Objective To compare the application of three automated microbiology identification and rapid susceptibility assessment systems,Vitek1,Viket2 and BD Phoenix,in detection of extended-spectrum beta-lactamases(ESBLs).Methods The genotypes of 67 clinical isolates of ESBLs-producing E.coli and K.phneumoniae,which were collected from the 1st and 2nd Affiliated Hospital of Zhejiang University,were determined by PCR amplification and sequencing.Meanwhile,these isolates were analyzed by the three automatic microbiology identification system,and the minimum inhibitory concentration(MIC)s for antibiotics were determined.Results Majority of the 67 isolates produced CTXM type of ESBL.Among them,CTX-M-14,CTX-M-22,CTX-M24 and CTX-M-3 were the most frequently detectable types.By using Vitek-AMS,60 isolates(89.6%)were detected to be ESBLs-producing.By using Vitek2,62 isolates(92.5%) were ESBLsproducing.No SHV-12,CTX-M-14 and SHV-28 were detected by the 3 automatic systems.The results of MICs analysis indicated these isolates showed either high resistance for multiple antibiotics or around the boundary areas of MICs test.Conclusions The three automatic analysis systems are able to provide a detectable rate of more than 98% for ESBLs.However,it is needed to further improve the detection for high resistant strain or the strains carrying multiple resistant genes.

OBJECTIVE To detect AmpC ?-lactamases from isolates of extended spectrum ?-lactamases(ESBLs) positive Escherichia coli and Klebsiella pneumoniae,which were isolated from four hospitals in Hangzhou from 2005 to 2006,and to analyze the antimicrobial activity of clinically commonly used antibiotics in vitro.METHODS Amount of 324 ESBLs positive isolates including E.coli and K.pneumoniae were collected from Zhejiang Provincial People′s Hospital;1st Affiliated Hospital of Zhejiang University;2nd Affiliated Hospital of Zhejiang University and Hangzhou Traditional Chinese Medicine Hospital perspectivly.AmpC ?-lactamase was identified by disc screening testing and three dimensional test,and the genotypes of AmpC ?-lactamase were also determined by multiplex polymerase chain reaction(Multi-PCR).Minimal inhibitory concentrations(MICs) of ten clinically commonly used antibiotics were determined by agar dilution for AmpC ?-lactamases positive isolates,and the data were analyzed by WHONET 5.3.RESULTS AmpC ?-lactamase phenotype test revealed that 76 AmpC ?-lactamase positive isolates(23.5%) were identified among 324 ESBLs positive isolates of E.coli and K.pneumoniae from four hospitals in Huzhou.69.7% Of the AmpC ?-lactamase phenotype positive isolates were positively amplified by Multi-PCR.The value of MIC50 for carbapenem was lower 0.25 ?g/ml.We also found four carbapenem-resistant strains in this study.CONCLUSIONS We found that the incidence rate of AmpC ?-lactamase is high among the ESBLs producing E.coli and K.pneumoniae strains in Hangzhou.Carbapenem antibiotics have higher antimicrobial activity than other tested antibiotics.

Objective To construct lentiviral vector carrying rat′s calcitonin gene-related peptide(CGRP) gene for the following-up study on the function of CGRP .Methods CGRP gene segment was subcloned into shuttle plasmid ,become Puc57-CGRP .The pLenO-DCE-CGRP expression vector was be constructed by double digests .The pLenO-DCE-CGRP and 4 auxiliary packaging plas-mids were co-transfected into 293T cells .Cells were cultured for 48 hours .The supernant was collected and concentrated ,and then the viral titers were tested by multiple proportions dilution method and flow cytometer .The expression levels of CGRP were detec-ted in CGRP-modified 293T cells by Real-time PCR .Results The results of digestion and sequencing show that the pLenO-DCE-CGRP vector was constructed successfully .The titer of the lentiviral particles was 5 .1 × 108 TU /mL .Conclusion The high-titer lentvirus vector containing CGRP gene is constructed successfully ,which lay a foundation for transfecting mesenchymal stem cell (MSC) and studying the function of CGRP .

Objective To investigate the photodynamic effect induced by laser and use it as a method for purging of minimal residual tumor cells from autologous peripheral blood stem cell grafts. Methods We studied the effect of various concentrations of hematoporphyrin derivative (HPD) combined with of different time of laser irradiation on human leukemia cell line K562, breast tumor cell line MCF-7 and human umbilical cord blood-derived hematopoietic stem/progenitor cells by using MTT assay to compare the photodynamic effect between cell lines of each group. Results HPD or laser irradiation alone had no apparent cytotoxicity while the application of HPD plus laser exposure caused photosensitization. HPD plus laser irradiation was more efficient for killing K562 cells and MCF-7 cells than doing the normal human umbilical cord blood-derived hematopoietic stem/progenitor cells. The photodynamic effect on K562 cells and MCF-7 cells was positively correlated with the increase of treatment duration and HPD concentration. Conclusion These results indicated that the laser photodynamic effect mediated by HPD might be used for purging autologous peripheral blood stem cell grafts in vitro.

BACKGROUND:The good rotational alignment of femoral prosthesis was very important in total knee arthroplasty. The research has shown that the posterior condylar angle was important to determine the alignment. The posterior condylar angle is the angle between the posterior condylar axis and the femoral epicondylar axis. MRI can clearly show the condylar cartilage, the projections of lateral epicondyle and the medial epicondyle depression, thus ensuring accuracy of measurement data. OBJECTIVE:To measure the posterior condylar angle of knee joint in the northern part of Baoding City in China, and to provide image evidence for identifying the rotational alignment of femoral prosthesis during total knee arthroplasty. METHODS:The knee was extended on a neutral position when MRI machine was applied to scan knee joint. The scanning plane was perpendicular to the mechanical axis of the knee. The best T1 axial plane of the knee was chosen, and two observers analyzed images independently. Existence rate of femoral medial epicondyle was observed using Bravo viewer 6.0 imaging software. The posterior condylar angle between posterior condylar axis and the femoral condyle axis was measured. RESULTS AND CONCLUSION:The posterior condylar angle was (2.73±1.28)° in males and (2.35±1.37)° in females on average, which did not show significant difference. The results showed that the MRI had great superiority in measuring the posterior condylar angle. The variability of the epicondylar axis was smal in total knee arthroplasty. Posterior condylar angle can be referenced to position femoral prosthesis and to avoid the complications after knee replacement.

Objective To explore in vitro mesenchymal stem cell (MSC) transfection of lentiviral vector mediated calcitonin gene‐related peptide(CGRP) gene and its effects on biological properties of MSC .Methods MSC were isolated ,cultured and identi‐fied .MSC were infected by lentivirus encoding recombinant enhanced green fluorescent protein (EGFP) gene and CGRP (Lv‐EG‐FP‐CGRP) .The transfection efficiency was determined by the inverted fluorescence microscope and flow cytometry .The expression levels of CGRP were detected in CGRP‐modified MSC by using real‐time PCR ,immunocytochemistry and enzyme‐linked immu‐nosorbent assay (ELISA) .The proliferation ,aging and differentiation ability of MSC were evaluated by MTT ,β‐galactosidase stai‐ning and inducing differentiation respectively .Results After 48 h of MSC transfection by Lv‐EGFP‐CGRP ,EGFP/CGRP could be expressed stably .When multiplicity of infection (MOI) was 30 ,the transfection efficiency reached more than 80% .Compared with the MSC group and the MSC‐EGFP group ,the mRNA and protein expression levels of CGRP in CGRP‐modified MSC(MSC‐CGRP group) were markedly increased(all P<0 .01) .The results of MTT ,β‐galactosidase staining and inducing differentiation assay dem‐onstrated that the transfected CGRP basically had no effect on the proliferation ,aging and endotheliocyte differerntiation of MSC . Conclusion MSC is a kind of ideal genetic vector cell ,which can serve as the target cell of CGRP gene transduction for the applica‐tion of gene therapy and lays the foundation for follow‐up in vitro and vivo experiments .

AIM: To examine whether Akt signal pathway proteins, including Akt, NF-?B and I?B?, are activated in kidney tissue of murine chronic graft-versus -host disease (GvHD) lupus nephritis in vivo , and whether prednisone suppres ses activation of them. METHODS: Akt activity and phosphorylated I?B? were detected by Weste rn-blot. Activation of NF-?B was detected by electropheretic mobilit y shift assay (EMSA). RESULTS: Activity of Akt, NF-?B and phosp horylated I?B ? were significantly increased in kidney tissue of murine chronic graft-versus -ho st disease (GvHD) in 8th week and 12th week after monocell injection, respective ly. However, they were no significant elevation in 16th week, when compared with controls. Prednisone treatment significantly prevented the increase in serum an ti-dsDNA antibody level, urinary protein excretion and glomerular cell prolif eration in GvHD mice, indicating the beneficial effects of prednisone on t his model. Prednisone also significantly suppressed the increase in the activities o f glomerular Akt, NF-?B and phosphorylated I?B?. CONCLUSION: T his study provides t he first evidence of marked increase in glomerular Akt-NF-?B signal pathway act ivities in murine chronic graft-versus-host disease lupus nephritis. The benefic ial effect of prednisone on this lupus nephritis model may be partially mediated by the suppression of abnormal Akt- NF-?B activation.

@@

Objective To investigate the effect of transplantation of CXC receptor 4 (CXCR4)gene-modified bone marrow mesenchymal stem cells (BMSCs) on repairment of carotid injure in rats.Methods BMSCs were cultured and transfected with lentivirus vector carrying CXCR4 gene to generate CXCR4 gene-modified BMSCs (CXCR4-BMSCs).CXCR4 expression was detected by Western blot.Rat model of carotid artery balloon injury was established.Rats were randomly divided into the PBS control group (n=12),CXCR4-BMSCs group (n=12) and BMSCs group (n=12).Two weeks after transplantation,the injured arteries were obtained.The homing of BMSCs was detected by immunofluorescence with green fluorescent protein (GFP).Platelet endothelial cell adhesion molecule (CD31) expression was detected by immunofluorescence staining.At 4 weeks after transplantation,proliferating cell nuclear antigen (PCNA) expression was determined by immunohistochemical staining,and the vascular morphological changes were observed by hematoxylineosin staining (HE).Results Compared with the control and BMSCs groups,the protein level of CXCR4 was increased in CXCR4-BMSCs group (both P＜0.05).The percentage of GFP-positive cells homing were much more in CXCR4-BMSCs group than in BMSCs group [(58.8±4.4)％ vs.(36.2±5.0) ％,P＜0.05].The CD31 expression were higher in CXCR4-BMSCs group than in BMSCs group [(58.8±4.3)％ vs.(28.8±4.2)％,P＜0.05].Compared to the control group,the PCNA expression was decreased in CXCR4-BMSCs and BMSCs groups [(21.0±4.2) ％,(36.5±4.9) ％vs.(78.3±3.5) ％,both P＜0.05].There was a significant difference in PCNA expression between the CXCR4-BMSCsgroupandBMSCs group [(21.0±4.2)％vs.(36.5±4.9)％,P＜0.05].The neointimal area and the ratio of neointimal/medial area were decreased in CXCR4 BMSCs and BMSCs group as compared with the control group [(0.205±0.018) mm2,(0.323±0.071) mm2 vs.(0.536 ± ±0.054) mm2; (1.039±0.123),(1.660±0.404) vs.(2.460±0.328); all P＜0.05],and there were significant differences in neointimal area and the ratio of neointimal/medial area in CXCR4-BMSCs group and BMSCs group [[(0.205±0.018) mm2 vs.(0.323±0.071) mm2,(1.039±0.123)vs.(1.660±0.404),both P＜0.05].Conclusions CXCR4 gene-modified BMSCs may increase the CXCR4 expression in BMSCs.CXCR4-BMSCs transplantation is more effective than BMSCs transplantation in increasing BMSCs homing capacity,reducing the reendothelialization and vascular restenosis.

Objective To observe the effect of adenovirus- receptor activity modifying protein-1 (RAMP1) on nuclear factor(NF κB) and myocardial fibrosis in heart of rabbit with myocardial infarction. Methods Myocardial infarction (MI) models were developed in 54 rabbits and they were randomly divided into RAMP1 group,EGFP group and control group according to whether pAd2-EGFP-RAMP1,pAd2-EGFP or PBS was injected into infarction region and its border.At 7 d,14 d and 28 d after injection,Left ventricular function indices such as LVEF,LVSd,LVDd and LVFS were estimated by echocardiogram,the expression of NF-κB in myocardium was detected by Western blot,the plasma level of TNF-α was measured by ELISA,and fibrosis and collagen content was measured by Masson stain. Results At 7 d after adenovirus injection,the expression of RAMP1was significantly increased in RAMP1 group (67.33 ± 3.97)％ as compared to EGFP group(20.59 ±3.26) ％ and PBS group ( 23.80 ± 5.08) ％ ( P ＜ 0.05 ).The expression of NF κB was decreased in RAMP1 group ( 26.54 ± 5.13 ) ％ versus EGFP group (62.60 ± 6.18) ％ and PBS group (62.95 ±5.17)％ (P＜0.05).The plasma level of TNF-α was lower in RAMP1 group than in EGFP group and in PBS group at different time[7 d:( 136.74 ± 5.42) μg/L vs.( 196.97 ± 14.17) μg/L,(203.67 ±13.90)μg/L; 14 d:( 154.51 ± 13.61 )]μg/L vs.( 112.22±6.74 )μg/L,(160.46± 14.27)μg/L ;28 d;(51.10± 5.62)μg/L vs.( 95.55 ± 9.94 )μg/L,( 98.96 ± 12.68) μg/L,all P＜ 0.05].The collagen content was reduced in RAMP1 group as compared with EGFP group and PBS group at 14 d and 28 d [14 d:(7.10±0.98)％ vs.(19.52±2.32)％,(17.91±0.96)％ ;28 d:(17.04±2.44)vs,(34.10±5.59) ％,(33.98±4.33)％,all P＜0.01].At 28 d after infarction,the infarct size was decreased in RAMP1 group (26.54 ± 5.13) ％ compared with EGFP group (32.20 ± 3.73) ％ and control group (35.58±2.65) ％ (P＜0.01),and better heart function appeared in RAMP1 group. Conclusions The high-expression of RAMP1 could decrease the collagen deposition and fibrosis in the border of infarction and improve heart function through lower expression of NF-κB and decreasing the plasma level of TNF-α.