Objective To investigate the effect of Hyaluronic acid(HA) on the expression of matrix metalloproteinases(MMPs)-3,9,13 and tissue inhibitor of MMPs(TIMP)-1 mRNA stimulated by interleukin (IL)-1β in chondrocytes from arthritis model in vitro.Methods Different levels of Sodium Hyaluronate(10,20,40μg/ml)were added to culture medium of rat chondrocytes.The ehondrocytes were isolated from rat osteoarthritis model.One hour later,10 ng/ml IL-1β were added to each sample and co-cultured for 24 hours.The expressions of MMP-3,9,13 and TIMP-1 mRNA were detected bv RT-PCR.The concentration of nitric oxide (NO) in the supernatants were measured by Griess reaction.Results HA could down-regulate MMP-3,9,13 mRNA expression but had no effect on TIMP-1 mRNA expression.Moreover,it also had a dose-dependent down regulate effect on nitric oxide synthesis.Conclusion This study demonstrates that HA can down-regulate the TIMP-1/MMPs expression by inhibiting NO production.

BACKGROUND:Pathogenesis of osteoarthritis is poorly understood,however,studies have demonstrated that vascular endothelial growth factor(VEGF)Is involved in the progression of osteoarthritis.OBJECTIVE:To detect the changes of VEGF mRNA expression of synovium in rabbit osteoarthritis and to evaluate the effect of sodium hyaluronate on its expression.METHODS:Twenty four white rabbits were divided into the normal control,physiologic saline,and sodium hyaluronate groups.The unilateral anterior cruciate ligament transection(ACLT)was performed in the physiologic saline and sodium hyaluronate groups.At weeks 4 after operation,rabbits in the physiologic saline group were injected 0.3 mL physiologic saline and in the sodium hyaluronate group received 10 g/L sodium hyaluronate injection,once per week for 5 successive weeks.All the animals were sacrificed at week 10 after operation.The cartilage changes on the medial femoral condyles were graded separately VEGF express Jon of synovium was detected by using teal time polymerase chain reaction(RT-PCR).RESULTS AND CONCLUSION:The macroscopic score showed that the cartilage degeneration in the physiologic sailne group was significantly more severe than that of the normal control and sodium hyaluronate groups(P＜0 05)The expression of VEGF mRNA was obviously decreased in the physiologic saline group than that of the normal control group(P＜0 05).of which was increased in thesodium hyaluronate group,but still smaller than the normal control group(P＜0 05).The results demonstrated that the decreased VEGF expression in synovium may involved in the progression of osteoarthritis,and sodium hyaluronate has protective effect on articular cartilage by up-regulating the VEGF expression.

Objective To observe the effect of treating acute spinal cord injury in rats by transplantation of autologous activated Schwann cells(AASCs). Methods Unilateral saphenous nerves were ligated directly, free it and culture Schwann cells 1 week later using the tissue clot method. Nerve growth factor(NGF) and brain-derived neurotrophic factor(BDNF) in medium were detected in different periods. 90 female Wistar rats[(200?30) g] were randomly assigned to 3 different study groups as follows: control group A(n=30): 20% DMEM injection; research group B(n=30): autologous Schwann cells(ASCs) transplantation; research group C (n=30): AASCs transplantation. The cells were purified before transplantion to the injuried T10 spinal cord site of rats (New York University type weight drop apparatus, NYU). The recoveries of the lower extremity were observed using Basso-Beattie-Bresnahan(BBB) locomotor scoring system and somatosensory evoked potential and motor evoked potentials(SEP & MEP). And then observe the coticospinal tract(CST) using the biotinylated dextran amine(BDA) tracing. Results BBB score was higher in research group than the control group 4 weeks after injury, the statistical difference was significant(P

Objective To observe the role of baicalin on the expression of phosphorylated protein of signal transduc-ers and activators of transcription signaling proteins (STATs) during the process that neural stem cells (NSCs) differentiating into neurons. Methods NSCs were isolated from the embryonic cerebral cortex of the 14-15-day pregnant SD rats, which were cultured and passaged in vitro. The 3rd generation of NSCs was used in the experiment. NSCs were randomized into nat-ural differentiation control group, three different doses of baicalin groups (7.5μmol/L, 15μmol/L and 30μmol/L), leukemia inhibitory factor (LIF)+basic fibroblast growth factor (bFGF) group and baicalin+LIF+bFGF group. After 6 d culture in vi-tro, the immunohistochemical method was used to observe the expressions of microtubule-associated protein 2(MAP-2) and glial fibrillary acidic protein (GFAP) in different groups. The expression levels of phosphorylation protein of STAT 3 in NSCs were detected by Western blotting method after 2 h and 6 d of culture. Results The expression of MAP-2 in NSCs was in-creased by baicalin, but the expression of GFAP in NSCs decreased. The expression of GFAP in NSCs was enhanced in LIF+bFGF group, which was inhibited by baicalin+LIF+bFGF. The phosphorylation level of STAT3 in NSCs was downregulat-ed by baicalin, but the phosphorylation level of STAT3 was upregulated in LIF+bFGF group. The upregulated phosphoryla-tion level of STAT3 was inhibited in baicalin+LIF+bFGF group(P＜0.05). Conclusion Baicalin can induce NSCs to dif-ferentiate into neurons, which may be caused by the downregulation of the phosphorylation level of STAT3 in NSCs.

Objective To observe the protective effect of sodium hyaluronate (Na-HA),and its effects on mRNA expression of peroxisome proliferators-activated receptor gamma (PPAR-γ) in cartilage of rabbit osteoarthritis (OA) model.Methods Forty eight white rabbits were divided into A,B,C groups randomly.Group A were normal controls,groups B and C were underwent unilateral anterior cruciate ligament transection (ACLT).The rabbits in group B were injected normal saline after ACLT;Group C rabbits received intra-articular 1% sodium hyaluronate (HA) injections 5 weeks after surgery,0.3 ml once a week.At week 11 after the surgery,all rabbits were sacrificed.The cartilage changes on the medial femoral condyles were graded.Cartilage sections were stained with safranin-O and HE,mRNA expression of PPAR-γ was detected by real time polymerase chain reaction (Real Time-PCR).Results Cartilage degeneration in group B was significantly more severe than in groups A and C.The grey value of Safranin-O of B group were higher than groups A and C.Expression of PPAR-γ mRNA in group B was higher than that in groups A and C.Conclusion NaHA has a protective effect on articular cartilage degeneration,and the inhibitory effect on PPAR-γ mRNA expression may be one of the therapeutic mechanism of Na-HA.

Objective To study the effect of interleukin (IL)-1β on mitochondria of chondrocytes and reactive oxygen species.Methods Rat chondrocytes were isolated and cultured in vitro,10 ng/ml IL-1β was added for establishing model of osteoarthritis.Then,ratio of apoptosis was surveyed by Annexin V-FITC and PI flow-cytometry.After Hoechst 33342 dyeing,morphology of nucleus was observed by fluorescence microscope.After rhodamine-123 luorescent staining applied,change of mitochondria membrane potential was observed by confocal microscopy.We evaluated the ability of ATP synthesizing in mitochondria by luciferase reaction.Content of active oxygen was observed by confocal microscopy.T test was used for statistical analysis.Results IL-1β could obviously reduce the mitochondrial membrane potential of chondrocytes and its ability to synthesize ATP,and could promote the expression of reactive oxygen species in cells.The values were changed to (24±4) U,(4.1±0.8) pmol/106 cells and (89±7) U from (86±10) U,(13.3±3.0) pmol/106 cells and (17±3) U.The difference had statistical significance.Conclusion IL-1β can induce chondrocyte differentiation by destroying the mitochondrial membrane integrity of chondrocytes and promoting the expression of reactive oxygen species.It can also promote the articular cartilage degeneration.

Objective To explore the difference of DNA methylation levels between normal Schwann cells (NSCs) and activated Schwann cells (ASCs) in rats. Methods The adult Wistar rats were received sciatic nerve ligation and fed for 7 days. The ASCs and NSCs were separated from ligated sciatic nerves and brachial plexus respectively. Immunocytochemical staining of S-100 antibody was used to identify the cells. The growth condition of cells was detected by CCK-8 method. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) was applied to filter the differentially methylated regions in ASCs and NSCs. The distribution of differentially methylated genes related with axonal regeneration in chromosome was analyzed, and Gene ontology(GO)and PATHWAY analysis were also conducted. Results High purity of ASCs and NSCs were obtained successfully, which were both positive for S-100 antibody. In the same culture condition, ASCs showed a faster proliferation than that of NSCs. A total of 177 176 differentially methylated regions were found by MeDIP-Seq. Among them, 1 097 were located in the promoter (≤1 kb), 1 136 in the promoter (1-2 kb) and 567 on the CpG. After functional annotation of differentially methylated genes, 214 differentially methylated genes related with axonal regeneration were found in ASCs and NSCs. Compared with NSCs, 191 genes were up-regulated and 23 genes were down-regulated in ASCs. These genes were located on different chromosomes, most of which on chromosome 12 (22 genes) and the least on chromosomes M (2 genes). GO analysis indicated that the differential methylated genes were involved in axon growth, axon formation, axon elongation and axon guidance. The MAPK, cell adhesion molecules, Ras signaling pathway may be related with the differential methylated genes. Conclusion The methylation levels between ASCs and NSCs are significantly different, which are probably related with axon regeneration.

Objective To observe whether immature Brain Derived Neurotrophic Factor (proBDNF)can affect the activities of OPCs in the fields of cell proliferation and migration after SCI,and to investigate the relationship between proBDNF and p75NTR signal pathway on OPCs.Methods OLN-93 cell line was cultured and maintained for in vitro experiments.Immunofluorescence were used to check the expression of endogenous proBDNF,p75NTR and sortilin on OPCs.MTT assay was used to illustrate the inhibitory effect of proBDNF.The effects of anti-proBDNF was also observed by BrdU staining to find a probably signal pathway for proBDNF on OPCs.The Sprague-Dawley rats were administered for T9 spinal cord injury animal model.BBB score was applied to observe the situation of functional recovery after treated by anti-proBDNF.BrdU staining was managed to observe the situation of OPCs proliferation and migration after SCI.Results Endogenous proBDNF inhibited proliferation and migration of OPCs after SCI.BrdU staining showed that population of proliferative OPCs in lesion site of spinal cord was less in proBDNF in treated group than that in control group and anti-proBDNF group.While anti-proBDNF could inhibit proBDNF specifically and might induced a better functional recovery which was illustrated by BBB scores.The in vitro experiments found the inhibitory effect of proBDNF is dose-dependent and can be neutralized by anti-proBDNF properly.Moreover,the expression levels of p75NTR and sortilin are down regulated by proBDNF antibody treated group.This indicated that proBDNF may inhibit OPCs via p75NTR pathway.Conclusion Endogenous proBDNF can inhibit cell proliferation of OPCs after SCI and can be neutralized by specific antibodies of proBDNF.This kind of detrimental effect may be induced by p75NTR-sortilin pathway.Furthermore,proBDNF antibody treatment is effective to block proBDNF and promote the functional recovery.

BACKGROUND:Since cannulated screw has been applied to femoral neck fracture, it is not uncommon that the screw wear penetrates or refunds. What factors affect the stability of cannulated screw for treatment of femoral neck fractures in adults remains unclear. ＜br> OBJECTIVE:To explore factors related to internal fixation failure by cannulated screws in treatment of adult femoral neck fracture and improve the stability of the adult femoral neck fracture by cannulated screws. ＜br> METHODS:A total of 92 adult patients of femoral neck fracture were treated by cannulated screws in our department between June 2007 and June 2011. Their data were retrospectively analyzed. According to clinical information and fol ow-ups, we selected factors such as age, gender, Garden type of fracture, preoperative skeletal traction, timing of surgery, Garden index, standards of pedicle screws, pedicle screw shapes, partial weight bearing time and postoperative complications, which may affect the success rate of cannulated screws for ＜br> treating femoral neck fracture. The selected factors were then grouped and assigned, after unrelated factors were excluded by one-way χ2 analysis, multiariable Logistic regression analysis was performed. ＜br> RESULTS AND CONCLUSION:The involved 92 patients were fol owed up for 18-72 months. According to Harris assessment criteria, hip function was excellent in 28 cases, good in 25 cases, fair in 17 cases, and poor in 22 cases at the final fol ow-up, the excellent and good rate was 58%. Radiographic results showed that, the patients were divided into two groups according to the presence of the displacement, GardenⅠ (n=22) and GardenⅡ (n=29) as a group, and Garden Ⅲ (n=25) and Garden Ⅳ (n=16) as the other group, the fixation failure rate was 12%and 39%, respectively. In normol and abnormal Garden Index groups, the fixation failure rate was 16%and 59%, respectively. In nail position standards and non-attainment standards groups, the fixation failure rate was 19%and 70%, respectively. In the complication and non-complication groups, the fixation failure rate was 14%and 55%, respectively. These factor groups showed significant differences (P＜0.05). Multiariable Logistic regression analysis showed that, Garden type of fracture, Garden index, standards of pedicle screws, and postoperative complications are the risk factors for internal fixation failure using cannulated screws in treatment of the adult femoral neck fracture.

Objective To seek an optimal method for the separation,culture of mouse embryonic germ cells (EGCs) in vitro,and to observe the influence of Activated Schwann cells (ASCs)-derived neurotrophins on the differentiation capability of mouse EGCs into neurogenic cells.Methods The gonadal ridges and a few abdominal tissues of the 11-day postcoitum (dpc) mouse embryos were isolated and disaggregated by 0.125％ trypsin-0.02％ EDTA,followed by culture of the mouse EGCs on mouse embryonic fibroblast (MEF) feeders.Monoclonal formation of the mouse EGCs was observed,and the staining of stage specificity embryo antigen-1 (SSEA-1),alkaline phosphatase (AKP),periodic acid-Schiff staining (PAS) were applied to identify the mouse EGCs.Two groups were divided as followed:mouse EGCs+basic medium (control group) and mouse EGCs+ASCs (experimental group).Immunofluorescence (NeuN,MBP,GFAP)analysis was used to evaluate the neurogenic differentiation of mouse EGCs and then to calculate the statistical positive rates of cell staining.All experimental results were analyzed statistically.Results (1) Identification ofmouse EGCs:Mouse EGCs were characterized by a dome-shaped colony containing a large nucleus and a relatively small amount of cytoplasm.All mouse EGCs were positive staining of SSEA-1,AKP,and PAS;(2)The neural induction of mouse EGCs:After one week induction,there were few round or oval cells with long axon-like processes migrating from the edge of the EGCs clones.3 weeks later,the neurogenic-like cells increased quickly.The results of immunofluorescence (NeuN,MBP,GFAP)staining demonstrated that mouse EGCs could differentiate into neurogenic cells under the influence of ASCs.The positive rate of cell staining was significant.Conclusion In this study,a simple,economical method was applied to successfully separate the mouse EGCs in vitro; mouse EGCs can differentiate into neurogenic cells under the influence of ASCs-derived neurotrophins.