Objective To establish the methods of isolating and culturing human ovarian endometriosis-derived microvascular endothelial cells (OEMEC).Methods The tissues of human endometriotic cyst of ovary were finely minced with scissors,then digested by collagenase Ⅰ,Ⅱ and trypsinethylene diamine tetraacetic acid (EDTA).The cells were purified by using centrifugation of 2000 r/min speed.OEMEC were identified by light microscope and transmission electron microscope observing CD34,FⅧ-Rag and Weibel-Palade in microvascular endothelial cells.Results The OEMEC grew as confluent monolayer like cobblestones under light microscope.CD34 and FⅧ-Rag were expressed strongly,and the percentages of CD34 and FⅧ-Rag positive cells were 91.4％ and 92.5％.Weibel-Palade bodies could be observed under transmission electron microscope.The time of cell doubling proliferation was 4.5 days.Conclusion The established system of isolating OEMEC would provide lab base for studying the mechanisms of angiogenesis in endometriosis lesions.

Objective To investigate the incidence and pregnancy outcomes of premature rupture of membranes (PROM) in pregnant women in Beijing.Methods A retrospective multicenter study of 18 534 cases delivered in Beijing Obstetrics and Gynecology Hospital,Beijing Friendship Hospital,Daxing MCH Hospital and Tongzhou MCH Hospital from January 2011 to December 2011,was conducted.Results Among 18 534 cases,PROM occurred in 4 504 cases (24.30％),including 3 910 cases of in term PROM (21.10％) and 594 cases of preterm PROM (3.20％).The incidence of premature delivery was 6.17％ (1 144/18 534),and among 1 144 cases of premature delivery 547 cases were PROM (47.81％);the incidence of PROM was 22.75％ (3 957/17 390) in term delivery.The overall cesarean section (CS) rate was 48.50％ (8 989/18 534) and that in pregnant women with PROM was 35.55％ (1 601/4 504),but the CS rate in pregnant women without PROM was 52.66％ (7 388/14 030).The rate of postpartum hemorrhage was 13.12％ (210/1 601)in CS cases and 4.17％ (121/2 903) in vaginal delivery cases (x2 =121.361,P=0.000).The mean hospital stay for PROM was (5.3±2.9) d in CS cases and (4.3±2.3) d in vaginal delivery cases (t =-12.136,P =0.000).Conclusions Without severe maternal or fetal complications,the incidence of PROM is relatively high in Beijing region and PROM may not increase the maternal or fetal complications.Vaginal delivery is the main mode of delivery for PROM.Cesarean section may not cause less neonatal complications,but may lead to more postpartum hemorrhage and longer hospital stay.

Objective To investigate the antimicrobial resistance mechanisms and genetic homogeny of Salmonella from community acquired infections in Shenzhen,China.Methods Ninety-three of Salmonella were isolated from 2002 to 2007 at Shenzhen People's Hospital,China.PCR and DNA sequencing were used to investigate the mutation in QRDR of the gyrA,gyrB,parC and parE.Plasmid mediated quinolone resistance genes including qnr and aac(6')-Ib-cr,β-lactamase genes including blaTEM,blaSHV,blaOXA, blaCTX-M, and class 1 integron were detected. All isolates were typed by PFGE. Results S. enterica typhi and S. enterica paratyphi A were susceptible to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole, ceftriaxone and ciprofloxacin, with the susceptible rate of 96%-100%. Fifty-two percent (13/25) of S. enterica typhi and 95% (61/64) of S. enterica paratyphi A were resistant to nalidixic acid. Twenty-four percent (6/25) of nalidixic acid-resistant S. enterica typhi and 94% (60/64) of nalidixic acid-resistant S. enterica paratyphi A showed decreased susceptibility to ciprofloxacin (MIC of 0. 125-1 μg/ml).All nalidixic acid-resistant (susceptible to ciprofloxacin ) Salmonella (NARS) isolates had a single substitution in the QRDR of GyrA, and 91% (68/75) of these isolates carried the substitution Ser83Phe in GyrA. Two mutations in the QRDR of GyrA were detected in both of two ciprnfloxacin-resistant Salmonella,with the additional one mutation in the QRDR of parC. Plasmid mediated quinolone resistance genes including qnr and aac(6')-lb-cr were not detected in any isolate. The blaCTX-M-14 gene was detected in a ceftriaxoneresistant isolate of S. enterica paratyphi A, with ISEcpl located on the upstream of it. Three muhidrugresistant strains of Salmonella all carried one 1 900 bp classⅠ integron gene cassette dhfrⅫ-orfF-aadA2,with the additional one β-lactamase gene of blaTEM-1, or blaOXA-30. Twenty-two distinct PFGE patterns were observed among twenty-five S. enterica typhi. The PFGE patterns of sixty-four S. enterica paratyphi A showed limited genetic diversity (average similarity of 91% ). Ninety investigated inpatients were infected in the community. Six patients infected by S. enterica paratyphi A had a travel history before infection. Conclusions Nalidixic acid-resistant S. enterica typhi and S. enterica paratyphi A are highly prevalent in Shenzhen,China. The mutation in the QRDR of GyrA is the prevalent mechanism responsible for the resistance to nalidixic acid in Slmonella. The great genetic similarity among S. enterica paratyphi A isolates indicates endemic disease from the presence of a single clone over 6-year period.

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

OBJECTIVE To investigate the prevalence of multidrug resistance(MDR) mechanisms of Staphylococcus haemolyticus against oxacillin,gentamycin and erythromycin.METHODS Agar dilution method was performed to detect the minimal inhibition concentration(MIC) of 3 antimicrobial agents against 63 strains of S.haemolyticus,and the resistance genes of mecA,aac(6′)+aph(2″),ermA,ermB,ermC and msrA/msrB were investigated by PCR in all clinical isolates.RESULTS mecA Gene was detected in 62 isolates of meticillin-resistant S.haemolyticus(MRSH),and aac(6′)+aph(2″) gene was found in 50 isolates resistant to gentamicin,and the most prevalence erythromycin resistance gene in S.haemolyticus was msrA/msrB(58.7%),followed by ermC(31.7%).Among the 43 MDR strains,the more commonly encountered three genes were mecA,aac(6′)+aph(2″) and msrA/msrB(58.1%)or ermC(20.9%),and 8 isolates(18.6%) were found harboring four genes of mecA,aac(6′)+aph(2″),ermC and msrA/msrB.CONCLUSIONS The mecA,aac(6′)+aph(2″),msrA/msrB and ermC genes are main resistance mechanisms against oxacillin,gentamicin and erythromycin in mutidrug resistant S.haemolyticus.

OBJECTIVE To investigate the susceptibilities of Staphylococcus aureus and Enterococcus isolated from Shenzhen Hospital during 2005 to 2006 to 17 antimicrobial agents.METHODS The minimal inhibitory concentrations(MICs) of 17 antibacterial agents were determined by agar dilution method,WHONET5.3 software was used to analyze the data.RESULTS The prevalence of meticillin-resistant S.aureus(MRSA) was 20.7%.Vancomycin and teicoplanin remained very high activity against MRSA(100%),with the MIC50 and MIC90 of 0.5 and 1 ?g/ml,respectively.Fluoroquinolones(ciprofloxacin,levofloxacin and gatifloxacin),trimethoprim/sulfamethoxazole,erythromycin,and clindamycin showed very low activity against MRSA(0-33.3%).The most active agent against Enterococcus faecalis was vancomycin and teicoplanin(100.0%),followed by piperacillin/tazobactam,imipenem and ampicillin(97.2-100.0%).E.faecium showed high resistance to various agents,except to vancomycin and teicoplanin(susceptible rate 100.0%).Cross-resistance to fluoroquinolones was found among MRSA,MSSA,E.faecalis,and E.faecium.CONCLUSIONS MRSA and E.faecium isolated from our hospital showed high resistance to multiple antimicrobial agents except vancomycin and teicoplanin.

OBJECTIVE To investigate the antimicrobial susceptibility of 9 antimicrobial agents and multidrug(resistance)(MDR) phenotype among Acinetobacter baumannii isolated from hospitalized patients.METHODS Disk diffusion method was used to detect the inhibitory zone diameter of 9(antimicrobial) agents to 221 strains of A.baumannii.according to standard from NCCLS in South China during 2002 to 2004.RESULTS 25.3% and 26.4% of isolates from ICU patients showed resistance to(imipemem) and meropemem,respectively,and the resistance rates were greater among the(isolates) from ICU(patients) than those from non-ICU inpatients by 13.4% and 12.2 %(P

Objective To investigate the factors affecting the vaginal birth after cesarean (VBAC). Methods Totaly 298 women who underwent trial of labor after cesarean section (TOLAC) from Jan 2015 to Dec 2015 were recruited from Beijing Obstetrics and Gynecology Hospital, FuXing Hospital, Tongzhou Maternal and Child Health Hospital of Beijing, the Second Affiliated Hospital of Chongqing Medical University and the People′s Hospital of Chengyang District of Qingdao. The maternal age, the interval from the last cesarean section, the body mass index (BMI) before pregnancy, the weight gain during pregnancy, the way into labor, the Bishop score before labor, the gestational age and the birth weight of the neonate were recorded in a self-made form. The factors affecting VBAC were analyzed by univariate analysis and multivariable logistic regression. Results (1)The incidence of VBAC, uterine rupture, postpartum hemorrhage and neonatal asphyxia were 70.5%(210/298), 2.7%(8/298), 9.4% (28/298) and 1.3% (4/298), respectively. No maternal death and perinatal death occurred. (2)The univariate analysis suggested that the maternal age, the BMI before pregnancy, the Bishop score before labor, the labor induction, the gestational age at delivery and the neonatal weight were factors affecting VBAC. The maternal age and the Bishop score before labor were significantly higher in the VBAC group than in the unsuccessful TOLAC group(P<0.05). While the BMI before pregnancy, the induction rate, the gestational weeks at delivery and the birth weight of the neonate were significantly lower in the VBAC group than in the unsuccessful TOLAC group (P<0.05). Multivariable logistic regression analysis showed that successful VBAC was affected by the maternal age, the BMI before pregnancy, the Bishop score before labor and the birth weight of the neonates(P<0.05). Conclusion The maternal age, the BMI before pregnancy, the Bishop score before labor and the birth weight of neonate are the main factors affecting VBAC.

Objective To study the epidemiological features of Cryptococcus neoformans and Cryptococcus gattii isolated from clinical samples in Shenzhen and to elucidate the distribution of species,varieties,genotypes and mating types within the strains tested.Methods The strains involved in this study were 55 cryptococcal strains isolated from our clinical samples.The canavanine-glycine bromthymolblue (CGB) culture was performed to distinguish Cryptococcus neoformans from Cryptococcus gattii.The genotype was characterized by polymerase chain reaction (PCR) fingerprinting with primer M13.The Cryptococcus gattii species and varieties of grubii and neoformans together with two opposite mating type α and a were identified by PCR with variety-specific and mating type-specific primers.The GEF1-restriction fragment length polymorphism analysis was conducted to simultaneously determine the genotype and mating types of strains tested.The sequence type of IGS1 region was analyzed for the VG Ⅱ genotype.Results Of the 55 tested cryptococcal strains,52 were Cryptococcus neoformans,all of which were var.grubii,genotype VN Ⅰ and mating type α.The remaining 3 strains were Cryptococcus gattii,among which,one was genotype VG Ⅰ and mating type α,and two were genotype VG Ⅱ and mating type α.The two VGⅡ genotype strains belonged to the sequence type Ⅱ.Conclusions The strains belonging to the Cryptococcus neoformans var.grubii,genotype VN Ⅰ and mating type α predominate in causative pathogens of cryptococcosis in Shenzhen.Cryptococcus gattii accounts for minority of the cryptococcal isolates,and the highly pathogenic VG Ⅱ genotypes in foreign countries are also characterized.The sequence types of IGS1 region of the two VG Ⅱ strains are in accord with VG Ⅱb sub-genotype.