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Objective To observe the inhibiting activities of fibronectin (FN) and citrullinated fibronectin (cFN) on disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4),and to explore the extracellular regulative mechanisms of ADAMTS-4.Methods FN was incubated with peptidylarginine deaminase type 4 (PADI4).Western blotting analysis was used to verify the citrullination of FN.The binding activity of FN and cFN to ADAMTS-4 were investigated by enzyme-linked immunosorbent assay (ELISA).The proteolytic ability of ADAMTS-4 after binding to FN and cFN were measured with the aggrecanase activity assay kit.One-way ANOVA,LSD-t test and t-test were used for statistical analysis.Results The immunosignal of citrulline was detected in FN after incubated with PADI4,but not in the absence of PADI4.A higher absorbance at 405 nm was detected when the full-length ADAMTS-4 protein was incubated with FN (2.182±0.042) than cFN (0.624±0.033; t=50.522,P＜0.01).Additionally,the recombinant ADAMTS-4 protein with a truncation at the C-terminus displayed low absorbance at 405 nm when the enzyme was incubated with both FN(0.971±0.024) and cFN(0.934±0.012; t=2.388,P＞0.05).Large amounts of ARGxx peptide were detected with full-length ADAMTS-4 in aggrecanase activity assay [(0.908±0.088) nmol/L],but significantly less when in the presence of FN and ADAMTS-4 [(0.573±0.000) nmol/L,P＜0.05].The production of this peptide was more when full-length ADAMTS-4 was incubated with cFN [(0.830±0.020) nmol/L,P＜0.05] than with FN.The reaction containing the truncated ADAMTS-4 without FN or cFN yielded the highest concentration of ARGxx peptide [(36.420±3.673) nmol/L],peptide production was not significantly altered when FN [(41.099±0.101) nmol/L] or eFN [(41.064±0.083) nmol/L] were added to the reaction.Conclusion FN could bind to ADAMTS-4 and inhibit its proteolytic activity.After citrullinated by PADI4,the binding activity of cFN is weakened and less inhibition to ADAMTS-4.
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Objective The present study investigated the expression of heat shock cognate protein 70 (Hsc70) in the synovial tissues and blood samples of patients with rheumatoid arthritis (RA) to determine the pathological role of this protein in the pathogenesis of the disease.Methods The expression of Hsc70 in synovial membranes was quantitatively analyzed by immunohistochemistry,real-time quantitative PCR and western blotting.The samples from osteoarthritis (OA) and ankylosing spondylitis (AS) were used as controls.The levels of Hsc70 in blood of patients with RA were determined using enzyme linked immunosorbent assay (ELISA) with the samples of the healthy subjects as controls.Statistical analysis was conducted with one-way ANOVA,LSD test and Spearmen's correlation.Results Immunohistochemistry showed that Hsc70 had significantly increased expression in synovial tissues of RA than in the samples of OA and AS.Real-time PCR and western blotting confirmed the above findings.ELISA detected significantly elevated level of Hsc70 in blood of patients with RA as compared with samples from the controls (P＜0.01).Conclusion The study suggests that the up-regulation of Hsc70 may be involved in the pathogenic process of RA.
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Objective To investigate the changes of bone tunnel and differences of clinical outcomes after anterior cruciate ligament (ACL) reconstruction with hamstring tendons from autografts and allografts. Methods The study involved in 61 patients with ACL injury undergone arthroscopic ACL reconstruction from June 2008 to November 2009.According to ACL graft differences,61 patients were assigned to two groups,ie,Group A ( reconstruction with tendon allografts,n =27 ) and Group B ( reconstruction with tendon autografts,n =34).MRI examination was performed one week and 6-12 months post-operatively to measure the sagittal bone tunnel diameters at the aperture location,at the location of 1cm away from the aperture and at the widest location of tunnel.Taking the bone tunnel diameter at each location one week post-operatively as the measurement criteria,the differences of bone tunnel diameters at each corresponding location were analyzed statistically.Lysholm score was used to evaluate the clinical effects in the two groups. Results Group A and B were followed up for average 8.4 months and 8.5 months,respectively.Pre-operative clinical data of the two groups had no significant difference.MRI follow-up showed that the tunnel diameters at femoral side and tibial side both had different degree of enlargement.Meanwhile,the enlargement degree at the tibial side was larger than that at the femoral side,with no statistical differences of the two groups regarding tunnel enlargement of the three measurement locations at femoral and tibial sides.Joint stability in all patients was good.The two groups showed no significant difference in Lysholm score after operation ( P ＞ 0.05 ). Conclusions Graft difference is a factor for bone tunnel enlargement after ACL surgery.Autologous and allogenic tendons show no difference in their role in bone tunnel enlargement after ACL surgery or correlation with clinical effects.
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ObjectiveTo investigate the expression of ATA1 in the synovial tissue from patients with ankylosing spondylitis (AS) and to localize expression of ATA1 in AS synovial membranes.In addition,tag SNPs were genotyped to determine the possible association of this gene with AS risk.MethodsWestern blotting analysis was applied to determine the expression of ATA1 in the synovial tissues by comparing the expression profiles of AS(n=8),rheumatoid arthritis(RA,n=9) and osteoarthritis(OA,n=9) samples.Immuno-histochemistry was used to localize the expression of ATA1 in the synovial membrane.The levels of ATA1 in the synovial fluid of patients with AS were determined using ELISA with OA and RA as controls.Taqman method was used to genotype tag SNPs (rs2753934,rs2749531 and rs6575424) in 56 AS cases,260RA cases and 160 healthy controls.ANOVA,LSD test andx2 test were used for statistical analysis.Results Increased expression of ATA1 in synovial membranes of AS was found when compared with samples from RA and OA.ELISA results showed significantly elevated level of ATA1 in the synovial fluid of patients with AS (1.6+0.6),but not in samples of RA(1.4±0.5) and OA (1.2±0.5)(P＜0.05).Haplotype analysis did not reveal a haplotype association in AS or RA(P＞0.05).ConclusionThe current findings suggest that upregulation of ATA1 may play an important role in the pathogenesis of AS.
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Objective To investigate the expression of thiredoxin domain containing 5 (TXNDC5) in the synovial tissues and blood samples of various arthritic conditions and autoimmune diseases to further confirm the previous findings, investigate the relations between the expression level of TXNDC5 and clinical parameters of RA. Methods The expression of TXNDC5 in the synovium was quantitatively analyzed by immunohistochemistry, real-time quantitative PCR and Western blotting. The levels of TXNDC5 in blood and synovial fluid was determined using sandwich ELISA in patients with RA, osteoarthritis (OA), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS) and normal controls. One-way ANOVA, LSD test and Spearmen' s correlation were used for statistical analysis. Results Immunohistochemistry indicated that TXNDC5 expression was significantly higher in the synovial tissues of RA (100%, 40±9) than in those of OA and AS(200%,4±4). Real time PCR and western blotting confirmed the above findings (P<0.01). Sandwich-ELISA indicated significantly elevated level of TXNDC5 in the blood and synovial fluid of patients with RA (A=1.31±0.37), but not in those of OA, SLE, and AS, the healthy controls (P<0.01). The level of TXNDC5 in the blood of RA patients (A=0.8185±0.299) was positively correlated with the level of anti-CCP (r=0.350, P =0.027). Conclusion The results suggest that the pronounced increase of TXNDC5 expression may stimulate synovial pannus formation in the hypoxic environment of RA.
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[Objective]To observe the clinical outcome of rotator cuff-reserved calcium deposit removal on extensive shoulder calcific tendinitis.To discuss if it is necessary to sacrifce the cuff integrity for a completely removal of calcium deposit.[Method]From Oct.2004 to Apr.2006,15 cases(15 shoulders)of extansive shoulder rotator cuff calcific tendinitis were treated with calcium removal under arthroseopy.Among them there were 6 males and 9 females with an average age of 44.7.The average pre-operative evaluation grade with Constant-Murley were 56(49~62).During the operation,the rotator cuff integrity were reserved in 8 cases and most of the deposits were removed with some calcium left in the reserved cuff layer.In the other 7 cases,complete deposit clearance were performed and the rotator cuff was repaired subsequently,average follow-up were 16.7 months(3~25).The Constant-Murley score and X-ray were taken after operation to evaluate shoulder function and the changes of remnant calcium were observed.[Result]13 cases reported significant pain dissolved and 2 reported pain relieved.The average postoperative Constant-Murley score of cuff-reserved group was 91(81~95),while the cuff-repaired group was 90.3(80~94).On postoperative radiograph,only 3 cases demonstrated remnant deposit,and disappeared within 3 months after operation.[Conclusion]Both of the two kinds of operations showed good clinical outcome.The rotator cuff-reserved calcium deposit removal has simplified operation techniques,less costs and fewer complications.It is unnecessary to sacrifice cuffintegrity for complete calcium removal.The remnant deposit could be absorbed when it is opened after operation.
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Objective To evaluate the effect of ?-lipoic acid(ALA)as a antioxidant on the histomorphologic changes of the kidney in type 2 diabetic Goto Kakisaki rats.Methods Four healthy Wistar rats(normal control group)and 10 GK rats were divided randomly into two groups:diabetic control group and lipoic acid-treatment diabetic group(?-lipoic acid 35 mg/kg intraperitoneal injection qd alt),who were both fed for 12 weeks.Indexes of oxidative stress of kidney were measured.Sections of renal tissue were evaluated using both a light and electron microscope to assess histomorphologic changes.Paraffin sections of the left kidneys were prepared and stained with H&E,PAS and Masson stains to compare the histomorphologic changes.The image analysis software of Motic Image Advanced 3.1 was used to measure the area of glomerulus and mesangial region,and the thickness of the glomerular basement membrane(GBM).Results Compared with normal control group,there were significant decreases in GSH,SOD,VitC and VitE levels in diabetic control group.The kidneys of the normal control group exhibited normal cortical morphology as determined by light microscope.Mild glomerulosclerosis,characterized by widening of the mesangial region,mesangial expansion and intercapillary cells proliferation,and GBM thickening under electron microscope were observed in the diabetic control group.In the ALA-treatment group,all the above pathological changes were alleviated.Conclusion In the kidney of type 2 diabetic rat,there exists oxidative stress which is associated with diabetic nephropathy.?-Lipoic acid can alleviate the abnormal histomorphologic changes of the kidney via antioxidative effects.