Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet).Methods By the template Src-biosensor, the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5′ end. With the intermediate vector pDMTM-18T, an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and expression of fusion proteins were observed after 24 h.Fluorescence resonance energy transfer (FRET)was observed by the inversion fluorescence microscope and measured by the MetaFlour FRET 4.6 software. Results The uPA biosensor vector was confirmed by the fragment of PCR and double restriction enzyme digestion.The transfection efficiency was nearly 40%.The immunofluorescence detection results displayed that uPA biosensor fusion protein expressed in the 293T cells membrane and the FRET of uPA biosensor in the living 293T cells was observed after incubation with the recombinant human uPA (rhuPA).Conclusion uPA biosensor is successfully constructed and it could be used as a molecular probe to study the temporal and spatial variation of uPA in living cells.

Objective To investigate the incidence of surgical site infection (SSI)in patients undergoing orthopedic surgery,analyze the risk factors,and provide basis for the prevention and control of SSI.Methods All hospitalized orthopedic patients undergoing surgery in a hospital from January 2010 to December 2014 were retrospectively sur-veyed,questionnaires were designed,patients’medical records were reviewed,incidence of SSI was analyzed,risk factors for SSI were analyzed with univariate and logistic regression methods.Results A total of 14 300 orthopedic patients undergoing surgery were investigated,576 (4.03%)patients had SSI,predominantly were superficial inci-sion infection (n=429,74.48%),615 strains of pathogenic bacteria were isolated from 576 patients,mainly were Staphylococcus aureus (n=137,22.28%),Escherichia coli (n=84,13.66%),and Enterobacter cloacae (n=73, 11 .87%).The incidence of SSI decreased year by year in patients undergoing orthopedics surgery(χ2 =24.706,P <0.001);the incidence of SSI in patients with amputation was the highest (22.67%),followed by patients with de-bridement (7.16%);multivariate logistic regression analysis indicated that long duration of operation,long length of hospital stay,underlying diseases,use of implants,contaminated incision,more intraoperative blood loss,irra-tional perioperative use of antimicrobial agents,and without using negative pressure drainage were independent risk factors for SSI in patients undergoing orthopedic surgery.Conclusion The incidence of SSI is high in orthopedic pa-tients undergoing surgery,effective preventive measures should be actively taken according to the related risk factors of SSI,so as to reduce the occurrence of SSI.

Objective To explore the clinical efficacy of polyetheretherketone( PEEK) cages and titanium cages combined with pedicle screw fixation operation in treatment of lumbar degenerative disease,and provide reference for clinic. Methods The data of 50 patients with lumbar degenerative disease in our hospital from March 2011 to March 2013 were retrospectively analyzed. All patients were treated with pos-terior decompression,bone graft and transpedicular screw internal fixation,according to the different cages,they were divided into PEEK group and titanium group. The PEEK group with 25 patients used polyetheretherketone cages,and the titanium group with 25 patients used titanium cages. The JOA scores of patient before surgery,1 month,6 months and 1 year after surgery between two groups were recorded and compared. The ROM of fusion levels and adjacent segment of patients before and after operation between two groups were compared. Results There was no statistical significance of difference in JOA score,Oswestry score and fusion rate between two groups(P>0. 05). There were statistical significance of differences intervertebral disc height change between 2 groups(P<0. 05). The ROM of fusion levels and adjacent segment of patients in PEEK group was better than those in titanium group(P<0. 05). Conclusion The polyetheretherketone cages combined with pedicle screw fixation operation have a good effect in the treatment of patients with lumbar degenerative disease,especially in matter of lumbar spinal motion and intervertebral height lost.

Objective:To study the construction of matrix metalloproteinase 3 (MMP3)biosensor vector,and to illuminate the activated process of MMP3 in the living cells.Methods:The ECFP-MMP3-YPet biosensor vector anchored on cellular surface was constructed and identified.The MMP3 biosensor was transfected into the 293T cells.The transfection efficiency was observed 24 h after transfection.The flurorescence resonance energy transfer (FRET )-based MMP3 biosensor was observed by inversion fluorescence microscope. Results:The MMP3 biosensor vector was successfully constructed.The length of MMP3-YPet identified by double enzyme digestion and PCR was about 780 bp.The transfection efficiency of MMP3 biosensor was about 40%,and which was evenly presented in cytoplasm of 293T cells.And the FRET ratio of MMP3 biosensor was decreased after stimulation with uPA on the 293T cells. The FRET ratio reached its minimum about 30 min later. Conclusion:The MMP3 biosensor can sensitively and reliably monitor the MMP3 activation in living cells.