Comparative Proteomic Study Of Proteins In Ligament Of Ankylosing Spondylitis And Biological Function Analysis Of Annexin A2

Ankylosing spondylitis(AS) is a chronic inflammation and abnormal ossification as the main feature of autoimmune diseases. There are many AS patients across the world. In our country, the incidence of AS is about 0.3%, which means 4 million AS patients because of the the huge population base. The main clinical features AS patients were chronic inflammation, new bone formation and abnormal ossification. AS patients suffer long-term back pain, back pain, joint pain, the body of the axial skeleton and limbs joint structure destruction, culminating in joint stiffness of bone, which will lead to neck, waist and leg diminished capacity to act, or even complete loss of activity. In which, 73% of the AS patients will be peripheral joint involvement, while the percentage of hip ligament ossification was 25%-50%, of these patients 50%-90% patients will involve bilateral hip. In addition, AS associated with systemic complications, it may involve a number of important tissues and organs eye, intestine, lung, kidney, heart, blood vessels, skin, bone and central nervous system and so on. Currently, the treatment of AS can only be relievable but the mechanism is not yet clear. Also, the new bone and the abnormal ossification treatment were still unresolved. Thus, more researches and strategies are needed to understand AS disease.Proteomics has become one of the frontiers of life sciences in recent years. Proteomics are using large-scale, high-throughput and high sensitivity of the techniques to reveal the essence of life activities through global genome expression profiles and expression in different spectral function of time and space, panoramic. Using 2D-Nano-LC-ESI-MS/MS proteomics technology can greatly improve the detection rate of the protein, which is much higher than the traditional solid 2D proteomics, and 2D-Nano-LC-ESI-MS/MS has now become the latest proteomic study means. Proteomics study of AS international now only at a preliminary stage, with only a few reports of blood cells as experimental material. The purpose of the study is the analysis of comparative proteomics in patients with AS and normal differences in protein expression ligament fibroblast-like cells to clarify ankylosing spondylitis(AS) in the pathogenesis.Chapter 1 Comparative proteomic study of proteins in ligament of Ankylosing SpondylitisThe use of tissue and ligament lesions occurred in the late normal tissue ligament ligament ossification or with a tendency AS patients. First, we cultured primary fibroblasts, which were obtained 35 samples of patients with AS and 10 normal control samples. Then we applied comparative proteomics research of ankylosing spondylitis ligament fibroblasts using shot-gun and lable-free methods, to determine the differences in protein expression profiles between AS patients and healthy controls. Which means the "disease-specific protein molecules" was identified using proteomics technology to analyze the differences in protein expression profiles between AS patients and healthy controls. First, all proteins were seperated through two-dimensional liquid chromatography- mass spectrometry and then Delta Vue software were used to identify the differences in protein expression between disease and normal control group of "quality"(acetylation and phosphorylation) and "quantity"(expression). After identifying the differential proteins, the protein was identified based on amino acid sequence, then bioinformatics were applied to predict the spatial structure of proteins and may function screening candidate genes; In our experiment, it was found that 6 proteins(HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were significant upregulated in the β fatty acid oxidation pathway, while 4 proteins(INSR, IRS1, PI3 K and PKC) were down-regulated in the INSR pathway. Then we took the real-time PCR, Western blot and MS and other technical expression to verify the expressions of these proteins. At the same time, the BMIZ and BMI scores of AS patients were significantly lower than normal based on the clinical data analysis. These results suggest that β fatty acid oxidation pathway reduces body fat in patients with AS, which is the first demonstration by the experimental data base.Chapter 2 Biological function analysis of Annexin A2 in Ankylosing SpondylitisAnkylosing spondylitis in the structure is mainly due to the formation of new bone and cause structural damage, and thus to make the spine occurs ossification stiffness. Bone tissue development requires a variety of factors to participate in the process, these factors include Osterix protein(OSX), runt-related gene 2(Runx2), transforming growth factor p, osteocalcin(OCN), alkaline phosphatase(ALP) and bone morphogenetic protein 2(BMP2) and the like. Regulation of osteoclast differentiation cytokines include: osteoprotegerin(osteoprotegerin, OPG), receptor activator of nuclear factor-κB(receptoractivator of nuclear factor-kappa B, RANK) and receptor activator of nuclear factor-κB factor ligand(receptoractivator of nuclear factor-kappa B ligand, RANKL).Currently, there are few reports for ankylosing spondylitis in vitro. We found high expression of Annexin A2 in AS patients, which may be the index of AS. Thus we used IL-6-induced primary ligament fibroblasts to analyze the function of Annexin A2 in vitro. We use Annexin A2 interference sequence transfection method and western blot detection method blot to examine BMP2, OCN, OPG, OSX, ERK1/2, RANK, and the expression levels of RANKL and found Annexin interference sequence A2 can inhibit BMP2, OCN, OPG, OSX and high expression of ERK1/2 phosphorylation, but also can promote the expression of RANK, RANKL, the three functions of Annexin A2 sequence interference are: 1) inhibiting the phosphorylation of ERK1/2; 2) adjusting the bone formation factor(BMP2, OCN, OPG, OSX) to inhibit new bone formation; 3) increasing osteoclast factor RANK, RANKL expression, and promoting the generation of osteoclasts, thereby inhibiting the proliferation and differentiation of osteoclasts. We also designed and synthesized Annexin A2 overexpressed sequence to investigate Annexin A2 overexpression sequence on IL-6-induced primary ligament fibroblasts, and found that Annexin A2 could induce osteoblasts factor(BMP2, OCN, OPG, OSX) high expression and inhibited osteoclast factor(RANK, RANKL) expression. Osteoblasts factor(BMP2, OCN, OPG, OSX) expressions were increased while the osteoclast factor(RANK, RANKL) expression has been decreased in the over-expressed in Annexin A2 combined with ERK1/2 inhibition group. Therefore, we speculated that Annexin A2 regulated osteoblast and osteoclast factor factor may be through the ERK1/2 signaling pathway.