OBJECTIVETo identify and characterize mesenchymal stem cells from synovial membrane of temporomandibular joint in vitro.

METHODSSynovial mesenchymal stem cells (SMSCs) were obtained by limited dilution method and expanded in 25 ml flasks. Methyl thiazolyl tetrazolium (MTT) method was used to determine the cell growth cycles. The expressions of vimentin and keratin were respectively detected with immunocytochemistry, while the expressions of CD8, CD34, CD44, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were determined by flow cytometry.

RESULTSPure mesenchymal stem cells were of spindle shape and uniform in size, which were intensively positive in vimentin, but negative in keratin. The expression of CD44, VCAM-1 and ICAM-1 were also verified by flow cytometry.

CONCLUSIONSMesenchymal stem cells could be purified from adult synovial membrane of temporomandibular joint.

Cell Differentiation ; Cell Separation ; methods ; Cells, Cultured ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Synovial Membrane ; cytology ; Temporomandibular Joint

OBJECTIVETo investigate the role of natural killer-22 (NK-22) cells in the synovial fluid in the proliferation of fibroblast-like synoviocytes (FLS) in patients with rheumatoid arthritis (RA) and explore the possible signal pathway involved.

METHODSNK-22 cells in the SF of RA patients were sorted by flow cytometry. NK-22 cells were cultured for two weeks and the purity was detected by flow cytometry before stimulation with 20 ng/ml phorbol 12-myristate 13-acetate and 0.5 µmol/L ionomycin for 4 h. The level of interleukin-22 (IL-22) in the culture medium supernatant was then measured with ELISA. The proliferation of FLS in the presence of the culture supernatant of NK-22 cells was assessed with MTT assay at 24, 48 and 72 h, and the effect of IL-22 antibody on FLS proliferation was also observed. Real-time PCR and Western blotting were used to detect Stat3 mRNA and p-Stat3 protein levels, respectively, in the FLS exposed to rhIL-22 and AG490.

RESULTSNK-22 cells were successfully sorted by flow cytometry with a purity exceeding 90%. The levels of IL-22 in the supernatant of NKp44(+)NK cell culture averaged 1273.42∓254.48 pg/ml. The FLS proliferated rapidly 24, 48, and 72 h after the addition of culture supernatant of NK-22 cells (P<0.05). IL-22 antibody obviously inhibited the proliferation of FLS induced by NK-22 cell culture supernatant (P<0.05). Exposure of the FLS to rhIL-22 obviously increased cellular Stat3 expression levels, which were significantly lowered by the addition of AG490 (P<0.05).

CONCLUSIONNK-22 cells in the SF of RA patients can produce high concentrations of IL-22 to promote the proliferation of FLS through the STAT3 signal pathway.

Arthritis, Rheumatoid ; metabolism ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; Humans ; Interleukins ; metabolism ; Killer Cells, Natural ; cytology ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Synovial Fluid ; cytology ; Synovial Membrane ; cytology

Animals ; Arthritis, Experimental ; metabolism ; pathology ; Breast Neoplasms ; pathology ; Cadherins ; metabolism ; physiology ; Cell Movement ; Female ; Fibroblasts ; cytology ; pathology ; Humans ; Macrophages ; cytology ; pathology ; Neoplasm Invasiveness ; Synovial Membrane ; cytology ; metabolism ; pathology

The progress of research on the the anterior cruciate ligament (ACL) wound healing demonstrates that the synovial tissue in the knee joint plays a very important role in the healing process of injured ACL. Therefore, the molecular response mechanisms of lysyl oxidase (LOX) and matrix metalloproteina (MMP) in normal/injured ACL fibroblast cells could be considered to perform the major analysis function of injured ACL healing mechanism. The mRNA expressions of LOXs and MMPs and the activity expressions of MMP-2 in ACL fibroblasts co-cultured with synovial cells were analyzed by quantitative real-time PCR and zymography. The results showed that co-culture could regulate the mRNA expressions of LOXs and MMPs in the ACL fibroblasts cells. These results suggest that the differential expressions of LOXs and MMP-1, 2, 3 in co-cultured ACL indicate that interaction crosstalk do exist between ACL cells and synovial cells and provide a theoretical basis for subsequent exploration of the mechanisms and treatment of ACL injury and repair.
Anterior Cruciate Ligament ; cytology ; Anterior Cruciate Ligament Injuries ; Coculture Techniques ; Fibroblasts ; cytology ; metabolism ; Humans ; Knee Injuries ; physiopathology ; Knee Joint ; cytology ; Matrix Metalloproteinases ; genetics ; metabolism ; Protein-Lysine 6-Oxidase ; genetics ; metabolism ; Synovial Membrane ; cytology ; Wound Healing ; physiology

OBJECTIVETo investigate the effects of sinomenine (SIN) and methotrexate (MTX) on the proliferation and apoptosis of in vitro cultured fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients, as well as the expression of osteoclast differentiation factor in FLS.

METHODSFLS were isolated from the synovium of RA patients and cultured in vitro. FLS were incubated with different concentrations of SIN and MTX respectively or combined: 0.001, 0.010, 0.100, 1.000 mg/mL SIN; 0.001, 0.010, 0.100, 1.000 mg/mL MTX; 0.001 mg/mL SIN + 0.001 mg/mL MTX, 0.010 mg/mL SIN + 0.010 mg/mL MTX, 0.100 mg/mL SIN + 0.100 mg/mL MTX, 1.000 mg/mL SIN + 1.000 mg/mL MTX, namely SIN1, 2, 3, 4 groups; MTX1, 2, 3, 4 groups and the combination 1, 2, 3, 4 groups. The medium without drugs was used as a control group. There was a total of 13 groups, each group with 3 complex holes. MTT was applied to detect the growth of FLS. The flow cytometry was applied to detect the apoptosis of FLS. The expressions of FLS receptor activator of nuclear factor kappa B ligand (RANKL) mRNA and osteoprotegerin (OPG) mRNA were observed by semi-quantitative RT-PCR.

RESULTSCompared with the control group, RA FLS proliferation OD values of all the drug groups were lower (P < 0.05). The RA FLS apoptosis OD value of the combination 3 group increased, the OPG mRNA expression increased, the expression of RANKL mRNA decreased with statistical difference (P < 0.05). The RA proliferation OD values of the SIN3 group and the MTX3 group increased when compared with the combination 3 group (P < 0.05).

CONCLUSIONSSIN and MTX had synergistic effects in inhibiting FLS. This might be one of the mechanisms for inhibiting RA bone damage.

Apoptosis ; drug effects ; Arthritis, Rheumatoid ; pathology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; Humans ; Methotrexate ; pharmacology ; Morphinans ; pharmacology ; Synovial Membrane ; cytology ; drug effects

OBJECTIVETo investigate the expression of anaphylatoxin receptor C5aR (CD88) in synoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).

METHODSThe expression of C5aR was assessed in synoviocytes isolated from 27 RA and 12 OA patients using reverse transcription-polymerase chain reactions (RT-PCR), flow cytometry, and immunofluorescence analysis. The effects of C5a on the release of tumor necrosis factor alpha (TNF alpha) from synoviocytes were assayed using enzyme-linked immunosorbent assays (ELISA).

RESULTSC5aR mRNA was detected in 24 of 27 samples from RA patients, and 10 of 12 samples from OA patients. Flow cytometric analysis and immunofluorescence study demonstrated the cell surface expression of C5aR in a significant proportion of synoviocytes from both RA and OA patients, and the level of C5aR expression in synoviocytes was significantly correlated with joint swelling, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in RA patients. Finally, interaction of C5aR with its ligand C5a was shown to enhance lipopolysaccharide (LPS)-induced TNF alpha release from synoviocytes.

CONCLUSIONSThe expression of C5aR in synoviocytes from inflammatory joint diseases and also the induction of TNF alpha release in activated synoviocytes by the interaction of C5a and C5aR suggest that the C5a/C5aR system may play an important role in joint inflammation process.

Adult ; Aged ; Arthritis, Rheumatoid ; metabolism ; Cells, Cultured ; Female ; Humans ; Male ; Middle Aged ; Osteoarthritis ; metabolism ; Receptor, Anaphylatoxin C5a ; analysis ; Synovial Membrane ; chemistry ; cytology

Objective To explore the role of multidrug resistance gene-1(MDR1)gene in methotrexate(MTX)resistance in patients with rheumatoid arthritis(RA).Methods Fibroblast-like synoviocytes(FLS)from RA patients were infected with recombinant adenovirus Ad-EGFP-MDR1 to obtain MDR1 over-expressed RA FLS.The transcription level of MDR1 gene and the expression level of its coding product P-glycoprotein(P-gp) rotein were detected by real-time PCR and Western blot analysis.The efflux function was verified by rhodamine 123 efflux assay.The resistance to MTX was detected by MTT assay.Results RA FLS were infected with recombinant adenovirus Ad-EGFP-MDR1;72 hours later,the particles size in MDR1 over-expressed RA FLS increased,the cell volume became larger,and the growth rate decreased.The transcription level of MDR1(1.4325±0.3924 0.0650±0.0070;=6.035,=0.004),the expression level of P-gp protein(1.8667±0.2857 0.9367±0.0551;=5.536,=0.005),and the ability of extracellular rhodamine 123(979.43±196.81 1680.06±147.04;=-4.940,=0.008) in MDR1 over-expressed RA FLS were significantly higher than those of negative virus control RA-FLS,and the survival rate of MDR1 over-expressed RA FLS was significantly increased at each concentration of MTX(<0.05).Conclusion The high expression of MDR1 can affect the efflux ability to MTX by up-regulating the expression of P-gp,thus enhancing the drug resistance to MTX in RA FLS.
ATP Binding Cassette Transporter, Subfamily B ; genetics ; Arthritis, Rheumatoid ; drug therapy ; genetics ; Cells, Cultured ; Drug Resistance ; Fibroblasts ; drug effects ; Humans ; Methotrexate ; pharmacology ; Synovial Membrane ; cytology

BACKGROUNDSynovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system.

METHODSThe clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II, aggrecan, SOX9, link-protein, collagen type X and BMP receptor II.

RESULTSCells isolated under the optimized culturing density (10(4)/60 cm(2)) showed clonogenicity and multi-differentiation potential. These cells were positive (> 99%) for CD44, CD90, CD105 and negative (< 10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type II was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type II and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone.

CONCLUSIONSSDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

Aged ; Bone Morphogenetic Protein 2 ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrogenesis ; drug effects ; Female ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Middle Aged ; Synovial Membrane ; cytology ; Transforming Growth Factor beta3 ; pharmacology

OBJECTIVETo investigate the effects of Danshen Injection (DSI) on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs) cultured in RA patients' serum.

METHODSThe RA FLSs harvested from RA patients' synovial fluid were primarily cultured by routines. The cells were cultured with 10% inactivated human serum (the healthy human serum and the RA patients' serum) for 24 h. Then DSI at the final concentration of 0. 4 mg/mL was added in the cells for further 24 h culture. By taking 10% fetal calf serum as the control, the morphological changes were observed under optical microscope. The proliferation was analyzed by MTT. The apoptosis was detected by flow cytometry. The total RNA was extracted and reverse transcription was performed. The Bax mRNA expression was detected by fluorescent quantitative PCR.

RESULTS(1) After human serum was added in the healthy human serum and RA patients' serum, cells could grow adhering to the wall. Compared with the fetal calf serum group (FCS), the cell density was higher in the healthy human serum group than in the fetal calf serum group, with no obvious morphological changes. (2) MTT results showed that, compared with the fetal calf serum group, the absorbance value (OD) obviously increased in the healthy human serum group and the RA patients' serum group, showing statistical difference (P <0.01). After adding DSI at the final concentration of 0.4 mg/mL, cells from different serums were inhibited to various degrees (with OD significantly decreased, P <0.05). The OD value significantly increased more in the healthy human serum group and the RA patients' serum group than in the fetal calf serum group, showing statistical difference (P <0.01). There was statistical difference between the healthy human serum group and the RA patients' serum group (P <0.01). (3) The apoptosis rate in the RA patients' serum group obviously decreased with statistical difference, when compared with the Salvia miltiorrhiza free fetal calf serum group (P >0. 01). The apoptosis rate in the fetal calf serum group and the RA patients' serum group significantly increased after adding 0.4 mg/mL Salvia miltiorrhiza, showing statistical difference when compared with the Salvia miltiorrhiza free fetal calf serum group and the Salvia miltiorrhiza free RA patients' serum group (P <0.05). The FLSs were effected by 0.4 mg/mL Salvia miltiorrhiza, the apoptosis rate significantly decreased in the healthy human serum group and the RA patients' serum group, showing statistical difference when compared with the fetal calf serum group (P <0. 05, P <0.01). (4) The expression of Bax gene significantly increased in the RA patients' serum group and the fetal calf serum group after action of 0.4 mg/mL Salvia miltiorrhiza, showing statistical difference (P <0. 01). When 0.4 mg/mL Salvia miltiorrhiza was added, the expression of Bax mRNA obviously increased in the healthy human serum group and the RA patients' serum group, showing statistical difference when compared with the fetal calf serum group (P <0.01).

CONCLUSIONS(1) Although healthy human serum can be favorable to the growth of RA FLSs, the fetal calf serum could reflect the actual results better in the cyto biological research on specific diseases (if there is no serum from patients with corresponding disease). (2) DSI could inhibit the proliferation of RA FLSs through promoting their apoptosis.

Apoptosis ; drug effects ; Arthritis, Rheumatoid ; pathology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; cytology ; drug effects ; Humans ; Phenanthrolines ; pharmacology ; Salvia miltiorrhiza ; Synovial Membrane ; cytology ; drug effects

OBJECTIVETo study the effect and related mechanism of seaweed polysaccharide (SP) on apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis (RA-FLS).

METHODSRA-FLS were in vitro cultured using modified tissue culture method. Effect of SP (0, 15, 20, and 25 mg/mL, respectively) at different time points (0, 3, 4, and 5 days, respectively) on the proliferation and apoptosis of RA-FLS, and protein expressions of Caspase-3, Bax, and Bcl-2 was detected by cell counting kit-8 (CCK-8) assay, Hoechst 33258 staining assay, TUNEL assay, and Western blot, respectively.

RESULTSCompared with 0 mg/mL SP at the same time point, the proliferation of RA-FLS was inhibited, and the apoptosis was promoted 3, 4, and 5 days after intervened by 15, 20, and 25 mg/mL SP, respectively (P<0.01) in time- and dose-dependent manners. RA-FLS Bax protein expression was up-regulated, Bcl-2 protein expression down-regulated, Caspase-3 activated and split by 15, 20, and 25 mg/mL SP, respectively for 4 days (P<0.05, P<0.01). Besides, the changes were in a dose-dependent manner.

CONCLUSIONSSP could inhibit RA-FLS proliferation and induce its apoptosis in dose- and time-dependent manners. Its apoptosis mechanism might be through up-regulating intracellular Bax protein expression and down-regulating Bcl-2 protein expression, thus influencing the mitochondrion signaling pathway, further promoting Caspase-3 activation and split, resulting in the apoptosis of RA-FLS.

Apoptosis ; drug effects ; Arthritis, Rheumatoid ; pathology ; Caspase 3 ; metabolism ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Humans ; Polysaccharides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Seaweed ; Synovial Membrane ; cytology ; drug effects ; bcl-2-Associated X Protein ; metabolism