| BackgroundAnkylosing spondylitis, a kind of chronic and progressive inflammatory disease which will occur on skeleton and soft tissue, will mainly damage each joint of spine as well as neighboring tendon and ligament. In the meantime, Chronic and progressive inflammatory reaction will also occur on sacroiliac joint and neighboring joints to some extent. Among ankylosing spondylitis patients, above 60% of them have peripheral joints diseases, in terms of hip joint in particular. During the course of disease, the bone substance around joint will be damaged and the bony ankylosis will appear until lose its activity function, which will greatly affect the patient’s life quality. The early phase of AS pathology mainly focuses on ligament attachment point inflammation, such as tendon, ligament and joint capsule. Then, soft tissue is affected and the neighboring bones will be corroded and damaged. In the later phase, the new bone of attachment point forms osteophyte, and becomes bony bridge in the gap of joints, gradually developing toward arthrofibrosis and bony ankylosis. Finally, it will lead to joint deformity and lose the function of normal activity.The specific pathogenesis of ankylosing spondylitis peripheral joint ossification is still not clear yet. The early viewpoint believed that it is disorder of bone formation mechanisms caused by inflammation, leading to the improvement of osteogenic capacity on certain parts. With the in-depth research on pathogenesis and process, peripheral joint ossification on ankylosing spondylitis patient is considered as a complicated process with interaction and multi-stages. Currently, the speculations on its pathogenesis are as follows:1. signaling abnormalities 2. inflammation effect 3. stress effect 4. gene dysimmunity. However, the research on ankylosing spondylitis peripheral joint bony ankylosis mainly focuses on how to adjust and control inflammation and cell metabolism, so as to stop inflammation path, reduce cell stimulation and delay the ectopic change of bony ankylosis.As the main synovial joint of human body, peripheral joint is the joint that is frequently affected. The synovial tissue covers on the internal surface of articular cavity and surrounding of femoral neck. It wraps the blood vessel of joint capsule, generating synovial fluid so as to reduce articular surface friction and provide articular cartilage with ingredient and transport metabolic product. However, the lesion of synovium will also lead to significant changes of intra-articular environment. Hence, the peripheral joint pathogenesis of ankylosing spondylitis is closely related to the pathology of intra-anticular synovium. According to the early investigation of ankylosing spondylitis peripheral joint pathogenesis, it shows that synovitis, which is the main clinical manifestation, appears in intra-anticular. When joint surgery is conducted under arthoscopy or direct view, synovium hyperplasia along with synovial inflammation is discovered in the patient’s intra-articular. In the meantime, auxiliary examination in terms of iconography and ultrasound has also provided reference basis for ankylosing spondylitis early diagnosis, proving the function of synovial pathological inflammation in changing joint pathogenesis. With the course of disease and induction of various cytokines, part of the tissues is under vascularization, making the synovium gather and differentiate various potential stem progenitors. The joint synovial cells differentiate toward bony cells, evolving as osteoblast, osteoclast, etc. Under the interaction of these cytokines released by these cells, synostosis metaplasia gradually occurs in synovium, becoming hyperplasia position of new bone and osteophyte.SOST is a kind of glycoprotein coded by SOST gene. Due to "cystine structure" exists in SOST, it has higher affinity with co-receptor in wnt signal transduction path, which can competitively bind LRP5/6 receptor and reach the effect of inhibiting signal transduction path. From the research on wnt induced protein aggregation and oligomerization, it finds out that the mediate LRP6 receptor can affect or enhance the activity of wnt signal. SOST is closely connected to wnt signal path and can affect the wnt signal path through LRP receptor. The competitive binding and antagonism of SOST and LRP5/6 wnt/β-catenin signal path cannot completely explain the inhibition activity of SOST. With the in-depth research on SOST in recent years, however, it is discovered that the osteocyte activity can be adjusted by increasing RANKL expression. Hence, SOST serves as an extensive participant in the course of bone formation.This research conducts a staining comparison on synovial tissue which is under ankylosing spondylitis peripheral joint ossification ankylosing and normal people’s synovial tissue with immumohistochemical technique. It detects the difference of SOST and β-catenin expression in ossification ankylosing joint synovium from gene and protein expression level through pcr and wb testing method as well as the relation expressed by wnt signal path, which has further ensured the possible existing function in ossification development process. Gene transfection technology is applied to set up overexpression SOST adenovirus vector to transfect normal people’s synovial cells, and study changes on the ossification capacity of synovial cells after soft over-expression as well as the differences and similarities of its physiological function.The first section Expression and function of SOST and β-catenin in the synovial tissues of peripheral joint ossification of ankylosing spondylitisObjectiveTo investigate the expression and correlation of sclerostin (SOST) and β-catenin in the synovial tissues of the ossification of peripheral joint with ankylosing spondylitis patients. To explore the relationship between SOST、 β-catenin and ossification of peripheral joint with ankylosing spondylitis.Metheods1.Synovium of ankylosed hip joint of 15 ankylosing spondylitis patients were in experimental group, Synovium of hip joint of 18 femoral neck fracture were in control group.2.1mmunohistochemical technique was used to detect the expression of SOST and β-catenin.3.Expression of SOST and β-catenin genes in Synovium were detected by PCR technique.4.Expression of SOST and β-catenin protein in Synovium were detected by Western-blot.5. Immunohistochemistry judgment of the results:SOST and β-catenin positive cells were defined as brown granule deposited in the plasma. Using Image-pro plus 6.0 image processing analysis system for quantitative analysis of immunohistochemistry, then examined by a microscope with enlargement of 200 time, Each slice were observed 6 horizons whichever is compared with the Integrated option density.6.Real time-PCR judgment of the results:Detection of expression of SOST and β-catenin genes in Synovium by using relative quantitative method.7. Western blot judgment of the results:Detection of expression of SOST and β-catenin protein in Synovium by using semiquantitative analysis,and compared with the a-tubulin gray scales,and calculating the ratio.8 Statistic analysis. All data were expressed by mean and standard error, Software SPSS 19.0 was used for the statistical analysis. Quantitative variables were compared by means of the student t test. Correlation analysis used Spearman rank correlation analysis. P<0.05 was accepted as statistically significant.Result1. Immunohistochemical result shows that SOST and β-catenin are strongly positively expressed in ankylosing spondylitis synovial membrane group, and are mainly distributed in fibroblast-like synoviocytes and its IOD value is higher than that of normal synovial membrane group. The difference is of great significance (P< 0.01). The detection index Ankylosing spondylitis group is considered different from normal synovial group, the Ankylosing spondylitis group is higher than the normal group; SOST is positively related to β-catenin expression in ankylosing spondylitis synovial tissue.2. Through Real time-PCR detection, it shows that the SOST and β-catenin gene content is greatly expressed in ankylosing spondylitis synovial membrane. From relative quantitative analysis, it shows that the SOST and β-catenin protein level in AS synovial membrane group is significantly higher than that of normal synovial membrane group. (P<0.05).3. From Western blot detection, it discovers that SOST and β-catenin protein is greatly expressed in ankylosing spondylitis synovial membrane group. After gray value analysis and semi-quantitative result, it shows that the SOST and β-catenin protein level in ankylosing spondylitis synovial membrane group is significantly higher than that of normal synovial membrane group. (P<0.05)Conclusion1. The abnormal expression of SOST and β-catenin in ankylosing spondylitis peripheral ossification joint synovial tissues is related to ossification.2. The ossification capacity in AS peripheral ossification joint synovial tissues is mainly embodied in synovial fibroblasts.3. The synovial tissues of AS peripheral ossification may rely on wnt/β-catenin signal to exert its function.The Second Section Expression Relevance of Osteogenic Effects and Osteoclast Effects in Ankylosing Spondylitis Ossification Joint Synovial TissuesObjectiveTo investigate the expression and correlation of OPG and RANKL, and osteoclast specificity tartrate-resistant acid phosphatase (TRAP) stain, in the synovial tissues of the ossification of peripheral joint with ankylosing spondylitis patients. To explore the relevance between OPG、RANKL and ossification of peripheral joint with ankylosing spondylitis.Metheods1.Synovium of ankylosed hip joint of 15 ankylosing spondylitis patients were in experimental group, Synovium of hip joint of 18 femoral neck fracture were in control group.2.1mmunohistochemical technique was used to detect the expression of OPG and RANKL. The OCs were observed by the TRAP stain.3.Expression of OPG and RANKL genes in Synovium were detected by PCR technique.4.Expression of OPG and RANKL protein in Synovium were detected by Western-blot.5 Immunohistochemistry judgment of the results:OPG and RANKL positive cells were defined as brown granule deposited in the plasma. TARP stain judgment of the results:positive cells were defined as wine red granule deposited in the plasma Using Image-pro plus 6.0 image processing analysis system for quantitative analysis of immunohistochemistry, then examined by a microscope with enlargement of 200 time, Each slice were observed 6 horizons whichever is compared with the Integrated option density.6.Real time-PCR judgment of the results:Detection of expression of OPG and RANKL genes in Synovium by using relative quantitative method.7. Western blot judgment of the results:Detection of expression of OPG and RANKL protein in Synovium by using semiquantitative analysis,and compared with the a-tubulin gray scales,and calculating the ratio.8 Statistic analysis. All data were expressed by mean and standard error, Software SPSS 19.0 was used for the statistical analysis. Quantitative variables were compared by means of the student t test. Correlation analysis used Spearman rank correlation analysis. P<0.05 was accepted as statistically significant.ResultImmunohistochemical result shows that OPG and RANKL are positively expressed in ankylosing spondylitis synovial membrane group, and are mainly distributed in fibroblast-like synoviocytes and its IOD value is higher than that of normal synovial membrane group. The difference is of great significance (P<0.05). The detection index ossification AS group is considered different from normal, synovial group, the AS group is higher than the normal group2. Osteoclast specificity TRAP staining in AS group synovial membrane is significantly higher compared with the normal synovial membrane group.3. Through Real time-PCR detection, it shows that the OPG gene content is greatly expressed in ankylosing spondylitis synovial membrane. From relative quantitative analysis, it shows that the OPG protein level in AS synovial membrane group is significantly higher than that of normal synovial membrane group (P<0.05).4. From Western blot detection, it discovers that OPG protein is greatly expressed in ankylosing spondylitis synovial membrane group. After gray value analysis and semi-quantitative result, it shows that the OPG protein level in ankylosing spondylitis synovial membrane group is significantly higher than that of normal synovial membrane group (P<0.05).5. Through Western blot and Real time-PCR detection, it shows that there is no significant rising in RANKL. From relative quantitative analysis as well as gray value analysis and semi-quantitative result, it shows that there is no significant difference between the RANKL gene expressions level of AS synovial group and normal synovial group. It is not considered that the detection index will be different in Ankylosing spondylitis group and normal synovial membrane group.Conclusion1. The ossification process of ankylosing spondylitis peripheral joint can be closely related to osteoblast and osteoclast, among which osteoblast plays a leading role in ossification.2. The expression of ankylosing spondylitis peripheral ossification joint in synovial tissues takes osteogenic capability as a priority.3. The osteoclastic ability of AS peripheral ossification joint in synovial tissues may be inhibited.The third section Influence on WNT Signal Pathway Function of Human Fibroblast-like Synoviocytes after Transfecting SOST Gene and Relevance Research on the Expression of Osteogenic effects and Osteoclast effectsObjectiveThis article probes into the changes of human fibroblast-like synovial cell function after over expressing osteosclerosis protein as well as the influence of wnt signal transduction pathway, which SOST relies on, on fibroblast-like synovial cell. It also studies the functional changes of human fibroblast-like synovial cell after over expressing SOST as well as its expression relevance with osteogenic function and osteoclast function.Metheods1. Culture HFLS cell in DMEM growth medium which contains 15% of fetal calf serum, and subculture it in incubator in which the saturated hygrometer contains 5% CO2 under 37℃.2. Take SOST gene-carrying adenovirus to infect human fibroblast-like synoviocytes as overexpression group. In the meantime, set up empty medium group and empty group as control groups. Detect the gene expression condition after 72h with fluorescence microscope and confirm the transfection efficiency.5 days later, detect the changes of expression of SOST, β-catenin, OPG, RANKL gene and protein.3.Expression of SOST、β-catenin、OPG and RANKL genes in Synovium were detected by PCR technique.4.Expression of SOST、β-catenin、OPG and RANKL protein in Synovium were detected by Western-blot.5..Real time-PCR judgment of the results:Detection of expression of SOST、 β-catenin、OPG and RANKL genes in Synovium by using relative quantitative method.6. Western blot judgment of the results:Detection of expression of SOST、 β-catenin、OPG and RANKL protein in Synovium by using semiquantitative analysis,and compared with the a-tubulin gray scales,and calculating the ratio.7. Statistic analysis. All data were expressed by mean and standard error, Software SPSS 19.0 was used for the statistical analysis. Quantitative variables were compared by means of the student t test. Correlation analysis used Spearman rank correlation analysis. P<0.05 was accepted as statistically significant.ResultPCR result shows that compared with empty group and empty medium group, the gene expression of SOST, β-catenin, OPG in overexpression group is significantly different from empty group and empty medium group. The overexpression group is higher than the empty medium group and empty control group, and the difference is of significance (P<0.05). The RANKL gene expression of overexpression group is significantly different from that of empty group and control group, and is lower than empty medium group and empty control group. (p< 0.05)Western Blot result shows that compared with empty group and empty medium group, the gene expression of SOST, β-catenin, OPG in overexpression group is significantly different from empty group and empty medium group. The overexpression group is higher than the empty medium group and empty control group, and the difference is of significance (P<0.05). The RANKL gene expression of overexpression group is significantly different from that of empty group and control group, and is lower than empty medium group and empty control group. (p< 0.05)Conclusion1. SOST overexpression in synovial tissues may lead to activation of wnt/β-catenin signal.2. SOST overexpression in synovial tissues may enhance osteogenic function and osteoclast function.3. Fibroblast-like synoviocytes is probably the main cell of AS synovial membrane changed by ossification and ankylosing. |
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