Objective To study the effect of silencing Nek2 via RNAi on cell cycle of ovarian cancer SKOV 3 cells and the re‐lated molecular mechanism .Methods The Nek2‐siRNA was transfected into the ovarian cancer SKOV3 cells .The change of cell cy‐cle of SKOV3 cells at 48 h after transfection was examined by the flow cytometry technique ;Western blot assay was used to deter‐mine the change of level of the cell cycle related factors cyclinB1 ,CDK1 ,P27 and the phosphorylation level of the ERK1/2 after Nek2‐siRNA transfection for 48 h .Results The flow cytometry detection results showed that the proportion of the cells in G 2/M stage in the blank control group ,negative control group and RNAi group was 13 .72% ,12 .27% and 1 .56% respectively .Compared with the control group ,the number of the cells in G2/M stage in the transfected group was reduced obviously (P< 0 .05) .The Western blot detection results showed that compared with the control group ,the expression of cyclinB1 and CDK1 protein in SK‐OV3 cells was significantly reduced ,the expression of P27 was increased after silencing Nek2 and the phosphorylation level of ERK1/2 in SKOV3 cells was significantly reduced after silencing Nek2 gene(P< 0 .05) .Conclusion Silencing Nek2 gene might block the ovarian cancer cell line SKOV 3 initiating mitosis ,thus inhibit their proliferation .

Objective To explore the effects of alendronate on adipogenic differentiation of bone marrow stremal cells (BMSCs) and the role of mitogen-activated protein kinases (MAPK) signal pathway in this process. Methods BMSCs were derived from 9-month-old ovariectomized SD rats and exposed to 0.01, 0.1, 1, 10 μmol/L of alendronate for 2 weeks. The number of BMSCs was counted under light microscope after oil red O staining. The expression of peroxisome proliferators activated receptor-γ, 2 (PPAR-γ2) was measured by RT-PCR. The effect of alendronate on MAPK signal pathway was detected by Western blot. Results After two weeks of induction of BMSCs by alendronate, BMSCs with positive oil red O staining significantly decreased as the increase of the concentration of alendronate (P <0.01), so did the expression of PPAR-γ2. The expression level of PPAR 2 increased when exposing BMSCs to ERK1/2 or JNK specific inhibitors, PD98059 and SP600125 for two weeks. However, the expression level of PPAR 2 decreased when exposing BMSCs to SB203580 (an inhibitor of p38) for two weeks. Condusion Alendronate can inhibit adipogenic differentiation of BMSCs derived from ovariectomized rats in a doso-dependent manner by activating ERK1/2 and JNK rather than p38.

BACKGROUND: Clinical studies demonstrate that the durable stability of the prosthesis depends on a close geometric fit between the prosthesis and femoral medullary cavity.OBJECTIVE: To study the law of the parameters of medullary canal section shape in proximal femur DESIGN: Repeated measurement observation.SETTING: Department of Orthopaedics, Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University MATERIALS: Ten femoral samples without any damage was obtained from corpse in the Staff Room of Anatomy of the Shanghai Second Medical University METHODS: The experiment was carried out in the Department of Or thopaedics, Ninth People's Hospital between January 2000 and March 2000. Section morphology of medullary cavity of proximal femur was dealt with image-processing, and conus curve fitted parameter mathematical method was proposed; and at the same time, section of medullary cavity of proximal femur of 10 patients was measured manually.MAIN OUTCOME MEASURES: Computer-aided method and manual method were applied to measure the coordination at the end of extrude and the coordinate of link joint RESULTS: 10 femoral samples entered the stage of result analysis. Com puter was used to measure the coordination of the end of extrude of the section medullary cavity of proximal femur (X,Y) and the coordination of the connection point (X,Y).There was no significant difference of the measuring results between computer-aided method and manual CONCLUSION: Computer-aided imaging-processing method not only reduces the error but also can be completed by computer automatically in measuring section morphology of medually cavity in proximal femur. It is suitable for a variety of morphology measurement.

Objective To investigate fluoride-induced inflammation and nuclear factor-κB (NF-κB) signaling pathway in cultured human acute monocytic leukemia cells (THP-1).Methods In vitro cultured THP-1 cells were used as a model of microglia.After cultured with different concentrations of [0 (negative control group),10,50,100,500,1 000 and 5 000 μmol/L] sodium fluoride (NaF) for 48 h,the survival of cells was detected by CCK8.THP-1 cells were divided into 3 groups:control group,low dose and high dose fluoride groups according to the results of CCK8 assay,and then treated with different concentrations of sodium fluoride (0,500,5 000 μmol/L) for 48 h,concentrations of inflammatory cytokines,such as Interleukin-lβ (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) in THP-1 mononuclear cell culture medium.The protein levels of IκBα,phospho-NF-κB p65 and phospho-IκB-α were detected by Western blotting.Results THP-1 cells were treated with different concentrations of sodium fluoride (500,1 000,5 000 μ mol/L) for 48 h.Fluoride group THP-1 cell survival rate [(73.21 ± 3.67)％,(31.40 ± 4.56)％,(0.40 ± 0.24)％] was lower than that of the negative control group [(100.00 ± 0.00)％,all P ＜ 0.01].Compared to the control groups [(0.36 ± 0.07),(31.07 ± 0.81)ng/L],significant increases of the inflammatory cytokines IL-1β [(1.42 ± 0.79),(19.47 ± 2.90)ng/L] and TNF-α [(61.06 ± 2.20),(172.72 ± 2.29)ng/L] were detected in culture medium in low-fluoride and high fluoride groups,respectively.Interestingly,compared to the control groups [(100.00 ± 5.48)％,(100.00 ± 14.82)％],significant increases of phospho-NF-κB p65 [(113.71 ± 8.99)％,(134.74 ± 1.93)％] and phospho-IκB-α [(152.61 ± 14.16)％,(176.91 ± 7.95)％] were observed in both low-fluoride and high fluoride groups.Meanwhile,the protein level of IκBα in high fluoride group [(63.53 ± 9.67)％] was significantly lower than that of the control group [(100.00 ± 10.99)％,P ＜ 0.01].Furthermore,significant positive correlation was detected between increased IL-1β,TNF-α and phospho-NF-κB p65 (r =0.74,0.72,all P ＜ 0.05).Conclusions Excessive fluoride can induce microglial cells to release inflammatory cytokines and activate nuclear factor-κB signaling pathway.The release of inflammatory cytokines and activation of the signaling pathway may be one of the mechanisms of the damage of the central nervous system caused by sodium fluoride.

Objective To evaluate osteogenetic effectiveness of hyaluronic acid (HA) mixed with adenovirus mediated human bone morphogenetic protein-2 gene(Adv-hBMP-2) transfected bone marrow derived mesenchymal stem cells(BMSCs) of rabbits in the repair of radial shaft defects of rabbits. Methods The BMSCs were collected from bone marrow of rabbits, cultured and transfected by Adv-hBMP-2 in vitro. The radial shaft defects (1.5 cm) models of 20 rabbits (40 sides) were created and then respectively injected the cells and/or materials according to the following four groups (n=10): Ⅰ, Adv-hBMP-2 transfected BMSCs+HA; Ⅱ, Adv-hBMP-2 transfected BMSCs; Ⅲ, HA; Ⅳ, control. Results The bone defects of 6 out of 8 sides in the group Ⅰ were repaired completely and partial bone marrow cavities were revasculized. Only 3 sides were repaired completely in the group Ⅱ and no side was repaired completely in the groups Ⅲ and control. There were statistic significance between each groups in X-ray scores. In the groups ofⅠand Ⅱ, much woven callus formed in the sites of the bone defects in the 4-8th week after injection. Partial bone marrow cavities were revasculized in the 12th week after injection; while in the groups of HA and control, the sites of bone defects were mainly full of fibrous tissue and no healing happened. Both ends of the bone defects were hardened. There was significant difference statistically in the new trabecular bone area in the groups ofⅠand Ⅱ. The maximum vertical loading and elastic modulus were significant differenct in the group ⅠandⅡ. The ratios of average maximum vertical loading/normal value of radial shaft in the groups ofⅠandⅡwere 75.86% and 45.45%, respectively. Conclusion The tissue-engineered bone made by HA mixed with Adv-hBMP-2 transfected BMSCs can repair the bone defects in the radial shaft of rabbits. HA is a safe, effective carrier for tissue engineering.

Objective To repair the experimental femoral head necrosis with the implantation of tricalcium phosphate (TCP) loaded with human bone morphogenetic protein-2(hBMP-2) gene transfected bone marrow derived mesenchymal stem cells(BMSCs). Methods Established the animal models of femoral head necrosis in 22 goats by the ligation of the lateral and medial circumflex femoral arteries and delivery of liquid nitrogen into femoral head. 4 goats served as the control group without any treatment. 3 weeks after surgery, necrotic bone in the other 18 goats were removed and TCP loaded gene transfer cells were implanted into the femoral head with BMP-2 gene transfected BMSCs in right side and ?-gal gene transfected BMSCs in left side. BMP-2 concentrations in femoral head were tested by the ELISA at 1st, 2nd, 3rd week after implantation. Histological observation was done before and at 6th, 10th, 16th week after implantation. New bone volumes (NBV) were measured at 16th week after implantation by the histomorphometry method. X-ray was taken at 16th week after operation in untreated group and after treatment in BMP-2 group and ?-gal group. Results The concentration of BMP-2 in femoral head of BMP-2 group was higher than that of ?-gal group at different time point. Empty lacunae and fibrotic marrow were demonstrated before implantation. New bone was evident in the implantation field of BMP-2 group while fibrous tissues were evident in that of ?-gal group at 6th, 10th and 16th week after treatment. Histomorphology analysis indicated that the NBV in BMP-2 group was significantly higher than that in ?-gal group. Articular surface collapse were observed in the X-ray photograph of untreated group. Regular shape and normal density of femoral head in BMP-2 group compared with the irregular shape and low density of femoral head in ?-gal group could be demonstrated in the X-ray photograph 16 weeks after treatment. Conclusion Implantation of the TCP loaded with human BMP-2 gene transfected BMSCs could repair early-stage experimental femoral head necrosis.

Objective　To explore the influence of stress-relaxation plate(SRP) fixation on the remodeling of cortex under plate. Methods　Twenty-eight New Zealand rabbits were used in this study, the bilateral tibia were osteotomized in the middle and fixed with SRP (experimental group) and rigid plate (control group) respectively. The scanning electron microscopy was used to observe the bone remodeling process from 2 to 48 weeks after operation. Results　There was cortex osteoporosis beneath plate in different degree in both experimental and control groups before 8 weeks, it showed as the disorganization of collagen fiber structure and formation of resorption cavities. In comparison, the osteoporosis degree in experimental group showed milder than that of the control group. After 12 weeks, the resorption cavities became smaller, and the structure of collagen fibers became regular with the alignment parallel to the long axis of cortex. In contrast to the experimental group, the bone osteoporosis under plate of control group exacerbated continuously. Conclusion　Without removal of the bone plate, SRP fixation not only reduce the degree of plated bone osteoporosis, but also make the osteoporosic bone return to normal.

[Objective]In present study an in vivo wear particles induced mouse calvarial osteolysis model was used to investigate the inhibitory effect of erythromycin over wear particles induced osteolysis.[Method]Twenty-four male eight-week-old C57BL/J6 mice were allocated into 4 groups: PMMA group receiving 30 mg PMMA particles implantation onto the calvariae,PMMA+2EM group receiving 30 mg PMMA particles implantation and EM 2mg/kg ip injection,PMMA+10EM group receiving 30 mg PMMA particles implantation and EM 10mg/kg ip injection,control group receiving sham operation.Seven days later,mice calvariae were harvested for pathology analysis.[Result]Middle suture area in the control group was 0.079 mm2?0.011 mm2,in PMMA group 0.335 mm2?0.129 mm2.In PMMA+2EM group the area was 0.094 mm2?0.019 mm2,PMMA+10EM group was 0.091 mm2?0.028 mm2.Osteoclasts number in osteolysis area in the control group were 5.3?1.0,in PMMA group 19.2?5.3,PMMA+2EM group was 6.6?1.1,PMMA+10EM group 6.1?1.9.Compared with control group,PMMA particles induced significant osteolysis(P

ObjectiveTo study the influence of stress－ relaxation plate on disorganization andrepair of the cortex under the plate. MethodsA washer made of viscoelastic polyethylene was placedbetween the screw and the screw hole of a conventional stainless rigid plate(RP) to form a stress－ relax-ation plate(SRP). Both SRP and RP were applied to the osteotomized New Zealand rabbit tibia, thefracture healing process of either fixing with SRP or RP(control) was put under comparative study bypolarized light microscopy, in situ hybridization of collagen mRNA and immunohistochemistry tech-nique from 2 to 36 weeks postoperatively. ResultsThe study of plated bone remodeling showed thatthe degree of cortex osteoporosis beneath the plate was similar between the SRP and RP group 12 － weekpostoperatively. The organization of the bone structure in the SRP group happened later and milder thanthat of the RP group, and the repair process began 12 weeks after implantation. So the resorption cavi-ties became smaller and the structure of collagen fibers became well oriented along, and the osteoblastslying on the surface of resorption cavities expressed and synthesized type Ⅰ collagen 8 to 12 weeks afterimplantation. The changes above increased significantly in most cavities by 36 weeks. No expression andsynthesis of any kind of collagen could be observed during 12 to 36 weeks after implantation in the RPgroup. ConclusionWithout removal of the bone plate, the SRP fixation not only reduces the degreeof plated bone osteoporosis, but also makes the disorganized bone structure return to normal by the ex-pression and synthesis of type Ⅰ collagen mRNA of osteoblasts lying on the surface of resorption cavities.

[Objective]To design a method of in vitro preparation and seperation for metallic wear particles around joint prosthesis and evaluate its feasibility in medical experiments.[Method]Ti-6Al-4V and Co-Gr-Mo alloys were used to make two friction jars respectirely. National inventive patent applied number 03142073.7.Lots of quadrate blocks made of the same materials are put into the jars respectively,which were then.lubri cated by man-made body fluid and vibrated on a bottle shaker.After 21 days the fluid was harvested and centrifuged to get the produced wear particles.The collected particles were studied by using element trace analysis,laser countersizer and scanning electron microscopy.The J774.A1 macrophages cultured together with these particles for 24 hours were observed under inverted phase contrast microscopy and transmission electron microscopy.[Result]A got great amounts of metallic particles with 1?m diameter coned beproduce using this method.The aver age diameter of titanium alloys(Dv90) is 1.011 and that of Co-Gr-Mo is 1.010.Particle size distribution had good consistency in different materials.Under scanning electron microscopy ,the particles had irregular shapes just like those got from revision operations.The particles taken into the J774.A1 macrophages could be seen under inverted p hase contrast microscopy and transmission electron microscopy.[Conclusion]This method is good enough to producl lots of metallic wear particles mosth like those around total joint prosthesis and can be used in further in vivo and in vitro studies about joint prosthesis loosening.