Objective To investigate the correlation between human parvovirus (HPV) B19 and spontaneous abortion.Methods Participants included 30 cases of spontaneous abortion patients (patients group) and 30 normal pregnancy (control group).The potential risk of spontaneous abortion factors were recorded.Extracting all subjects cubits venous blood,HPV B19 DNA was tested by nested polymerase chain reaction,and HPV B19 IgM antibodies were determined by ELISA.The more factors analysis were performed by Logistic multiple regression analysis.Results There were significant differences in gravidity more than 2 times,combined with hypertension,combined with diabetes,HPV B19 DNA positive expression between patients group and control group [43.3％(13/30) vs.13.3％(4/30),33.3％(10/30) vs.6.7％(2/30),30.0％ (9/30) vs.6.7％ (2/30),36.7％ ( 11/30 ) vs.3.3％ ( 1/30)] (P ＜ 0.05 ).Logistic multiple regression analysis showed that gravidity more than 2 times,combined with hypertension,HPV B19 DNA positive expression were the independent risk factors of spontaneous abortion (OR =1.85,1.95,4.85,P ＜ 0.05).There was significant difference in the risk between early spontaneous abortion and lately spontaneous abortion in HPV B19 DNA positive expression patients (P ＜ 0.05 ).Conclusions HPV B19 infection is the natural abortion independent risk factors,need to early detection of pregnancy,and carry out necessary intervention in pregnant women,in order to reduce the occurrence of natural abortion.

Objective:To investigate the clinical effects of levonorgestrel-releasing intrauterine device(LNG-IUD) in the treatment of adenomyosis in perimenopausal period. Methods: A total of 92 patients with adenomyosis in perim enopausal period were treated with LNG-IUD and followed up at the 3rd,6th, 12th,24th month. Change of menses, uterine volume,visual analogue scale (VAS) of dysmenorrhea,the serum CA_(125) level and degree of satisfaction with the treatment were observed. Results:The dysmenorrhea was alleviated along with the follow-up time in all of the pa tients after insertion of the LNG-IUD ( P<0.05). The menstrual volume, uterine volume and serum CA_(125) level were decreased significantly(P<0.05) .Conclusions:LNG-IUD is a safe, effective and simple treatment of adenomyosis in perimenopausal period.

Objective To assess the predictive value of neutrophil gelatinase associated protein lipocalin (uNGAL) in urine for detection of acute kidney injury(AKI) in patients with severe traumatic brain injury. Methods Patients with severe traumatic brain injury from the ICU were collected from Jan. 2011 to May. 2013 in our hospital. 43 cases that met the RIFLE criteria for diagnosis of AKI in the ICU within 7 days were selected as AKI group. Another 43 cases that were matched for age ,gender ,illness severity , surgery method with AKI cases ,selected as non‐AKI group. The levels of uNGAL and Scr were measured when the patients admit‐ted in the ICU with 15 min ,at 24 h ,48 h ,72 h. the sensitivity and specificity of uNGAL and Scr for diagnosis for AKI were evalua‐ted by ROC curve. Results The incidence of severe traumatic brain injury AKI was 42. 16% (43/102). The uNGAL levels in the AKI group were higher when the patient stayed in the ICU longer and no obvious in the non AKI group. When admitted to the ICU 24 h ,the level of uNGAL(720. 32 ± 684. 25)ng/mL in AKI group was significantly higher than that (421. 92 ± 351. 20)ng/mL in non AKI group. The difference was statistically significant (P< 0. 05). The levels of Scr between two groups were not statistically significant. The area under ROC curve of uNGAL and Scr were 0. 879 (95% CI :0. 807 - 0. 949) and 0. 612 (95% CI :0. 493 -0. 731). When the cutoff value of uNGAL was 180 ng/mL ,the sensitivity and specificity were 0. 890 and 0. 823 respectively. The sensitivity was superior to Scr. Conclusion uNGALis superior to Scr for early diagnosis of AKI in patients with severe traumatic brain injury and it could be used as a biomarker for early diagnosis of AKI.

Objective:To investigate the protective effects of Ghrelin on LPS-induced apoptosis of human alveolar epithelial A549 cells,along with the possible molecular mechanisms.Methods: CCK-8 assay was used to examine the cell viability of A549 treated by LPS.Apoptosis of A549 cells was measured by TUNEL.NO(Nitric oxide)production was detected by NO-specific fluorescent probe 3-Amino,4-aminomethyl-2′,7′-difluoresceindiacetate(DAF-FM DA).Western blot was also performed to examine the expressions of iNOS(inducible nitric oxide synthase),AKT,ERK,p-AKT,p-ERK and apoptotic proteins,such as Bcl-2,Bax,and cleaved caspase-3.Results: LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration-and time-dependent manners accompanied with increased Bax and cleaved caspase-3 production,coupled with decreased Bcl-2 levels.Meanwhile,LPS promoted iNOS expression and the production of NO.Ghrelin pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression.TUNEL analysis showed that Ghrelin could decrease the apoptosis induced by LPS in A549(P<0.05).Simultaneously,LPS remarkably decreased the expression of p-AKT and p-ERK in A549 cells,which was abrogated by Ghrelin pretreatment.However,Ghrelin had no significant effect on NO production induced by LPS.Conclusion: Ghrelin reduces LPS-induced apoptosis of human alveolar epithelial cells partly through activating the AKT and ERK pathway,but the level of iNOS derived NO could not be reduced.

BACKGROUND: Bone morphogenetic protein-2(BMP-2) production of targeted cells is promoted by transfection of adenoviral vectors containing gene, but there are some immune responses. Transfection with plasmid as vector holds promise.OBJECTIVE: To explore the feasibility to construct human bone morphogenetic protein-2 eukaryotic expression vector labeled with green fluorescent protein (GFP).DESIGN: Single sample observation.SETTING: Tianjin Hospital.MATERIALS: The experiment was performed at the Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2(R)-T Easy by Dingguo, China; restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.METHODS: With pcDNA3.1/CT-hBMP2 as template, hBMP2 target fragment was subcloned by PCR binding with designed specific primers. The fragment was bound with pTA2-T-easy and pSELECT-GFPzeo-MCS, separately, and transfected into DH5 α cells. pSELECT-GFPzeo-hBMP2 containing GFP was obtained after screening.MAIN OUTCOME MEASURES: hBMP2 sequence was identified by PCR; whether hBMP2 was cloned into pTA2-hBMP2 and pSELECT-GFPzeo-MCS was identified by digestion and sequencing.RESULTS: A target fragment of 1 216 bp was obtained by PCR amplification, and cloned into pTA2-T-easy and pSELECT-GFPZeo-MCS. The screening and sequencing results showed that the target fragment was 100% matched with BMP2cDNA sequence (NM-001200) from GenBank.CONCLUSION: hBMP2 eukaryotic expression vector labeled with green fluorescent protein is successfully constructed.

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector. Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase chain reaction (PCR). hBMP2 gene was inserted into pTA2-T-easy and pSELECT-GFPzeo-MCS eukaryotic expression vector, and then transferred into competence DHSα cells. After screening, pSELEC-GFPzeo-hBMP2 was obtained and identified by sequence analysis. The recombinant vector pSELECT-GFP zeo-rhBMP2 was transfected into CHO cells. The successful trasfection was verified by fluorescence microscope in 48-72 hours. The RT-PCR and immunofluorescence was used to confirm the hBMP2 expression. Western Blotting was used to detect the secretion of hBMP2.Results A 1216 bp fragment was obtained by PCR, the same as expectant fragment. The recombined pSE-LECT-GFPzeo-hBMP2 eukaryotic expression vector was identified by restriction mapping and sequence analysis. The results were identical with that of reported hBMP2 sequence (Genebank NM-001200). The successful transfection was verified by fluorescence microscope in 48-72 hours. The stable expression in eukaryotic cells was confirmed by immunofluorescence and RT-PCR which showed an obvious band between 1000-2000 bp. Western Blotting identified the immunogenicity of recombinant human BMP2 with the molecular weight of about 17×103. Conclusion The pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was constructed successfully.

Objective To elucidate the function way of micro RNA(miR)-155 in the differentiation of Th17 cells.Methods CD4+T cells were separated from mice spleens using MACS CD4+T cells separatinge kit and cultured with interleukins [interleukin (IL)-2, IL-23 and IL-6] which could induce CD4+ T cells differentiate into Th17 cells.IL-17 was detected by flow cytometry and enzyme linked immunosorbent assay (ELISA) after transfected with miR-155 mimics or inhibitor lentiviral vectors.The expression levels of miR-155, IL-17A mRNA and Ets-1 mRNA were detected using fluorescent quantitation real-time quantitative polymerase chain reaction (RT-PCR).The si-Ets and miR-155 co-function for Th17 differentiation was analyzed.Data analysis was perfoemed using one-way analysis of variance (ANOVA) test and Dunnett test for pair-wise comparison and t test.P＜0.05 was considered to be statistically significant.Results The CD4+T cells were divided into four groups (the untreated control untreat group, the treatment control treat group, the miR-155 mimnics group and miR-155 inhibitor group).IL-17 was scarcely expressed and secreted in the untreated control untreat group.The cells expression of IL-17 were significantly different among the four groups (F=160.549, P＜0.01).The cells expressing of IL-17 were higher in the miR-155 mimics group (39.86±4.62)％ than those at the miR-155 inhibitor group (22.02±2.81)％, P＜0.01) and in the treated control treat group [(19.44±1.49)％, P＜0.01].The level of IL-17 was also significantly different among the four groups (F=260.813, P＜0.01).The level of IL-17 was higher in the miR-155 mimics group [(1 509±136) pg/ml] than that in the miR-155 inhibitor group [(923± 42) pg/ml, P＜0.01);and in the treated control group [(767±94) pg/ml, P＜0.01).The expression of miR-155 (12.53±0.80 vs 1.78±0.14, 7.16±0.62, 6.47±0.92, P＜0.01) and IL-17A mRNA (46.55±6.71 vs 1.01±0.19,15.62±1.26, 14.20±2.73, P＜0.01) was significantly higher than that in the other three groups, while the expression of Ets-1 mRNA was significantly lower (0.66±0.10 vs 1.19±0.04, 1.01±0.16, 1.37±0.27, P＜0.01).si-Ets-2 was screened because it markedly inhibited the expression of Ets-1 mRNA among the three designed siRNAs.The expression of IL-17A mRNA was higher (17.19±3.58 vs 10.08±0.76, t=-3.361, P=0.028) and the expression of Ets-1 mRNA was lower (0.27±0.01 vs 0.74±0.03, t=-30.275, P＜0.01) in si-Ets-2 group than that in si-Con group when si-Ets-2 or si-Con was co-transfected with miR-155 mimics or inhibitor lentiviral vectors.The expression of Ets-1 protein was lower in si-Ets-2 group than that in si-Con group by Western blotting and the decrease was markedly obvious in the miR-155 mimics group.Conclusion miR-155 can induce CD4+T cells to differentiate into Th17 cells by inhibiting the gene expression of Ets-1.

Objective To compare the predictive value of 5 scoring systems for hemorrhagic transformation risk after intravenous thrombolysis in patients with acute ischemic stroke (AIS) in different therapeutic windows.Methods A single-center and retrospective study was performed for 243 AIS patients who underwent intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) in different therapeutic windows in our department during January 2014 and December 2016.Five scoring systems,including HAT model (hemorrhage after thrombolysis),MSS model (multicenter stoker survey),GRASPS model (glucose at presentation,race,age,sex,systolic blood pressure at presentation,severity of stroke at presentation),SEDAN model (baseline blood sugar,early infarct signs,hyperdense cerebral artery sign on admission CT,age,NIHSS on admission),and SITS model (safe implementation of thrombolysis in strokemonitoring study) were used to evaluate the risks for hemorrhagic transformation.The relationships between the 5 scoring systems and incidence rate of hemorrhagic transformation were analyzed among the patients in different therapeutic windows.The predictive values of the 5 scoring systems were compared using the areas (AUC) under the receiver operating characteristic (ROC) curve.Results When the AIS patients were treated by intravenous thrombolysis within 3 h,the AUC of GRASPS and HAT models were 0.698 and 0.619,respectively,higher than those of the other 3 systems.When the therapeutic window was between 3 to 4.5 h,HAT model and SEDAN model had highest AUC (0.719,0.744) than the other 3 systems (P ＜0.05).When the windows were ＞4.5 ～6 h,the HAT model had the highest AUC (0.676).Conclusion The 5 scoring systems show better predictive value for hemorrhagic transformation after intravenous thrombolysis.For the therapeutic window within 4.5 h,HAT model presents best predictive value than the other 4 scoring systems.

Objective To investigate the effects of sperm DNA fragmentation (SDF)and sperm morphology on the fertilization and embryo development in ICSI.Methods SDF and sperm morphology were detected in the meanwhile of taking eggs in 1 45 ICSI treatment cycle.On the basis of SDF index (DFI)divided into group A (DFI≤30%)and group B (DFI >30%).According to the normal sperm morphology divided into group C (NMSR≥4%), group D (1 %≤NMSR <4%),group E (NMSR <1 %).According to the sperm DFI and NMSR divided into group F (DFI≤30% and NMSR≥4%)and group G (DFI >30% and NMSR <4%).Statistically analyzed ICSI outcome of research group:fertilization rate,embryo utilization rate,good quality embryo rate,implantation rate and clinical pregnancy rate.Results (1 )The normal fertilization rate,embryos utilization rate,good quality embryo rate,implan-tation rate in group A were significantly higher than those in group B (χ2 =6.96,8.95,5.49,3.92,all P <0.05), but clinical pregnancy rate had no significant difference (χ2 =1 .08,P >0.05).(2)The normal fertilization rate was statistically significant in group C,group D,group E (χ2 =34.5,65.8,11 .8,all P <0.05),but there were no significant differences in embryo utilization rate,good quality embryo rate,implantation rate and clinical pregnancy rate (P >0.05).(3 )The normal fertilization rate,embryo utilization rate,good quality embryo rate,implantation rate and clinical pregnancy rate in group F were significantly higher than those in group G,and normal fertilization rate,embryos utilization rate,good quality embryo rate had statistically significant differences (χ2 =37.5,1 1 .0,4.3,all P <0.05). Conclusion Sperm abnormal morphology has negative effect on fertilization,and the high DNA fragments have negative effects on fertilization and embryo development.

Objective To investigate the treatment outcome of modified intracytoplasmic sperm injection (ICSI)using zona pellucida(ZP)-bound sperm.Methods 82 patients with less,weak,abnormal sperm disease who were conformed to ICSI,were divided into traditional ICSI group and the group of modified ICSI using ZP-bound sperm according to ICSI case number.The results of normal fertilization rate,cleavage rate,high-quality embryo rate, planting rate,clinical pregnancy rate and early abortion rate were compared.Results The women's age,the sterility year,mature egg rate,normal fertilization rate,cleavage rate in the two groups had no statistically significant differ-ences (all P>0.05).The planting rate and clinical pregnancy rate of the observation group (46.6%,63.3%)were higher than those of the control group(38.5%,53.6%),but there were no statistically significant differences(all P>0.05).The using embryo rate and high-quality embryo rate of the observation group (73.9%,51.0%)were signifi-cantly higher than those of control group(65.8%,38.6%),the differences were statistically significant(χ2 =5.84,χ2 =11.6,all P<0.05).Conclusion Modified ICSI using ZP-bound sperm can effectively improve the embryos quality in ICSI.