Objective To investigate the effects of 5-Aza-CdR( methylation transferase inhibitor) on the expression levels of leptin gene in chondrocytes and methylation states of leptin promoter region between osteoarthritis (OA) group and control. Methods The chondrocytes in osteoarthritis group were treated with 5-Aza-CdR with different doses and time-points, and the expression level of leptin was detected by real-time polymerase chain reaction for picking up the optimum dose and time-point. Next, the chondrocytes in 5 osteoarthritis patients and 5 control patients (amputation due to severe trauma) were treated with 5-Aza-CdR. Lastly, leptin mRNA expression levels in the four groups osteoarthritis and control chondrocytes treated with/without 5-Aza-CdR were measured by real-time PCR and the methylation state of promoter region ( - 280- + 79) was detected by epityper quantitative DNA methylation analysis. Results ( 1 ) After treating the chondrocytes in OA groups with 10 μmol/L 5-Aza-CdR for 72 h, the mRNA expression levels of leptin were increased significantly. ( 2 ) The mRNA expression levels of leptin were significantly different among the four groups ( P ＜ 0. 05 ), and the chondrocytes in osteoarthritis groups treated with 5-Aza-CdR showed a marked induction of leptin mRNA expression. (3) Analysis of quantitative methylation data using an unsupervised hierarchical clustering algorithm, showed that methylation patterns of leptin promoter was different between control and osteoarthritis chondrocyte treated with/without 5-Aza-CdR. Conclusion Demethylation of leptin promoter might up-regulate leptin gene expression level and it might contribute to osteoarthritis.

Objective To investigate the inhibitory effect of methylprednisolone (MP) on the development of coronary arteritis in a murine model of Kawasaki disease (KD) induced with a candida albicaus watersoluble fraction (CAWS).Methods Forty-five C57BIL/6mice were evenly divided into three groups (the control group,the CAWS group and the MP group).Mice in the CAWS group were intraperitoneally injected phosphate buffer saline (PBS) for 5 days.MP or PBS was administered to the different group.The animals were sacrificed at day 3,day 10 and day 28,and the status of vasculitis in the coronary arteries and the aortic root was investigated histologically.One-way analysis of variance (ANOVA) was used to compare the differences among three groups,and t-test for two independent groups.Results The mice in CAWS group and MP group,which induced by CAWS,showed that the body weight and heart weight decreased significantly,and the spleen weight was increased at day 10 and day 28 (P＜0.05).Vasculitis was induced in the mice of those two groups,and the severity score was the highest at day 10 (12.7±0.5).In addition,the severity of the inflammation of the aortic root and the coronary arteries were reduced in MP group (t=6.35,5.55,2.8,P＜0.05).Elastic fiber staining showed that the layers of vascular walls were in disorder and elastic fibers were broken in the CAWS group.However,there was no disruption or breakage in the MP group.Conclusion MP can suppress the progression of coronary arteritis in this CAWS-induced murine vasculitis model,which indicates the efficacy of MP in KD patients with coronary artery lesions.

Objective To investigate the effects of 5-Aza-CdR (methylation transferase inhibitor) on gene expression levels of interleukin-1β (IL-1β),transforming growth factor-β (TGF-β) and nitric oxide synthase (NOS) in chondrocytes.Methods Chondrocytes from patients with osteoarthritis (OA) (n=22),rheumatoid arthritis (RA) (n=3) or trauma without rheumatic diseases (n=10) were collected and cultured.The mRNA expression levels of IL-1β,TGF-β and NOS were detected by real-time polymerase chain reaction (RT-PCR).Chondrocytes in trauma group were treated with 5-Aza-CdR(10μmol/L) for 72 h,and the mRNA and protein expression levels of IL-1β,TGF-β and NOS were detected by RT-PCR and ELISA,respectively.Results The mRNA expression levels of IL-1β,TGF-β and NOS were increased in OA and RA group as compared with trauma group (P＜0.05),while they had no differences between OA and RA groups.After treated with 5-Aza-CdR,the mRNA and protein expression levels of IL-1β,TGF-β and NOS in chondrocytes rised in trauma group as compared with pretreatment (all P＜0.05).Conclusions The mRNA expression levels of IL-1β,TGF-β and NOS in chondroytes are higher in OA and RA patients.5-Aza-CdR could increase the mRNA and protein expression levels of IL-1β,TGF-β and NOS by inducing relative gene methylation,which suggests demethylation might play a role in OA pathogenesis by influncing the inflammatory signal pathway or cell apoptosis.

Objective To compare the cytokine expression profile of 3 CD8+, 3 CD4+ and 3 gamma delta-positive T cell clones derived from the synovial fluids of reactive arthritis (ReA) patients and to explore the immunological pathogenesis of ReA. Methods Complementary DNA-based microarrays containing genes for 56 cytokines were used for screening the expression profile of 3 CD8+, 3 CD4+ and 3 gamma delta-positive T cell clones derived from the synovial fluids of 3 reactive arthritis (ReA) patients. Results Transcripts encoding for interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha were expressed among all CD8+ and CD4+ T cell clones by microarray. Expression of these cytokines could be verified by RT-PCR in 14 out of 15 microarray experiments. Lymphotoxin (LT)-alpha and granulocyte macrophage colony-stimulating factor (GM-CSF) were also consistently expressed among CD4+ cells. However, ??+T cells revealed a different cytokine pattern, mainly expressing transforming growth factor (TGF)-beta 2 and GM-CSF. Conclusion CD8+ and CD4+ T cells mainly reveale a Th1-mediated profile, whereas ??+T cells expresse less pro-inflammatory cytokines resembling a Th3-driven pattern. T lymphocyte clones from the joint of ReA patients exhibit different cytokine expression profiles refelecting their different roles in pathogenesis.

Objective To explore the effect of different concentrations of iguratimod (IGU) on mouse model of bleomycin-induced pulmonary fibrosis.Methods A total of 108 female C57BL/6 mice were randomly divided into the control group,the model group,the low dose IGU group,the moderate dose IGU group,high dose with group and the methylprednisolone (MP) group (n=18 in each group).The mice in the control group were injected with 0.2 ml normal saline endotracheally,and others were injected with 0.2 ml bleomycin (5 mg/kg) from endotracheally to induce pulmonary fibrosis model.The next day,the mice in both control group and the model group were fed with 0.2 ml normal saline every day;The mice in the IGU groups and methylprednisolone group were fed with 0.2 ml iguratimod liquid the IGU (10,30,90 mg/kg) and 0.2 ml methylprednisolone (10 mg/kg) every dayrespectively.Finally the mice were sacrificed at day 7,day 14,day 28 respectively,and the lung tissue was examined by HE staining and Masson staining to evaluate the degree of alveolitis and fibrosis.Repeated measurement of variance analysis was used to compare the differences for time and group,and multi-factor analysis of variance LSD test was used for the comparison between groups.Results ① The body mass of mice in bleomycin-induced groups were decreased compared to the control group.② The pathological alveolitis scores in the high dose IGU group and methylprednisolone group were significantly decreased compared to those of the model IGU group at day 7 and day 14 (P＜0.05),and the pathological fibrosis scores were decreased dramatically compared with the model group at day 14 and day 28 (P＜0.05).Conclusion High concentration of IGU and methylprednisolone can reduce and inhibit lung inflammation and fibrosis of bleomycin-induced pulmonary fibrosis in mice.

Objective To search for the genetic and molecular immunity basis of CXCR-1 associated pathogenesis in ankylosing spondylitis (AS) patients. Methods Sequencing analysis was used to detect mutation in the exonic, junctional and promoter sequences of CXCR-1 which might be related with ankylosing spondylitis; the hydrophobicity, conservation and evolutionary distance of the mutated amino acids were also analyzed. Results Six affected individuals in the family were detected with a novel mutation Arg192Gly. The glycine at 192 codon was highly conserved in different species. Arginine and glycine had quite distinct hydrophobicity and BLOSUM score. Conclusion The mutation CXCR-1 (Arg192Gly) detected in these patients might be involved in genetic and molecular immunity mechnisms of ankylosing spondylitis.

Objective To investigate an effective endoscopic management of biliary anastomotic stric-tures (AS) following orthotopic liver transplantation (OLT) and to evaluate the factors which may effect the ontcome. Methods Sixty-five patients, who were diagnosed as AS 3 months after OLT, underwent ERCP. Af-ter adequate dilation of the narrowing bile ducts, plastic stents, as many as possible, were inserted across the strictures and kept in place for at least six months. Results A total of 90 consecutive endoscopic procedures were performed in 65 patients. Before stents placement, the strictures were dilated by catheter or balloon (di-ameter range: 6-10 mm), or not dilated, according to the status of the bile ducts. An average of 3 (ranging from 2 to 6) plastic stents were placed with mean total size of 22.8 F (range 14-42 F), and the stents were kept for 8. 0 months on average (range 0.2-37.8 months). Of 90 procedures of stents placement, 54 (60%) were followed by stents removal and cholangiography, which confirmed stricture resolution in 26 (48.1%). The stricture resolution rate was 81.0% (17/21) in patients who underwent balloon dilation followed by more than 3 stents (> 21 F) for at least 3 months. Stricture re-occurred in 3 patients after stents removal, in whom stents were kept less than six months. Conclusion Endoscopic sequential intervention is effective for post-OLT biliary strictures according to the stage and grade. Radical dilation with maximal stenting can lead to complete resolution of AS. To achieve better result, if possible, balloon dilatation followed by three or mere endoprothe-ses (of at least 21 F) sustaining for more than 6 months is necessary.

Objective To investigate the elasticity of the radial artery wall in type 2 diabetes mellitus (T2DM) patients with elastosonography. Methods A total of 37 patients with T2DM and 42 normal subjects were studied with elastosonography. The systolic diameter (Ds) and diastolic diameter (Dd) of the radial artery were measured, and the strain ratio of the blood in the radial artery to the wall of the radial artery was calculated. Results The strain ratio of the radial artery in T2DM group was significantly higher than that in normal group (P＜0.05). There was no significant difference in Ds and Dd of the radial artery between T2DM group and the control group (P＞0.05). Conclusion The early change of the radial artery wall elasticity in patients with T2DM can be assessed with elastosonography.

Objective To evaluate left atrial(LA) function in patients with prophase type 2 diabetes mellitus(T2DM) combinated with or without hypertension using left atrial volume tracking method(LAVT). Methods Thirty-one simple T2DM(T2DMI group) ,21 T2DM accompany with hypertension(T2DM2 group) and forty-five healthy subjects (control group) were enrolled in this study. Ultrasound LAVT(EUB-6500, Hitachi Medical Corporation) was applied to display and analyzed the LA volume loop imaging on the standard LV apical two and four chamber views. The maximal and the minimal LA volume (LAVmax, LAVmin) and the volume before LA contraction (LAVp) were recorded from the LA volume loop. The body surface area was used to correct these volume indexs. The LA reservoir function was assessed by calculating the total of LA filling volume (LAVItotal) and the expansion index(iLAVIe). The passive and active emptying percentage of the total emptying volume(LAVIpass, LAVIact) and the emptying index(iLAVIpass,iLAVIact) were caculated as the parameters of the LA conduit and booster pump function. Results Compared with the values in the control group, the LAVhotal,LAVIact were significantly higher and the LAVlpass,iLAVlpass were lower in the T2DM group (all P<0.05) ,while the iLAVIact was higher only seen in the T2DM2 group(P<0. 05). The LAVlact, iLAVIact were higher and the LAVIpass was lower in T2DM2 group than those in the T2DM1 group (all P<0.05). Conclusions The LA conduit hypofunction in primary in the prophase T2DM,when combinated with hypertension the LA constriction function compensatorily increased, LAVT can evaluate the function of LA in patients with T2DM accurately and rapidly.

Objective To study the detection of cytomegalovirus (CMV)-DNA in different kinds of blood and urine sample .Methods The CMV-DNA loads in 3 different kinds of blood sample from 52 patients and 3 different kinds of urine sample from 85 patients were detected by real-time fluorescence quantitative polymer-ase chain reaction(FQ-PCR) .The differences in CMV-DNA detection rate and virus loads were compared a-mong different kinds of blood and urine samples .Results The CMV-DNA detection rates in serum ,whole blood ,plasma and peripheral blood mononuclear cell (PBMC) from 52 patients were 48 .08％ ,71 .15％ ,57 .69％ and 69 .23％ respectively .The CMV-DNA detection rates of whole blood and PBMC were higher ,the difference was statistically significant (P<0 .05) .The quantitative results of PBMC was higher .The CMV-DNA detec-tion rates of mixed urine ,urine supernatant and urine sediment from 85 patients were 72 .94％ ,62 .35％ and 84 .71％ respectively ,the CMV-DNA detection rate of urine sediment was higher ,while the quantitative re-sults of mixed urine was higher .Conclusion The CMV-DNA detection results of blood and urine have large difference ,therefore using the same kind of sample for conducting detection has an important clinical signifi-cance in the clinical diagnosis ,treatment and monitoring of CM V infection .