Objective To investigate the cytotoxic activities of γδT cells against methotrexate (MTX)-resistant osteosarcoma (OS) cells.Methods The MTX-resistant U2OS cell line (U2OS/MTX) was established by in vitro exposing the parental cells to MTX at stepwise-increasing concentrations.Periph-eral blood mononuclear cells ( PBMCs) were isolated from healthy subjects and stimulated with zoledronate ( ZOL) in combination with IL-2 to induce the proliferation ofγδT cells.The cytotoxicity ofγδT cells against U2OS/MTX cells was analyzed by using a standard lactate dehydrogenase release assay (LDH).Flow cy-tometry analysis and ELISA were performed to detect the expression of CD69 and IFN-γby γδT cells, re-spectively.Results The γδT cells derived from healthy subjects showed cytotoxicity against the U2OS/MTX cells.Moreover, stronger cytotoxic activities of γδT cells were detected after pretreatment of U2OS/MTX cells with ZOL (1 μmol/L) for 24 hours (P<0.01).Stimulation with U2OS or U2OS/MTX cells in-creased the expression of CD69 onγδT cells and enhanced the secretion of IFN-γbyγδT cells (P<0.05). Increased expression of CD69 and enhanced secretion of IFN-γwere induced in γδT cells in response to stimulation with ZOL-pretreated U2OS/MTX cells (P<0.01).Conclusion TheγδT cells showed cytotox-icity against U2OS/MTX cells.Pretreatment of U2OS/MTX cells with ZOL could enhance the cytotoxic ac-tivity of γδT cells.

Objective To discuss parallel fibular osteoseptocutaneous flap apply in heel defect reconstruction Methods From February,2006 to December,2011,parallel fibular osteoseptocutaneous flap were used to repair calcaneus and skin defects in 4 cases.The causes included road accident in 2 cases and strangulation in 2 cases.The defect locations were at the back of the calcaneus (1/2 of calcaneus in 2 cases and 2/3 in 2 cases,respectively).Results Followed up 24-72 months,all cases achieved bone union in allograft and had no complications of absorption,infection and repulsion.The flaps survived completely in 4 cases ; the distal flaps necrosed partly in 1 case and healed by dressing.According to USA foot-malleolus scores of AOFAS,1 case was excellent,2 good,and 1 fair.Conclusion Parallel fibular osteoseptocutaneous flap reconstruction heel defect can recover patients visage and heel function,improve their life quality.

Objective:To investigate the effect of typeⅠIFN on the cytotoxic activity ofγδT cells from peripheral blood mono-nuclear cells ( PBMCs) of healthy donors and osteosarcoma patients stimulated by zoledronate ( Zol) and IL-2 against OS.Methods:PBMCs from healthy donors and osteosarcoma patients were stimulated with Zol+IL-2 or Zol+IL-2+typeⅠIFN,respectively.After 14 days of culture,ex vivo expandedγδT cells were assessed by flow cytometry.γδT cell cytotoxicity against target cells was analyzed using a standard lactate dehydrogenase release assay.IFN-γsecreted fromγδT cells was determined by ELISA kits.Results:γδT cells from PBMCs of healthy donors and patients with OS were selectively expanded by Zol+IL-2 or Zol+IL-2+typeⅠIFN in vitro,respectively, and showed cytotoxicity against OS.In addition,γδT cells pre-treated by TypeⅠIFN showed significantly higher cytotoxicity against OS cells.Conclusion:Type I IFN can enhance humanγδT cells’ cytotoxic activity against OS.

Rifabutin(RBT) is a rifamycin derivative,like rifampicin(RIF),registered for the prophylaxis and treatment of mycobacterium avium complex (MAC)in patients with AIDS by FDA in 1992.Subsequently,the drug was approved by many other countries.But now,it is used not only in the prophlaxis and treatment of mycobacterium avium complex but also in the treatment of pulmonary tuberculosis and Helicobacter pylori.For its high lipophilic characteristic and weak inducing properties compare to other rifamycin derivative,it can be applied in treatment with many diseases successfully,especially when combine with other antibiotics,and can solve the problem of traditional antibiotics resistance and increase the clinical safety of combined medical treatment.This paper just shows the progress of clinical pharmacological study and related aspects on rifabutin in order to instruct prescription.

Objective:To investigate whether celastrol can increase the cytotoxic activity of γδ T cells against osteosarcoma (OS) cells line HOS through TRAIL way. Methods:The death receptors 4/5 (DR4/5) protein levels in the OS cell lines HOS was in-vestigated by Western blot analysis. Zoledronate ( ZOL) was used to induceγδT cells from PBMCs of healthy volunteers.γδT cell cy-totoxicity against HOS cells was investigated by a standard lactate dehydrogenase release assay ( LDH ) . Results:Celastrol increased DR4/5 protein expression in HOS ( P<0. 05 ) .γδ T cells from PBMCs of healthy volunteers showed cytotoxicity against HOS ( P<0. 05). Following co-culture with HOS pre-treated with celastrol (1 μmol/L) for 24 h,γδ T cells showed significantly higher cytotoxicity against HOS (P<0. 05). The induction of DR4/5 molecules increased lysis of HOS by γδ T cells which was abolished by addition of a blocking TRAIL antibody. Conclusion:Celastrol can enhance γδ T cells'cytotoxic activity against OS cells line HOS through TRAIL way.

The purpose of this study was to compare the biomechanical stability obtained by using our technique featured an anatomical plate and compression bolts versus that of the conventional anatomic plate and cancellous screws in the fixation of intraarticular calcaneal fractures. Eighteen fresh frozen lower limbs of cadavers were used to create a reproductive Sanders type-III calcaneal fracture model by using osteotomy. The calcaneus fractures were randomly selected to be fixed either using our anatomical plate and compression bolts or conventional anatomic plate and cancellous screws. Reduction of fracture was evaluated through X radiographs. Each calcaneus was successively loaded at a frequency of 1 Hz for 1000 cycles through the talus using an increasing axial force 20 N to 200 N and 20 N to 700 N, representing the partial weight bearing and full weight bearing, respectively, and then the specimens were loaded to failure. Data extracted from the mechanical testing machine were recorded and used to test for difference in the results with the Wilcoxon signed rank test. No significant difference was found between our fixation technique and conventional technique in displacement during 20-200 N cyclic loading (P=0.06), while the anatomical plate and compression bolts showed a great lower irreversible deformation during 20-700 N cyclic loading (P=0.008). The load achieved at loss of fixation of the constructs for the two groups had significant difference: anatomic plate and compression bolts at 3839.6±152.4 N and anatomic plate and cancellous screws at 3087.3±58.9 N (P=0.008). There was no significant difference between the ultimate displacements. Our technique featured anatomical plate and compression bolts for calcaneus fracture fixation was demonstrated to provide biomechanical stability as good as or better than the conventional anatomic plate and cancellous screws under the axial loading. The study supports the mechanical viability of using our plate and compression bolts for the fixation of calcaneal fracture.

Objective To study the expressions of aggrecan (Agc) Ⅰ and Ⅱ collagen and Sox9 by bone mar-row mesenchymal stem cells exposed to electromagnetic fields (EMFs) and it's mechanisms involved. Methods Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and cultured in vitro. The third passage cells were harvested and exposed to 15 Hz 1 mT EMFs for 8 h/d. The semi-quantitative reverse transcription-polyme-rase chain reaction (RT-PCR) technique was used to measure parathyroid hormon receptor related peptide (PTHrp) ,Agc Ⅰ and Ⅱ collagen and Sox9 mRNA. Western blotting was used to measure type Ⅱ collagen expression. After the inhibitor of protein kinase A (PKA) H-89 and the inhibitor of protein kinase C (PKC) Go-6976 ( 12 μm) were added, the effects of EMFs on Agc Ⅰ and Ⅱ collagen and Sox9 mRNA expressions were measured again by using RT-PCR, and Western blotting technique. Results The EMFs induced significant increase of mRNA expressions of PTHrp, Agc Ⅰ and Ⅱ collagen and Sox9 in comparison to the controls, and promoted type Ⅱ collagen protein expres-sion. The Agc Ⅰ and Ⅱ collagen expressions decreased after PKA pathway inhibitor H-89 and PKC inhibitor Go-6976 were added, but the mRNA expression of Sox9 was not affected. Conclusion This study shows 15Hz 1mT EMFs can promote mRNA expressions of Agc Ⅰ and Ⅱ collagan and Sox9 of cbondrogenesis differentiation markers in bone marrow mesenchymal stem cells. The effect is correlated with PKA and PKC pathways.

Objective To discuss the applied value of diffusion weighted imaging(DWI) in evaluating emboliza-tion effect of transcatheterhepatic arterial chemoembolization(TACE) for Hepatic Metastases. Methods DWI was performed in 17 Hepatic Metastases patients at the next day after TACE. Embolization effect was analyzed andcompared with DSA results. Results 17 lesions were revealed in DSA anA with complete iodinated oil accumulation,9with incomplete accumulation. 47 lesions were revealed in DWI and 33 with complete iodi, a oilaccumulation,9 with in-complete accumulation,5 without iodinated oil accumulation.The area of iodinated oll accumulation and necrosis appeared aslow signal on DWI,and residual tumor tissues appeared as high signal. Conclusion DWI may detect Hepatic Metastases lesions sensitivelyand can be used to evaluate the embolization effect of TACE.

OBJECTIVETo investigate the cytotoxic effect of γδ T cells from osteosarcoma patients against interferon-γ (IFN-γ)-treated osteosarcoma cells in vitro.

METHODSHuman γδ T cells were amplified by zoledronate from peripheral blood cells of osteosarcoma patients. The expression of Fas on the osteosarcoma cells were measured by flow cytometry and quantitative real-time PCR analysis before and after IFN-γ treatment. The cytotoxicity of γδ T cells against osteosarcoma cells was evaluated with LDH assay.

RESULTSIFN-γ significantly enhanced the susceptibility of the osteosarcoma cell lines HOS and U2OS to the cytotoxicity of γDelta; T cells from osteosarcoma patients (P<0.01). IFN-γ obviously up-regulated the expression of Fas in HOS and U2OS cells (P<0.01). Anti-FasL mAb failed to inhibit the cytotoxicity of γδ T cells in untreated osteosarcoma targets (P>0.05), but significantly impaired γδ T cell cytotoxicity in IFN-γ pre-treated osteosarcoma targets (P<0.01).

CONCLUSIONIFN-γ can enhance the cytotoxic effect of human γδ T cells from osteosarcoma patients against osteosarcoma cells in vitro.

Bone Neoplasms ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Humans ; Interferon-gamma ; pharmacology ; Osteosarcoma ; immunology ; metabolism ; Receptors, Antigen, T-Cell, gamma-delta ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects ; immunology ; fas Receptor ; metabolism

Objective To investigate the relationship between.serum 25-hydroxy vitamin D (25OHD) and parathyroid hormone (PTH) with the severity in elderly patients with osteoporosis.Methods A total of 160 patients with fractures treated in this hospital from January 2015 to January 2016 were selected and divided into the normal bone mass group(53 cases),osteopenia group (63 cases) and osteoporosis group(44 cases) according to the bone mineral density (BMD) in the lumbar vertebra 1-4,total hip and femoral neck any position.Then the serum levels of 25OHD,PTH,T value of BMD,serum osteocalcin (BGP) and bone alkaline phosphatase (ALP) were compared among 3 groups.Results The serum PTH detection value had no statistically significant difference among the normal group,osteopenia group and osteoporosis group (P＞0.05).The serum 25OHD level in the normal group was higher than that in the osteopenia group and osteoporosis group (P＜0.05);the serum BGP and.ALP levds in the normal group were significantly lower than those in the osteopenia group and osteoporosis group (P＜0.05).The level of serum 25OHD in the osteopenia group was higher than that in the osteoporosis group (P＜0.05).The level of serum BGP and ALP in the osteopenia group was significantly lower than that in the osteoporosis group (P＜0.05).The BMD value of the lumbar vertebra 1-4,total hip and femoral neck in the normal group was significantly higher than that in the steopenia group and osteoporosis group (P＜0.05).There was a significant positive correlation between serum 25OHD and the BMD value in lumbar vertebra 1-4,total hip joint and femoral neck (P＜0.05).Conclusion The level of serum 25OHD is significantly decreased in the patients with osteoporosis,and.is positively correlated with the BMD level.