myogenic cells in differentiation inducing agent BMP-2 induced differentiation into osteoblasts, inflammatory stimuli TNF-inhibit bone differentiation of C2C12 muscle into primary cells, NBD polypeptide in inflammatory stimulation effect to inhibit the reaction of inflammation of bone differentiation. The experimental cell culture after the completion of the effectiveness of histochemical staining of ALP activity and ALP fluorescence analysis in order to verify the model. As the next step of proteomic experiment analysis based.Part Two:NBD polypeptide into protein group improved TNF-inhibition of bone cell differentiation analysis based on iTRAQ technologyThis part of the study will use a chapter to TNF- alpha as inflammatory stimuli, NBD peptides as inhibitors of inflammation role in BMP2 induced myogenic precursor C2C12 cells to differentiate into osteoblast model, will use the iTRAQ chemical label protein samples were marked inflammatory stimulation group, NBD polypeptide group and normal group cell differentiation the separated, the use of multidimensional liquid chromatography separation to the identification of tandem mass spectrometry, to screen out the potential and inflammatory stimulation and inflammation by inhibiting osteoblast differentiation process under the sex related difference in protein.Part Three:Improvement of NBD polypeptide TNF-inhibited osteoblast differentiation related differences in protein expression of MRM verificationThis part studies using multiple reaction monitoring mass spectrometry （MRM） based on the analysis of differential protein technology, provides the basis for the screening of NBD polypeptide in the improvement of TNF-alpha and further study on Mechanism of differences in bone differentiation inhibitory protein.METHOD1.Establishment of cell model:the experimental cells from mouse muscle derived cell line C2C12. It is divided into 3 groups, with different stimuli to culture. The B groups, i.e. BMP group, joined the BMP-2 in experimental group cells in liquid culture, the concentration of 100 ng/ml; the BT groups, namely group BMP+TNF, in BMP-2 and TNF-while adding liquid in the experimental group cell culture, the concentration of BMP-2 was 100 ng/ml, the TNF-concentration is 5 ng/ml; group BTP, namely BMP+TNF+NBD multi peptide group. After alkaline phosphatase （ALP） staining assay determination and fluorescence activity:the detection of C2C12 osteogenic differentiation of. Be in 5*103/hole ratio were seeded into 24 well plate culture. After 3 days, ALP activity was examined by fluorescence detection kit, the determination of the ALP using the method of PNPP activity, the choice of wavelength 405 nm, determination of optical density value of each hole in the ELISA detection apparatus, recording the results. After 7 days of cultivation and dyeing experiments were conducted, in accordance with the Alkaline Phosphatase Staining Kit operating instructions.2.Using iTRAQ technology and bioinformatics analysis of screening differential protein:protein in a cell by extraction and quantification, the labeling and detection using iTRAQ reagent. Then a preliminary LC/MS/MS analysis, strong cation exchange （SCX）, and then after a reversed-phase liquid chromatography separation and ESI mass spectrometry. Finally, the analysis of data, the retrieval and identification of proteins, these proteins from.2..1. analysis of differential protein bioinformatics:to analyze GO, the differences of the protein COG annotations, GO enrichment and Pathway analysis of significance, to study differences in protein main biological function, closely related to the metabolism pathway and signal transduction pathway.3.MRM:Selecting the target protein Transition screening after validation of chromatographic peak transition in the MRM+EPI mode, and then after the peak strength the daughter ions of MRM detection, calculation of ion peak area integration, finally the comparison of different samples in the target protein expression abundance.4.Statistical analysis:Using the SPSS statistical software （version 13.0） for data processing. Analysis and comparison of multiple groups using one-way ANOVA. Homogeneity of variance test by LSD method, Kruskal-Wallis method, adopts unequal variances otherwise. The level of test for alpha=0.05.Result1.ALP staining results of determination results show:B group showed blue particles; compared with BT group and B group, the cell culture showed a weak blue particle hole, show that the group BT ALP activity decreased significantly; the blue granules of BTP group than in BT group increased distribution. The fluorescence of ALP activity determination result has statistical significance 3 osteoblast osteogenic differentiation activity difference （F=225.80, P=0.00）. Each multiple comparison, lower in group BT than B osteoblast osteogenic differentiation activity （P<0.05）, suggesting that bone cells to differentiate into TNF-inhibited BMP-2 induced; group BTP compared with group BT, osteogenic activity increased significantly （P<0.05）.2.ITRAQ kit to mark the 3 set of C2C12 myogenic cells （BMP-2, TNF-alpha, BMP-2+TNF-alpha +NBD）, according to the screening of LC MS/MS identification analysis results, B group and BT group （bB114-VS-eBT118） two groups to achieve quantitative standard （ratio was larger than 1.5 or less than 0.6） of the 76 proteins, including protein upregulation in BT group 59, in the BT group down regulated protein 17.BT group and BTP group （bBT118-VS-fBTP119） of two groups of quantitative standard （ratio was larger than 1.5 or less than 0.6） protein number 43, the upregulation of BTP group protein 25, BTP group down regulated protein 18. NPC1 proteins were identified by database 3 added, comparison and analysis of all the NPC1 protein. The B VS BT between group differences in protein and BT VS BTP between group differences in protein do intersection processing, screened 8 differentially expressed proteins, namely Periostin, SERCA3B, Scafl, Actn2, Atp5d, Elp2, Atp51 and Npcl.The peak intensity of MRM detection of target protein ions, the integration calculation of ionpeak area size, which can compare the differentially expressed target protein in differentsamples. Osteogenic differentiation is stimulated by inflammation was inhibited, the upregulation of the expression of protein Elp2, Atp5d. The biggest rise of down regulated expression of Elp2; for Actn2, Atp51 and Npc1, Atp2a3 （SERCA3B）; when adding NBD peptideimprove inflammatory reaction, Elp2, Atp5d expression appeared down, the rest of the trendlevel of protein.Conclusions1.NBD polypeptides to improve TNF-alpha inhibition of BMP-2 induced C2C12 cell osteogenic differentiation experiments can be considered to improve inflammation inhibit osteogenic differentiation model, validated.2.iTRAQ technology and bioinformatics analysis of screened 8 inflammatory stimuli into differences in process of bone cell differentiatio 51 and Npcl, and improve the suppression of inflammation is closely related to the differentiation into bone.3.Result of MRM and iTRAQ identification analysis results agree. |
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