ObjectiveTo analyze the homology of Citrobacter freundii strains and detect multidrug resistant genes.MethodsThe minimum inhibition concentration was tested with standard agar dilution method and the the homology of C.freundii strains was measured with pulsed field gel electrophoresis (PFGE) and the PCR was used to perform the drug resistant genes such as β-lactamase,disappearing of outer membrane channel protein.ResultsThe four strains ( No.1,2,4,5 ) showed nineteen banding patterns with PFGE.PCR experiment showed that there were 2 strains (No.1,4) with blaCTX-M,3 strains (No.2,3,5 ) with blaDHA,1 strains(No.3) with blaACT/MIR,and 4 strains(No.1,2,4,5) with blaKPC-2.PCR analysis comfirmed that No.2 and No.4 disappearing of OmpF and No.3 disappearing both of OmpC and OmpF.The expression levels of the chromosomal ampC gene of No.3 isolates was 106.7 times higher than the negative isolates.ConclusionThere is high homology in carbapenem-resistant isolates and the mechanism of drug resistance is complex including blaKPC,AmpC,ESBLs,disappearing of outer membrane channel protein.The spreading of drug resistance result from above genes.

Objective To investigate the clinical distribution and the drug resistance of carbapenemresistant Proteus mirabilis strains isolated from 2009 to 2012 ; and to study carbapenemase genotypes of the isolates.Methods A total of 15 non-repetitive carbapenem-resistant Proteus mirabilis strains were selected from 422 Proteus mirabilis strains isolated from Ningbo First Hospital during January 2009 to December 2012.The minimal inhibitory concentrations (MIC) of 6 antibacterial agents,including imipenem (IPM),meropenem (MEM),ertapenem (ETP),ciprofloxacin (CIP),amikacin (AK) and minocycline (MIN),against 15 isolates were determined by E-test.The modified Hodge test (MHT) was used to detect the carbapenemase production in isolates.The PCR assay was performed to detect drug resistance genes of blaKPC,blaNDM-1,blaGES,blaSME,blaIMI-1/NmcA and blaSHV-38.Plasmids were extracted from the blaKPC-positive strains and then transformed into Escherichia coli J53 strains by electroporation.The transformed and untransformed Escherichia coli J53 strains were tested for MIC values and blaKPC gene by E-test and PCR respectively.Results The resistance rates of the 15 carbapenem-resistant Proteus mirabilis strains to IPM,MEM,ETP,CIP,AK and MIN were 100％,100％,100％,86.7％ (13/15),33.3％ (5/15) and 80％ (12/15),respectively.7 out of 15 strains were Hodge test positive,and 11 strains were blaKPC-2 positive.Results of PCR amplification showed that the transformed Escherichia coli J53 strains,whose MIC values to IPM,MEM,and ETP were increased by 2 to 64 times,were blaKPC-2 gene positive.Conclusion The carbapenem-resistant Proteus mirabilis strains in this study were resistant to many commonly used antibiotics,however,the resistance rates to AK were relatively low.The dominant carbapenemase genotype was blaKPC-2 carried by the plasmid.Attention should be paid to its easily transmissible feature among the strains in clinic.

A method was developed for the quantification of human growth hormone ( hGH ) by protein purification and isotope dilution-high performance liquid chromatography mass spectrometry ( HPLC-IDMS ) . The hGH was purified and fractionated by fast protein liquid chromatography ( FPLC ) , then hGH molecular weight was accurately determined by Fourier transform ion cyclotron resonance mass spectrometer ( FTICR-MS). The purified hGH was hydrolyzed and the separation was performed on an KINETEX C18 column (150 mmí2 mm I. D. , 2. 6 μm) with water ( containing 0. 1% TFA) and acetonitrile isocratic solution as the mobile phase at a flow rate of 0 . 2 mL/min and 40℃. The electrospray source was operated in the positive ion mode, and monitored in the multiple reaction monitoring ( MRM) mode. The measured hGH molecular weight by FTICR-MS was only 0. 31 Da difference from theoretical value. Three amino ( proline, valine and phenylalanine) were clearly separated by isocratic elution within 5 min. Under the optimized conditions, the content of hGH was 186 . 80 μg/g with a RSD of 0 . 5%. The detection results of hGH in international comparison by this method were consistent with the reference value, which validated the feasibility of the established method. The developed method is simple, practical, accurate, reliable and reproducible, and can be used for the hGH quantitation of pure hGH CRM to provide reference for the routine detection of hGH.

Objective To identify the effects of androgen on mouse model of autoimmune ovary failure.MethodsFifty mice were randomly divided into five groups as control group,early therapeutic group and its model group,late therapeutic group and its model group.Autoimmune POF was induced in forty mice by Pzp3.Then,the animals were randomly divided into four groups: two were treated with androgen from the 15th and 20th day after immune.The control group was treated with distilled water.The therapeutic efficacy of each treatment was evaluated by the changes in sexual cycles of mice,the serum level of AzpAb,infiltration of cells positively expressing CD45 in the ovary as well as the percentage of growing follicles of the ovary.Results The sexual cycles,serum level of AzpAb,infiltration of CD45 cells in the ovary of each model group were more significant than those in control group;early therapeutic group was less than model group;and serum level of AzpAb in early therapeutic group was lower than that of late therapeutic group.The percentage of growing follicles in the ovary of each model group was lower than that of control group;early therapeutic group was higher than model group and late therapeutic group.Conclusion The use of androgen in mice with autoimmune POF may notably ameliorate the pathogenetic conditions of the disorder,and the treatment during prophase was apparently more effective than that during the advanced stage.

Objective To explore the changes of inflammatory factors in patients with different stage of rheumatoid arthritis (RA) ,and to study the changes of immune microenvironment in patients with RA .Methods The serum levels of tumor necrosis factor(TNF) ,interleukin‐10(IL‐10) ,interleukin‐6(IL‐6) ,interleukin‐1β(IL‐1β) and interleukin‐4(IL‐4) in patients with RA on ac‐tive stage(36 cases) ,patients with RA on remitting stage and healthy individuals (30 cases)were detected by using cytometric bead array .Results The serum levels of IL‐6 ,IL‐1βand TNF in active stage RA group were higher than those in control group and re‐mitting stage RA group ,while serum levels of IL‐4 and IL‐10 were lower than those in control group and remitting stage RA group , and the differences were statistically significant (P<0 .05) .The serum levels of IL‐6 and IL‐1βin remitting stage RA group were significantly higher than those in control group(P<0 .05) .There were no significant differences in levels of IL‐4 ,IL‐10 and TNF between remitting stage RA group and control group(P>0 .05) .As the disease develops ,except for IL‐6 which tended to be stable , the serum levels of IL‐4 and IL‐10 had a rising trend ,while serum levels of IL‐1βand TNF had a downward trend with the progres‐sion of RA .Conclusion There is an imbalance between Th1 and Th2 cytokines in patients with RA on active stage ,in which Th1 cytokines are dominantly expressed .Periodic detection of inflammatory factors according to the course of RA could provide a relia‐ble basis for the assessment of disease activity .

Objective To analyze the risk factors for postoperative stricture after endoscopic submucosal dissection (ESD) for early stage esophageal cancer.Methods The data of 362 patients with early esophageal cancer treated by ESD from January 2007 to February 2012 were reviewed to investigate the risk factors of postoperative stricture.Results Esophageal stricture after ESD occurred in 42 patients (11.6％)with a mean time from ESD to stricture of (58.5 ± 12.3) days.The rates of mild,median and severe stricture were 16.7％ (7/42),38.1％ (16/42) and 45.2％ (19/42),respectively.Multivariate analysis revealed that lesion range ＞ 3/4 esophageal circumference (odds ration [OR]:44.2 ; 95％ confidence interval [CI]:4.4-443.6) and tumor invasion beyond m2 (OR:14.2; 95 ％ CI:2.7-74.2) were independent risk factors.Stricture level was related to lesion's circumferential extension (relational coefficient (φ) =0.47,P ＜ 0.05) and tumor invasion depth (relational coefficient (φ) =0.647,P ＜ 0.05).Conclusion Circumferential extension and invasion depth of early esophageal cancer were independent risk factors for post-ESD esophageal stricture and related with the degree of stricture.

ObjectiveTo investigate the managements of complications during and after peroral endoscopic myotomy (POEM) for esophageal achalasia (EA).MethodsData of 119 EA patients who underwent POEM from October 2010 to July 2011 were collected.Complications during and after POEM and during follow-up were analyzed.Results Complications during operation included bleeding in 19 patients ( 16.0％ ),mucosa rupture in 9 (7.6％),mediastinal and subcutaneous emphysema in 27 (22.7％ ) and pneumothorax in 3 (2.5％ ).Complications occurred at the night of procedure included pain in 43 (36.1％ ) patients and pneumothorax in 7 ( 5.9％ ).Postoperative complications included asymptomatic pneumothorax in 23 patients (19.3％),delayed hemorrhage in 1(0.8％),pleural effusion in 58 (48.7％ ),minor pulmonary inflammation or segmental atelectasis in 59 (49.6％),emphysema of mediasti-na and subcutaneous tissue in 76 (63.9％ ),and gas under diaphragm or aeroperitoneum in 47 (39.5％ ).Complications during follow-up included one case of dysphagia caused by stricture of mucosa and one case of dehiscence at the tunnel entry with food retention.No POEM-correlated death occurred.All the complications were cured by conservative treatments.No additional surgery was needed.ConclusionMain complications as emphysema of mediastina and subcutaneous tissue,pneumothorax,aeroperitoneum and bleeding during and after POEM can be treated timely and effectively with conservative treatment.

ObjectiveTo evaluate a second endoscopy for prevention of delayed bleeding after ESD.MethodsData of 67 patients with gastric epithelial neoplasms undergoing ESD from May to November 2011 were reviewed.The median age was 63 ( 31 ～ 84) years.All patients were followed up by endoscopy on the first and the third day after ESD.ResultsOf 67 lesions,5 were located at cardia,6 at gastric body,3 at fundus,35 at antrum,16 at gastric angle,and 2 at residual stomach.The mean maximum diameter of the lesions was 3.73±4 1.24 (2.0 ～ 7.0) cm.There were no intraoperative complications.Post-ESD delayed bleeding was detected by endoscopy in 6 (9.0％ ) patients,with 5 on the third day and 1 on the fourth day.Forrest grading showed 2 cases of Ⅰ b,and 4 of Ⅱ b.All 6 cases were cured by endoscopy.The incidence of postoperative bleeding was far more than that evaluated based on the patients' clinical manifestations only.But therapeutic effect and saffety were the same according to the follow-up results.ConclusionIncidence of post-ESD bleeding is high,but there are no symptoms or severe consequences,so a second endoscopy after gastric ESD may contribute little to the prevention of delayed bleeding.

Objective To explore the clinical value of endoscopic treatment by summarizing the experience of endoscopic treatment of duodenal submucosal tumor (SMT).Methods The data of SMT patients with endoscopic treatment from May 2006 to May 2011 at Endoscopy Center of Zhongshan Hospital Fudan University were studied retrospectively,including the lesions characters,the procedure of performance,complications and recurrence after the treatment.Results A total of 67patients received 69 times of endoscopic treatment,including 36 males and 31 females.The median agewas 55 years,and the average maximum diameter of the lesions was (1.34±0.50) cm.Of these 69lesions,38 lesions located at the bulb,12 at the ball and descending junction,and 19 at the descending part.All lesions were treated by endoscopic treatment successfully.Eleven leisions were treated by polypectomy,12 by endoscopic mucosal resection (EMR),45 by endoscopic submucosal dissection (ESD) and 1 by nylon rope.Complication rate was 14.5％ (10/69),including 1 case of active bleeding,2 cases of perforation,3 cases of delayed bleeding,3 cases of transient increase in amylase level and 1 case of delayed perforation.Total 67 cases were with pathological diagnosis,and brunner′s glands adenoma (36 cases) was the most common.Sixty patients in all were followed up,and the median follow-up time was 13 months.After ESD,one carciniod case was indicated tumor cells in vascular by the pathologic diagnosis and then received extended resection.One case of brunner′s glands adenoma recurred 1 year after EMR and improved after ESD.Conclusion Endoscopic treatment is safe,minimally invasive and effective,which may be used of duodenal SMT.

Objective To investigate the effects of Annexin-A1 ( Anxa1 ) gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms.Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells.The efficiency of siRNA-in-duced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay.MTT assay was performed to measure the proliferation of BV-2 cells with si-lenced expression of Anxa1 gene.Flow cytometry with Annexin V-FITC/PI double staining was used to de-tect the apoptosis rate of BV-2 cells.Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells.Western blot assay was performed to detect the expres-sion of signaling proteins related to cell cycle and migration.Results Compared with the siRNA negative control ( siRNA-NC) group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [(16.9 ±2.1)%, (23.1±3.6)%and (42.4±1.7)%vs (1.35±0.5)%, (2.06±0.7)% and (8.65±0.9)%, P<0.05 ].The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was (18.4±2.1)%, which was significantly elevated as compared with that of the siRNA-NC group (5.2±0.3)%and control group (4.3±0.2)%.Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as com-pared with that of the siRNA-NC group (28.7±5.2 vs 173.4±11.4, P<0.01).Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells.Conclusion The growth and migration of BV-2 cells were signifi-cantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and the activation of p38 and JNK signaling pathways.