Objective To clone and study the antimicrobial-resistant gene in Neisseria gonorrhoeae.Methods The gene library,which contains the differential genes of antimicrobial resistant strains and stan-dard reference strains of Neisseria gonorrhoeae,was constructed using a technique known as suppression subtractive hybridization(SSH).Then the antimicrobial resistance associated genes were cloned and ana-lyzed.Results Subtractive gene library in antimicrobial-resistant Neisseria gonorrhoeae was successfully constructed,which contains2500positive clones.Sequence analysis was performed on5clones.The se-quences of these five clones were unknown previously.Conclusions The subtractive DNA library is succes-sively constructed which may provide an important clue for studying the mechanism of antimicrobial resis-tance in Neisseria gonorrhoeae.

Objective To study the role of the outer membrane protein Rmp of Neisseria gonor-rhoeae strain in immunosuppression and the strategy of eliminating it .Methods The rmp gene of Neisseria gonorrhoeae strain was amplified by PCR and inserted into pMD 19-T vector .The recombinant vector pMD 19△rmp∷Kan containing Kan and the 5′-and 3′-flanking regions of rmp (△rmp∷Kan) was constructed by replacing 200 nucleotide residues of pMD 19-rmp with kanamycin resistance gene Kan and transformed into Neisseria gonorrhoeae WHO-A strain.PCR and Western blot assay were used to screen and identify the re-combinant mutant strains that could not express Rmp .Mice were immunized with mutant strains and bacteri-cidal activities of the immune sera were detected by antibody-mediated complement-dependent cytotoxicity assay.Results The mutant strains that could not encode Rmp were successfully constructed .Antibodies in-duced by mutant strains showed stronger bactericidal activity against Neisseria gonorrhoeae in comparison with those induced by wild strains .Conclusion The recombinant Neisseria gonorrhoeae strain with rmp gene de-letion might eliminate the immunosuppressive effects of Rmp expressed in wild gonococcal strains , which provides a reference for further development of novel live attenuated whole-cell vaccines of Neisseria gonor-rhoeae.

OBJECTIVETo develop a LC-MS/MS method for determination of astragalin in rat plasma and study its pharmacokinetics in rats.

METHODSThe analytical column was packed with ZORBAX SB-C18, and the mobile phase was methanol-water containing 10 mmol/L ammonium acetate-formic acid (80:20:0.15, V/V/V). Quercetin was used as the internal standard (IS). Multiple reaction monitoring (MRM) mode was employed, and the transition of m/z was m/z 449.1→m/z 287.1 for astragalin, and m/z 301.1→m/z 151.1 for IS.

RESULTSA good liner relationship was obtained within the range of 1.00-1000 ng/ml (r(2)=0.9929), and the lower limit of quantification of astragalin was 1.00 ng/ml in rat plasma. The extraction recoveries were all above 93%. After oral administration of astragalin, the maximum plasma concentration of astragalin was 231.1∓67.3 ng/ml and the time to reach this value was 0.5∓0.1 h, with a half-life of 3.9∓1.3 h and an AUC of 782.6∓152.8 ng·h/ml.

CONCLUSIONThe method is highly sensitive, selective and rapid for determination of the concentration of astragalin in rat plasma to facilitates the study of its pharmacokinetics.

Objective To evaluate the efficacy of pegaspargase (PEG-ASP) combined with GEMOX regimen for the treatment of extranodal natural killer (NK) / T-cell lymphoma (ENKL),and to observe the changes of coagulation function.Methods 35 patients with histologically confirmed ENKL were enrolled from January 2010 to December 2014.All patients received 180 cycles of PEG-ASP combined with GEMOX chemotherapy and the efficacies were observed.The coagulation items such as prothrombin time (PT),activated partial thromboplastin time (APTT),fibrinogen (Fbg) and international normalized ratio (INR) were tested respectively on day 1st,day 8th and day 14th of every treatment cycle.Results Among 35 patients,11 patients (31.43 ％) were in stage Ⅰ-Ⅱ,and 24 patients (68.57 ％) were in stage Ⅲ-Ⅳ.All patients were subjected to 180 cycles of PEG-ASP combined with GEMOX chemotherapy,and each case was estimated to receive 6 cycles.The overall response (CR+PR) rate (ORR) was 71.43 ％ (25/35),the ORR was 81.82 ％ (9/11) in stage Ⅰ-Ⅱ group,and 66.67 ％ (16/24) in stage Ⅲ-Ⅳ group.The increased PT and APTT and decreased Fbg were observed on day 8th of the chemotherapy.The increased APTT and decreased Fbg were still observed on day 14th of the chemotherapy.Compared the data of patients one day before chemotherapy with healthy persons,the changes had statistical significance (P ＜ 0.05).Conclusions PEG-ASP combined with GEMOX regimen in the treatment of ENKL is safer and more effective compared with traditional chemotherapy,but the abnormal alternations of coagulation might be common during therapy.Dealing with the bleeding risk and supplement with plasma,PPSB or Fbg in time should be necessary.

Objective To explore the significance of the suppressor of cytokine signaling-1 (SOCS-1)gene methylation in the genesis, development and prognosis of diffuse large B-cell lymphoma (DLBCL).Methods The methylation state of CpG island in SOCS-1 gene were detected by methylation-specific polymerase chain reaction (PCR) and the level of SOCS-1 gene was measured by the real-time PCR. The clinical data of 30 patients with DLBCL were collected, and according to the international prognosis index (IPI), they were divided into low risk group and high risk group. Results Aberrant methylation of SOCS-1 in 17 DLBCL patients (56.7 %) were positive, however, in control group aberrant methylation was negative(P ＜0.01).The methylation level of DLBCL patients with positive SOCS-1 methylation was higher than that of patients with negative (P ＜0.05). Combined with the clinical data, the positive rate of methylation in patients with high level of serum LDH or the numbers of extra-nodal lesions＞l were significantly higher than that in patients with normal LDH level or the numbers of extra-nodal lesions ≤ 1, respectively. Hence, the positive rate of methylation in the high risk group of DLBCL was higher than that in the low risk group (P ＜0.05). Conclusion There were aberrant methylations of the SOCS-1 gene in the patients with DLBCL. The methylations of SOCS-1 can silence the gene expression, which indicates that SOCS-1 and its methylations play some role on genesis and development of DLBCL and can evaluate the prognosis of the patients with DLBCL.

Objective To establish a mouse model of bacterial vaginosis ( BV) by infecting estro-gen-treated mice with Prevotella bivia ( P. bivia) . Methods The mice were intraperitoneally injected with beta-estradiol 17 valerate which was suspended in sesame oil and then inoculated with different doses of P. bivia strains at the logarithmic phase. Samples of vaginal flushing fluid were collected at different time points after inoculation and used for the isolation of P. bivia strains and the detection of sialidase activities. Altogether 30 mice treated with estrogen and high dose of P. bivia were killed on days 2, 7 and 21 (n=10). Samples of cornua uteri, bladder and kidney were collected from those mice for P. bivia strains isolation. Re-sults Injection the estrogen-treated mice with P. bivia via vagina could cause the P. bivia infection for more than 14 days. The numbers of P. bivia strains isolated on day 21 decreased significantly. Enhanced sialidase activities and clue cells were observed in vaginal secretions of mice with P. bivia infection. Injection of mice with the high dose of P. bivia could spread the infection to cornua uteri. Conclusion Estrogen-treated mice could be used as an animal model for researches on BV.

Objective To assess the effect of ultrasound-guided percutaneous 125I seed implantation for the treatment of osteolytic metastases. Methods From February 2011 to December 2013, a total of 18 patients with advanced cancer complicated by osteolytic metastases received ultrasound-guided percutaneous implantation of 125I seeds. According to visual analog score (VAS) the pain was estimated before and 3 days as well as one month after the treatment. One month after the treatment follow-up CT scan was performed in all patients to check the tumor size and the distribution of 125I seeds, and the therapy was repeated if necessary. The efficacy was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). Results A total of 33 times of procedure were carried out in the 18 patients. Three days after the treatment, local pain was obviously relieved in all cases. Compared with preoperative VAS, the postoperative VAS was significantly improved (P=0.000). The pain was well controlled even one month after the treatment (P=0.000). One month after the last treatment the complete remission (CR) rate was 0% (n=0), partial remission (PR) rate 61.1% (n=11), stable disease (SD) 33.3% (n=6) and progress disease (PD) 5.6% (n=1), and local control rate (CR+PR) was 61.1%. No severe brachytheray-related complications occurred. Conclusion Ultrasound-guided percutaneous 125I seed implantation brachytherapy is technically simple and repeatable, it can safely and effectively guide the performance of percutaneous 125I seed implantation for the treatment of osteolytic metastases. Even in the circumstance of not using TPS, this technique can also obtain satisfactory local control rate and significant pain relief.

Objective To detect the significance of the SHP-1,p15 and SOCS-1 genes methylation status in the genesis and development of diffuse large B-cell lymphoma (DLBCL).Methods The methylation state of SHP-1,p15 and SOCS-1 genes CpG islands were measured by methylation-specific polymerase chain (MSP) reaction.Results The positive rates of aberrant methylation for SHP-1,p15 and SOCS-1 genes in 50 specimens of DLBCL were 96 ％ (48/50),52 ％ (26/50) and 56 ％ (28/50) respectively.In control group,however,15 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1,p15 and SOCS-1 genes promoter.Conclusion Aberrant methylations of the SHP-1,p15 and SOCS-1 genes exist in the patients with DLBCL.The methylations of SHP-1,p15 and SOCS-1 genes may be associated with the occurrence and development of DLBCL.This study provides a theoretical basis of treatment methylation for DLBCL.

Objective: To evaluate the clinical value of echocardiography in aortic valvuloplasty (AVP) in the low-age pediatric patients with congenital aortic valve stenosis. Methods: We retrospectively studied 39 low-age (at median of 23 months) patients with congenital aortic valve stenosis who received aortic valve repair in our hospital for their echocardiography information, and statistically analyzed the main indicator changes by 4 time points as pre-operation and 1 week, 1-3 months, 6-12 months after the operation respectively. Results: In our study, the bicuspid to tricuspid valve ratio was approximately at 5.5/1 and 2 patients died during peri-operative period. Compared with pre-operative time point, Doppler aortic valve peak velocity (Vmax ) and the mean aortic transvalvular pressure gradient (MPG) were reduced accordingly, for Vmax: (4.30 ± 0.73) m/s vs (2.65 ± 0.78) m/s, (2.93 ± 0.63) m/s, (3.01 ± 0.83) m/s,P<0.01, for MPG: (45.78 ± 15.19) mmHg vs (18.24 ± 10.08) mmHg, (21.01 ± 10.08) mmHg, (22.31 ± 13.41) mmHg. Compared with pre-operative time point, left ventricular ejection fractions (LVEF) were similar in 3 post-operative time points. Compared with 1 week post operative time point, left ventricular end-diastolic anteroposterior diameter (LVEDD) was increased at 6-12 months post-operative time point, the relative wall thickness (RWT) was decreased, bothP<0.05, and aorta valve regurgitation (AR) was increasedP<0.01. Pearson correlation analysis showed that aortic annulus (AA) inner diameter was positively related to LVEDD (r= 0.648,P<0.01), negatively related to Vmax (r= -0.205,P<0.05) and RWT was positively related to Vmax (r= 0.196,P<0.05). There were 6 patients with pre-operatively decreased LVEF, 1 of them died and the rest 5 with elevated LVEF at 6-12 months post-operative period,P<0.05. Conclusion: Echocardiograghy could be used as the ifrst choice of imaging method for diagnosing congenital aortic valve stenosis, it has the important role for in-operative monitoring and post-operative evaluation of AVP in relevant patients.

Objective To develop fast detection techniques for the diagnosis of gonococcal infections.Methods Prokaryotic expression vector for Neisserial surface protein A (NspA) was constructed using the NspA gene cloned by PCR.Mice were immunized with the renatured recombinant NspA (rrNspA)to prepare antibodies against NspA.Western blot and ELISA was used to analyze the binding of NspA antibodies to lysate of gonococcal cells or to intact gonococci.Results NspA antibodies that were prepared by the rrNspA expressed in E.coli could bind to rrNspA,the natural NspA existing in gonococcal cells,or intact gonococci.Conclusion RrNspA and its antibodies have potential value in developing fast diagnostic kits for gonococcal diseases.