AIM: To detect serum osteocalcin level during bone transplantation.METHODS: The animal model of the dog's bone transplantation was established.15 dogs were randomly divided into three groups: irradiation,non-irradiation and bone morphogenetic protein(BMP)-adding group.The allografts bones were treated by cryopreservation and lyophilization,respectively.The animal models were constructed by resecting 2 cm diaphysis and periosteam from the middle pane of both radius of the dogs and transplantation bone were fixed by triargle needle.X-ray examination was taken on the operating day and 1 month,3 months,5 months after operation,respectively.The serum osteocalcin level was detected preoperation,2 weeks,4 weeks,6 weeks,8 weeks and 10 weeks after operation.RESULTS: The level of serum osteocalcin significantly changed in the bone transplantation before and after operation.BMP made an effect on the changes of BGP.CONCLUSIONS: Serum osteocalcin is a peculiar sensitive and convenient biochemical index that reflects metabolism of bone transplantation.More over,it has some importance for differently processed allografts of bone.BMP increases the content of BGP in the serum and accelerates the bone formation.

Objective To characterize and compare the different biological behaviors of two novel human osteosarcoma cell lines, Zos and Zos-M, established respectively from the primary site and the skip metastasis of an osteosarcoma patient. Methods Two novel human osteosarcoma cell lines, Zos and Zos-M,were developed using tissue plant culture method. The vitro examinations included observations of morphology, analysis of karyotype and cell cycle, calculation of doubling time and growth curve, detection of osteoblastic markers and matrigel invasion assay. Subcutaneous, intratibial and intravenous inoculations into nude mice were performed to study the in vivo tumorigenicity and metastatic potentials of both cell lines.MTT were used to detect sensitivity of the cell lines to chemotherapeutic drugs. RT-PCR was performed to assess the expression of and some metastasis-related genes. Results Both cell lines proliferated actively and remained stable for more than 100 passages in vitro without interruption. The morphology and expression of osteoblastic markers of Zos and Zos-M were conformed to the characteristic of osteosarcoma. The karyotype analysis displayed aneuploidy and various structural abnormalities. The population doubling time of Zos and Zos-M were 33.65 h and 31.58 h respectively. Both cell lines were less sensitive to the current chemotherapy protocols compared to U-2OS. Zos and Zos-M were 100% tumorigenic by subcutaneous and othotopic injection. 37.5% of nude mice injected Zos-M and none of nude mice injected Zos developed lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis-related genes revealed that the decreased expression of cadherin-11 in Zos-M may correlate with a high potential of metastases. Conclusion The two novel established human osteosarcoma cell lines, Zos and Zos-M and related animal models could serve as models for the study of drug resistance and screening of new therapeutics for osteosarcoma. In addition, the study also provide tools for the study of metastasis because the same genetic background and different potential of metastasis of Zos and Zos-M.

Objective To study the growth inhibition,apoptosis induction effects of cinobufagin(CB)on human osteosarcoma(OS) cell line U2OS,MG63 and SaO2 in vitro and the underlying mechanism of action of cinobufagin in OS cells.Methods Cell viability was assessed by MTT assay.Cell-cycle status,apoptosis-inducing effects were evaluated by flow cytometry,fluorescent staining and DNA fragmentation assays.Inhibitors of apoptosis proteins (IAPs) and Bcl-2 family proteins including Bax,cleaved-PARP,xIAP,cIAP-1,survivin and p65 were tested by Western blot.Results MTT assay showed that CB could inhibited the growth of U2OS,MG63 and SaO2 cells in a dose-and time-dependent manner.The 48 h IC50 of CB on U2OS,MG63 and SaO2 cells were (104.83± 16.96) nmol/L,(47.07±7.5) nmol/L,and (136.72±10.08) nmol/L respectively.The induction of G2/M cell-cycle arrest was seen in the cells treated with CB.After cells were cultured for 12 h in the presence of 100 nmol/L CB,the percentages of cells in the G0/G1 phase were decreased,while G2/M phase were increased in U2OS,MG63 and SaOS2 cells,respectively.The results showed CB inhibited the proliferation of osteosarcoma cells through blocking the cell cycle in G2/M phase.Induction of apoptosis was confirmed by Hoechst 33258 and Annexin V/PI staining.After treating with 100 nmol/L CB for 48 h,the extents of apoptosis were 33.6％±6.4％,36.4％±7.8％ and 29.3％±5.1％,respectively.These results indicate that the anti-tumor activity of cinobufagin in osteosarcoma cells was due to a G2/M cell cycle arrest and apoptosis inducing effect.Western blot showed that CB could induce the apoptosis related family proteins Bax,cleaved-PARP up-regulation,xIAP,cIAP-1,survivin and p65 downregulation in OS cells.Conclusion CB can inhibit the cell viability and induce G2/M cell cycle arrest and apoptosis in U2OS,MG63 and SaO2 cells.The apoptosis-inducing effect of CB is confirmed by the regulation of apoptosis related proteins IAPs and Bcl-2 in vitro.

Objective To investigate the expression of inducible co-stimulator (ICOS) and inducible co-stimulator ligand (ICOSL) on PBMCs,and the plasma concentrations of soluble forms of ICOSL and their clinical relationship with systemic lupus erythematosus (SLE) patients.Methods Peripheral blood samples were collected from 45 SLE patients and 39 healthy subjects (HC).The expressions of ICOS and ICOSL on peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry.The concentrations of soluble ICOSL were assessed by measured by enzyme linked immunosorbent assay (ELISA).And the relationship between their expression levels and patients' clinical manifestations were analyzed.Levene F test was used for statistical analysis,the comparison between groups was conducted using t test,and the correlation between two variables were tested by Pearson correlation analysis.Results The expression of ICOS on CD4+ and CD8+ T cells were significantly higher than that of the HC [(19.1±1.7)％ vs (14.0±1.5)％,t=2.156,P=0.035],[(10.0± 1.0)％ vs (6.4±1.0)％,t=2.587,P=0.012].The expression of ICOSL on CD14+ mononuclear cells in SLE patients was significantly higher than that in the HC group [(2.94±0.88)％ vs (0.89 ±0.21)％,t=2.152,P=0.04].Plasma ICOSL concentrations in patients with active SLE were significantly higher than those of patients with inactive SLE [(362±25) ng/ml vs (278±15) ng/ml,t=2.356,P=0.025].We also found a significant negative correlation between the soluble ICOSL expression and the surface ICOSL expression on both mononuclear cells and B cells (r=-0.4243,P=0.022;r=-0.4099,P=0.025).MMPI induced an evident reduction in sICOSL levels released from the cells,which was statistically significant in comparison with untreated cells (P＜0.05).Conclusion The up-regulated expressions of ICOS and ICOSL on peripheral lymphocytes and the high levels of plasma concentration of soluble ICOSL are closely correlated with the severity of the disease,suggesting that ICOS/ICOSL pathway may play a critical role in the pathogenesis of SLE.

Objective To investigate treatment and prognosis of patients with osteosarcoma of the extremities.Methods A total of 311 patients with osteosarcoma of the extremities,who had undergone treatment in our institute from 1998 to 2008,were enrolled in this retrospective study.Kaplan-Meier survival curve and Cox regression model were used to analyze the correlation between survival rate and variables including patients' demographics,chemotherapy,surgery,complications,and tumor metastasis.Results Among 311 patients,there were 206 males and 105 females,aged from 5 to 56 years (average,18.6 years).A total of 282 patients underwent aggressive or radical surgery,including 149 cases of limb salvage surgery and 133 cases of amputation surgery.One hundred and five patients underwent standard chemotherapy and 206 patients underwent non-standard chemotherapy.The 5-year survival rate was 57.4％ in patients treated with standard chemotherapy,36.3％ in patients treated with non-standard chemotherapy,16.8％ in patients with lung metastasis,50.7％ in patients without lung metastasis,56.6％ in patients who underwent limb salvage surgery,31.8％ in patients who underwent amputation surgery,44.6％ in patients with Enneking stage Ⅱ B and 33.1％ in patients with Enneking stage Ⅲ.For patients treated by amputation surgery,because non-standard chemotherapy which was performed in most of them and other confounding factors,the 5-year survival rate of them was lower.The Cox regression analysis showed that lung metastasis and non-standard chemotherapy were associated with inferior outcomes.Conclusion Neoadjuvant chemotherapy combined with aggressive or radical surgery could cure about 60％ of patients with osteosarcoma of the extremities.Lung metastasis and non-standard chemotherapy are risk factors that severely affect prognosis.

Objective To evaluate the long following-up outcome of the medial gastrocnemius muscle transferring reconstruction the patella tendon after the wide resection of aggressive bone tumors in the proximal tibia. Methods With the 69 patients of the osteogenetic sarcoma in the proximal tibia were treated with the wide resection and reconstruction the patella tendon. After the long following up the knee extensor,function and complications were evaluated. Results With the 69 patients, the 45 survival patients were followed up for the average 68.6 (24-128) months. The local recurrence rate was 8.7%(6/69). The strength of knee extending was in the average of grade 4.2(3.6-5.0), the degree of knee flexion was in the average of 95°(75°-135°), the degree of knee extension was in the average of-2°(0°-12°), the knees of five patients cannot fully extension. The MSTS functional score was in the average of 77% (23.1/30). Conclusion During the limb salvage of the proximal tibial aggressive bone tumors, the medial gastrocnemius muscle transferring reconstruction the patella tendon could offer the knee extension strength; improve the soft tissue coverage and functional results.

Objective To study the affect and the related molecular mechanism of glycogen synthase kinase-3β in the proliferation of osteosarcomaand its value in the target therapy of osteosarcoma.Methods The expression level of p-GSK-3β(Ser9)and GSK-3β were detected in human osteoblast cell and osteosarcoma cells by western blot.Observe the effect of GSK-3β inhibitors and siRNA interference on the GSK-3β regulate osteosarcoma cells using apoptosis protein chip.Evaluate the valueof GSK-3β target therapy on osteosarcoma in vivo.Results The expression level of p-GSK-3β (Ser9)was lower in osteosarcoma cells.LiCL,GSK inhibitor Ⅸ,siRNA knockdown could inhibit the cell viability and up-regulated the apoptosis-related protein cleaved-caspase3.The results of the protein array showed that downstream proteins of NF-κB downregulated significantly.The results were validated by western blot,while the downregulation of p-Iκ-Bα and nuclear NF-κB p65 were also observed after LiCL treatment.Inhibition of GSK-3β by either LiCl or specific siRNA resulted in a significant reduction of NF-κB luciferase reporter activity.Furthermore,the NF-κB luciferase reporter activity was significantly increased in CA cell lines,but not in KD cell lines.By contrast,NF-κB-luciferase reporter activity was significantly decreased in stably GSK-3β knockdown cells.GSK3β inhibitor LiCL and shRNA knock down demonstrated a strong cytotoxicity effect on osteosarcoma cells in vivo.Conclusion GSK-3β is in the state of relative active in osteosarcoma in osteosarcoma and important in cell proliferation.GSK-3β regulates cell survival partially through the NF-κB pathway.It is a promising therapeutic target in osteosarcoma.

Objective To investigate the expression level and clinical significance of melanoma antigen gene A (MAGEA) in osteosarcoma patients. Methods Compare gene expression profiles in osteosarcoma cell lines and osteoblasts with gene microarrays. Validation of differentially expressed genes was carried out by real-time polymerase chain reaction analysis. Corresponding protein levels were measures by Western blot analysis in osteosarcoma cell lines and by immunohistochemistry in osteosarcoma tissues. The staining intensity of immuno-histochemistry was correlated with clinical outcome , and its prognostic significance was analyzed. Results Sev-eral genes belonging to MAGEA increased significantly in all osteosarcoma cell lines and tumor tissue , but not in normal osteoblast cell. Patients with MAGEA expression has higher risk of lung metastasis (relative risk 2.79, 95% confidence interval, 1.12-6.93; P = 0.028) and lower five-year survival rates (39.6% ± 8.4% vs. 80% ± 8.9%, P = 0.01) compared with patients without MAGEA expression. Conclusions The expression of MAGEA increased in osteosarcoma , which inversely correlating with outcome of osteosarcoma patients.

OBJECTIVETo study the effect and mechanisms of quercetin (Qu) on proliferation and apoptosis of human methotrexate resistant osteosarcoma cell U-2OS/MTX300.

METHODMTT assay was used to observe cell proliferation. The apoptosis was examined by using Annexin V/PI staining. Western blot of mitochondrial membrane potential and cytochrome c were used to detect mitochondria spoptosis pathway. The protein expressions related to Akt pathway was detected by continuous activated Akt transient transfection and western blot.

RESULTQu can obviously inhibit the growth of human MTX resistant osteosarcoma cell U-2OS/MTX300 cells in a dose- and time-dependent manner. Annexin V/PI staining showed obvious cell apoptosis. Reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to cytosol and dephoshorylation of Akt were observed after Qu treatment.

CONCLUSIONQu can inhibit proliferation and induce apoptosis of human MTX resistant osteosarcoma cell U-2OS/MTX300, which may be related with mitochondrial apoptosis pathway and Akt activity.

Apoptosis ; drug effects ; Bone Neoplasms ; drug therapy ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Osteosarcoma ; drug therapy ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Quercetin ; pharmacology

OBJECTIVETo detect the expression of CXCL14 in human osteosarcoma cell lines and tissues and investigate its association with the prognosis of the patients.

METHODSRT-PCR, enzyme-linked immunosorbent assay (ELISA) and real-time PCR were used to detect the expression of CXCL14 in 4 osteosarcoma cell lines and in 40 pairs of osteosarcoma tissues and adjacent muscular tissues. CCK8 assay and colony formation assay was used to assess the effect of CXCL14 suppression mediated by two specific siRNAs on the proliferation of U2OS osteosarcoma cells. Immunohistochemistry was performed to detect the expression of CXCL14 in 58 osteosarcoma tissues, and Kaplan-Meier method and log-rank test were performed for survival analysis of the patients.

RESULTSSignificant up-regulation of CXCL14 expression was found in the osteosarcoma cell lines and in osteosarcoma tissues compared with the adjacent muscles (P<0.01). In U2OS cell, suppression of CXCL14 expression by siRNA significantly inhibited the cell proliferation (P<0.01) and colony formation rate (P<0.05). Kaplan-Meier survival analysis indicated that patients with high CXCL14 expression had worse prognosis than those with low CXCL14 expression (P=0.02).

CONCLUSIONCXCL14 is up-regulated in both osteosarcoma cell lines and primary osteosarcoma tissues to promote the proliferation of osteosarcoma cells. A high CXCL14 expression in osteosarcoma tissues is associated with a poor prognosis, suggesting the that CXCL14 serve as a potential therapeutic target for osteosarcoma.

Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Chemokines, CXC ; metabolism ; Humans ; Osteosarcoma ; metabolism ; pathology ; Prognosis