Cloning and identification of differentially expressed genes or proteins is helpful not only for finding the functions of genes and proteins, but also for discovery of the mechanism of some diseases. Some methods have been developed for screening differentially expressed genes, such as differential display RT-PCR (DDRT PCR), subtractive hybridization (SH), DNA chip technique, and serial analysis of gene expression (SAGE). In subtractive hybridization, there have advanced three improved methods which include representational difference analysis (RDA), suppression subtractive hybridization (SSH), and full-length-gene-obtainable subtractive hybridization. For obtaining differentially expressed proteins, scientists have only two choices so far. One is two-dimentional gel electrophoresis. The other is phage display antibody repertoire library technique. Since all of the methods above have their own advantages and disadvantages, they should be used according to different needs.screening, differentially expressed genes, differentially expressed proteins

BACKGROUND:There are less studies on static magnetic field, and its biological effects are stil unclear. OBJECTIVE:To investigate the static magnetic field effect on the secretion of nerve growth factor from Schwann cel s. METHODS:Schwann cel s were randomly assigned into three groups:0.05 mT sciatic magnetic field group, 0.1 mT sciatic magnetic field group and control group. At 2 days of inoculation, the cel s were exposed to the sciatic magnetic field, 12 hours per day. Cel s in the control group were not exposed to the sciatic magnetic field. After 6 days of exposure, RT-PCR method was used to detect nerve growth factor mRNA expression in Schwann cel s, and ELISA was adopted to detect the level of nerve growth factor secreted from Schwann cel s. RESULTS AND CONCLUSION:The mRNA expression and secreted level of nerve growth factors in the culture supernatant were significantly higher in the two sciatic magnetic field groups than the control group (P<0.05), especial y in the 0.1 mT group. However, there was no difference between the 0.05 mT and 0.1 mT groups (P>0.05). These findings indicate that the static magnetic field with certain intensity can promote the secretion of nerve growth factors from Schwann cel s.

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.

Objective To study the preventive and therapeutic effect of dexamethasone on communicating hydrocephalus following subarachnoid hemorrhage (SAH), and explore the mechanism of communicating hydrocephalus following SAH. Methods SAH rat model was constructed by injecting auto blood through foramen magnum into subarachnoid space. There were two groups ( one control group and one experimental group) in this study with 16 rats each group. Sufficient doses of auto blood were injected into subarachnoid space of the rats of the control group and sufficient dose of auto blood together with sufficient dose of dexamethasone into subarachnoid space of the rats of the experimental group. The changes of rats transforming growth factor bata-1 (TGF-β_1 )in the cerebrospinal fluid (CSF)and thickness of pia mater collagen fibrosis( CF) following SAH were observed after 1 day and 20 days. The levels of TGF-β_1, in the CSF were quantitatively detected by the way of ABC-ELISA. The brain tissues were dyed through Massions technique, and then the average thicknesses of meningina CF were measured by the software of micrograph collection and analysis computer system. Results In later stage after SAH, TGF-β_1, concentrations of the experimental group was lower than control group( P <0. 01). At the same time, the average thicknesses of meningina CF of experimental groups was thinner than control group( P <0. 01). Moreover, the relationship between the level of TGF-β_1 in the CSF and the thickness of the CF of the rat's cerebral pia mate had positive correlation. Conclusions In the later stage after SAH, dexamethasone can inhibit the over expression of TGF-β_1 in rat CSF following SAH and also can inhibit the accrementition of rat collagenous fibers. Therefore, this study provided experimental and theoretical evidence for preventing and treating communicating hydrocephalus following SAH.

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.

BACKGROUND: Up to now, no universally successful therapy to treat substantial articular cartilage defects has been available. Numerous therapeutic approaches can only improve clinical symptoms of joint lesions, but not stimulate the regenerative and reactive capacity of the biological tissue in the defect, and not restore an articular surface capable of functional load bearing.OBJECTIVE: To investigate the curative effects of homograft of mesenchymal stem cells (MSCs) seeded onto cancellous demineralized bone matrix (DBM) on articular cartilage defects.DESIGN, TIME AND SETTING: A randomized controlled study which conducted in Orthopaedics Institute, the Second Hospital of Lanzhou University from January to March 2005 and Central Laboratory of Guilin Medical Collage from May to August 2008.MATERIALS: Bone metaphysis and vertebral cancellous bone were derived from rabbits to prepare DBM materials. MSCs were seed on DBM stent and cultured in vitro. All 36 rabbits were randomly divided into combination group (DBM/MSCs), DBM alone group, and blank control group, with 12 rabbits per group.METHODS: Full-thickness cartilage defect model of knee joint was frilled using a cylinder of 4 mm diameter and 3 mm thickness on intercondylar fossa. The cartilage defects in the intercondylar fossa were filled with MSCs/DBM in combination group A, with only DBM in the DBM group, and nothing was treated in the blank control group.MAIN OUTCOME MEASURES: Four rabbits were killed at three time points, which were 4, 8 and 12 weeks after the operation in each group, and the reparative tissue samples were evaluated grossly, histologically, immunohistochemically and graded according to gross and histological scale.RESULTS: Tirty-six rabbits were included in the final analysis. The defects of MSCs/DBM transplantation were repaired by byline-like tissue, and the other defects were repaired by fibrous tissue. Gross and histological grading scale was made on 12 weeks postoperatively. Gross and histological scores in the MSCs/DBM group were significantly lower than DBM group and control group (P<0.05); while, the scores in the DBM group was significantly lower than control group (P<0.05).CONCLUSION: The full-thickness cartilage defects of rabbits were repaired with homograft of mesenchymal stem cells seeded onto cancellous demineralized bone matrix, which is a promising way for the treatment of cartilage defects.

Objective To investigate the effects of metal particles produced by metal prosthesis on oxidative stress levels in human mononuclear cells . Methods Mononuclear cells obtained from peripheral blood of 15 healthy volunteers .Coculture mononu-clear cells with iron particles , cobalt particles , chromium particles , titanium particles and physiological saline , respectively .Levels of reactive oxygen species ( ROS) , malondialdehyde ( MDA) , superoxide dismutase ( SOD) and glutathione peroxidase ( GSH) were as-sessed in mononuclear cells at 6 h, 12 h, 24 h, 36 h, 48 h, respectively . Results Levels of ROS and MDA were higher in the parti-cle groups than that in the control group at 6 h, 12 h, 24 h, 36 h, 48 h, respectively (P<0.05).Levels of ROS and MDA were higher in cobalt particle, chromium particle and iron particle groups than that in the titanium particle group (P<0.05).Levels of SOD and GSH were lower in particle groups than that in the control group (P<0.05).Levels of SOD and GSH were lower in cobalt particle , chromium particle and iron particle groups than that in the titanium particle group (P<0.05). Conclusion Oxidative stress levels increased and anti-oxidant levels decreased in mononuclear cells when cocultured with metal debris .Cobalt particles , chromium parti-cles and iron particles induced higher changes than that in titanium particles .Oxidative stress may play an important role in metal deb-ris-mediated osteolysis .

AIM: To observe the preventive effect of DNA vaccine based on human calcium-activated chloride channel 1 (hCLCA1) on airway hyperresponsiveness in asthmatic mice.METHODS: The DNA vaccine was constructed by inserting the hCLCA1 gene into pSecTag-2A, and then BALB/c mice were vaccinated by im. once every two weeks. Serum antibody was checked with the antigen of mCLCA3 by ELISA analysis. Asthma was induced with ovalbumin in the vaccinated mice. The airway pressure time index (APTI), the contractile responsiveness of isolated tracheal rings and the number of eosinophil in bronchoalveolar lavage (BAL) were investigated. Mice injected with pSecTag-2A, saline or normal mice were regarded as control groups. RESULTS: The title of antiserum binding to mCLCA3 in vaccine group was 1∶800 to 1∶(1 000) after three times vaccination. Compared with normal group, APTI, contractile responsiveness and number of eosinophil in vaccine group, pSecTag-2A or saline group were increased markedly (P