Objective To study the isolation method and the culture method of primary human endometrial cells and to iden‐tify the purity and biological activity .Methods We digested human endometrial tissue by collagenase at first ,and then to separate , purify and culture the human glandular stromal cells(ESC) and endometrial epithelial cells(EEC)by differential centrifugation ,dab‐ble screen filtration and adhesion purification technology .Ultimately ,we identified the isolated cells with cytokeratin and vimentin immunocytochemical staining and immunofluorescence method .Results Stromal cells showed a parallel growth .The cells were spindle or polygonal ,and the vimentin antibody showed a positive immunohistochemical staining ,the purity was more than 95% .At the same time ,glandular epithelial cells grew in whorls .The cells were polygonal or tadpole shaped ,and the cytokeratin antibody immunohistochemical staining ,the purity were up to 90% .Conclusion The successful isolation and culture of high quantity ,viabili‐ty and purity by collagenase digestion and dabble screen filter method of endometriall cells make a strong operability .The laboratory which has the basic cell culture conditions can develop the experiment .

The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.

Objective To evaluate the application of scenario-based training and medical simulator in the orthopedics education. Methods A total of 60 students from Shanghai Jiao Tong University School of Medicine who finished their orthopedics internship from January 2016 to July 2016 were involved. They were randomly divided into the study group and control group with 30 students each. The study group re-ceived 2 classes of scenario-based training and medical simulator assisted education during their internship in the orthopedics department while the control group received 2 classes of traditional lessons instead. Sur-veys were conducted after the internship and the scores of internship were also recorded. Result The overall satisfaction was higher in the study group than the control group [(8.6±0.6) vs. (8.1±0.5), P=0.001]. On the part of learning interest, clinical thinking, clinical practice and group working, the study group also received better evaluation (P<0.05). The study group achieved better scores in the final examination than the control group [(84.4±2.6) vs. (82.5±3.4), P=0.018]. Conclusion The combination of scenario-based training and medical simulator can improve the ability of medical students in the orthopedics education, and receive higher satisfaction.

Objective To explore the associations of the genetic polymorphisms of cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) and cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9) with early-onset severe pre-eclampsia and the efficacy of labetalol therapy. Methods Totally 105 gravidas diagnosed with early-onset severe pre-eclampsia (experimental group) and 103 healthy gravidas (control group) were recruited from Beijing Obstetrics and Gynecology Hospital between August 2013 and July 2016. Labetalol was given to control blood pressures in gravidas with early-onset severe pre-eclampsia. If labetalol administration alone did not exceed the mean dose (100 mg, one dose per eight hours) and effectively controlled the blood pressures, it would be considered to be valid (n=75), otherwise it would be viewed as an invalid treatment. Genotype and allele frequencies of CYP2C9 gene (rs1057910 and rs4918758) and CYP2D6 gene (rs1065852, rs28371725, rs35742686 and rs3892097) in the gravidas were analyzed by TaqMan probe polymerase chain reaction. Differences in the genotype and allele frequencies were compared between the experimental and control groups, and the valid and invalid labetalol treatment groups. Chi-square test, analysis of variance and LSD test were used as statistical methods. Results The gravidas in both experimental and control groups were AA genotype in CYP2C9 gene rs1057910, TT genotype in CYP2D6 gene rs35742686 and CC genotype in CYP2D6 gene rs3892097. Frequencies of CC and CT genotypes in CYP2D6 gene rs28371725 in the experimental group were higher than those in the control group [18.1% (19/105) vs 14.6% (15/103);56.2% (59/105) vs 42.7% (44/103); χ2=6.707], and higher C allele frequency in CYP2D6 gene rs28371725 was also observed in the experimental group [46.2% (97/210) vs 35.9% (74/206), χ2=4.529] (all P<0.05). No statistical differences in maternal age, diastolic pressure, body mass index before pregnancy, serum triglyceride, creatinine and neonatal birth weight were observed among women with CC, CT or TT genotype of CYP2D6 gene rs28371725 in the experimental group (all P>0.05). Compared with the gravidas with CT or TT genotype of CYP2D6 gene rs28371725, those with CC genotype had longer gestational age [(32.5±2.1) vs (29.5±1.8) and (29.8±2.2) weeks] and higher plasma albumin [(27.2±9.3) vs (20.3±10.4) and (22.5±7.4) g/L], but lower systolic pressure and 24 hours urine protein (LSD test, all P<0.05). The G allele frequency in CYP2D6 gene rs1065852 in invalid labetalol treatment group was higher than that in valid labetalol treatment group [93.3% (56/60) vs 76.0% (114/150), χ2=8.351, P=0.004]. Conclusions The polymorphism of CYP2D6 gene rs28371725 may be associated with early-onset severe pre-eclampsia, and the allele of G in CYP2D6 gene rs1065852 may be associated with the efficacy of labetalol in treatment of early-onset severe pre-eclampsia.

Medical simulation training as a modern medical education has been used for decades in developed countries. Simulation based team training for obstetric emergency provides opportunities to re-hearse without risks to patients and helps overcome some limitations of the current medical education to improve the ability of medical staff to deal with obstetric emergencies, and improve adverse pregnancy out-comes. Simulation training is still at early stage in China. The Simulation Training Center of Beijing Ob-stetrics&Gynecology Hospital was established in June 2013. There are 100 medical staffs from our hospital and 406 from other hospitals who are trained using simulations to respond to acute obstetrical emergencies, including postpartum hemorrhage, neonatal recovery, shoulder dystocia and amniotic fluid embolism. Through introduction—on-scene simulation training—task report, we focus on learning, practical operation, emer-gency team and multidisciplinary coordination and communication skills training and through training, help learners understand the medical simulation training mode, and improve personal and team's ability to deal with the obstetric emergency. There is no doubt that simulation training will be further developed in China. Further research is required to investigate the application significance and feasibility of simulation training and access effectiveness of it.

Objective To investigate the performance of MicroScan WalkAway 96 Plus (MSW) system in detection of carbapenem-resistant Enterobacteriaceae (CRE).Methods A total of 81 stock CRE strains were used in this study. Bacterial identification and antimicrobial susceptibility test were performed by MSW system. Beta-lactamases genes blaKPC,blaIMP,blaVIM, blaOXA-48 and blaNDM were amplified by PCR and subjected to sequencing analysis. Disk diffusion method and PCR were used as gold standard to evaluate the performance and reliability of MSW system in identifying carbapenem-resistant and carbapenemase-producing Enterobacteriaceae.Results Overall, 69.1 % (56/81) of the Enterobacteriaceae strains were identified as CRE by the MSW system. The results of PCR showed that 48 strains were carbapenemase-producing Enterobacteriaceae. When carbapenemase-producing Enterobacteriaceae strains were identified by the instrument using an advanced expert system, the sensitivity was 93.8 % and specificity was 42.4 %. The positive predictive value was 70.3 %, the negative predictive value was 82.4 % and the predictive accuracy value was 72.8 %.Conclusions The MicroScan WalkAway 96 Plus system has shown good performance in detection of CRE.

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.

ObjectiveTo evaluate the clinical value of prenatal ultrasonography combined with maternal serology screening for chromosomal abnormality in 15 to 20+6 gestational weeks.MethodsSix hundred and twenty-eight pregnant women (628 fetuses) in 15 to 20+6 gestational weeks were selected to undergo prenatal ultrasonography, who were in critical risk of trisomy 21 or trisomy 18 by maternal serology screening. Transabdominal ultrasonography were performed and fetal nasal bone and nuchal fold were detected. Those who had nasal bone hypoplasia, thickened nuchal fold (NF>6 mm) and other abnormal fetal soft markers underwent amniocentesis for karyotyping analysis. ResultsThere were 6 cases of nasal bone hypoplasia (0.96%, 6/628), including one case of thickened nuchal fold, two cases of echogenic bowel, two cases of choroid plexus cysts and 1 case of echogenic cardiac focus. All these 6 cases underwent amniocentesis and 2 were trisomy 21 (33.3%, 2/6). The other 4 cases had no significant chromosomal abnormality.ConclusionsPrenatal ultrasonography may improve the detection rate of chromosomal abnormality for those pregnant women who are in critical risk of chromosomal abnormality prompted by serology screening. But invasive procedures are still needed to verify the chromosomal abnormality.

Objective To investigate the dynamic changes of uterine artery and umbilical artery in the first, second, and third trimester of normal pregnancy and hypertensive disorders in pregnancy (HDP).Methods A multi-center prospective study was conducted on 1098 women with normal singleton pregnancies at the first prenatal visit in the Second West China Hospital of Sichuan University, First Affiliated Hospital with Nanjing Medical University, Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University, Wuhan Union Hospital Affiliated to Medical School of Huazhong University of Science and Technology and Renji Hospital Affiliated to Medical School of Shanghai Jiao Tong University from April 2005 to July 2006, with the average age of (28. 3±3.3). The pulsatility indices (PI),resistance indices (RI) and systolic to diastolic velocity ratios (S/D) of uterine artery and umbilical artery were measured for all subjects in the first (10th -14th gestationa] weeks), second (20th-26th gestational weeks) and third trimester(30th -36th gestational weeks), respectively. In this longitudinal study, women who developed HDP were classified into HDP group, and the rest into normal pregnancy group. Results (1) Among the 1098 pregnant women, 44 developed HDP during the index pregnancy, including 20 gestational hypertension, 15 mild pre-eclampsia and 9 severe pre-eclampsia, giving an incidence of 4.17%,and the rest 1054 were normal until delivery.(2) In the normal pregnancy group, the RI, PI and S/D of uterine artery were decreased with the progress of pregnancy (RI: 0. 64, 0. 57, 0. 50; PI: 1.24, 0. 98,0. 80; S/D: 3.26, 2. 58, 2. 20; P<0.01). However, the above indices showed an increasing trend with the increase of gestations in the HDP group (RI: 0. 55, 0. 67, 0. 64; PI: 1.22, 1.36, 1.20; S/D: 3.18,3.41,3.05; P <0. 01). In the second and third trimester, the RI, PI and S/D of uterine artery in the HDP group were higher than those in the normal pregnancy group (P<0.01). (3) In the normal pregnancy group, the RI, PI and S/D of the umbilical artery decreased from the secondto the third trimester (RI:0. 71 and 0. 58; PI: 1.16 and 0. 87; S/D: 3.58 and 2. 48; P<0.01). However, no significant difference was found in the RI, PI and S/D value of umbilical artery in the second and third trimester between the normal and HDP group (RI:0.71 and 0.63; PI: 1.20 and 0.95; S/D: 3.71 and 2.69; P>0.05,respectively), despite the decreasing trend in the HDP group. Conclusions In uncomplicated pregnancies,the blood flow resistance of uterine artery decreases and the end-diastolic blood flow of uterine artery increases with the progress of pregnancy. However, in pregnant women with HDP, the blood flow resistance of uterine artery increases significantly with the increase of gestations which shows significant difference to that in normal pregnancies. The blood flow resistance of umbilical artery decreases in both normal and HDP pregnant women with the increasing gestational age.