Objective To study the relationship between tyrosine kinase receptor B (TrkB)expression and anoikis-suppression and invasion in OVCAR3 ovarian cancer ceils. Methods The expression of TrkB mRNA in OVCAR3 ovarian cancer cells under two culture conditions :adhesive cells and ceil-spheroids were evaluated by RT-PCR and real-time PCR.The relationship between TrkB expression and anoikis-suppression of OVCAR3 ovarian cancer ceils was examined by RNA interference (RNAi) technic,anchorage independent culture and fluorescence-activated ceil sorting analysis.The difference in invasion and metastatic ability of OVCAR3 cells under two culture conditions and with or without TrkB silenced by small interfering RNA (siRNA) was investigated by matrigel invasion assay and in vivo studies.Results The expression of TrkB mRNA was highest in OVCAR3 ovarian cancer ceils,0.0240 ~ 0.0017,compared with the other three cell lines,0.0030±0.0006,0.0027±0.0009 and 0.0087±0.0003 respectively,andthe expression in OVCAR3 multicellular spheroids was significantly higher than that in ceils under monolayer adhesive culture,0.0437±0.0021 versus 0.0240±0.0017 (P＜0.01) . TrkB mediated anoikissuppression in OVCAR3 ovarian cancer ceils.OVCAR3 multiceilular spheroids had a higher invasion ability than OVCAR3 cells under monolayer adhesive culture,and the penetrating cells of the two groups were 71.8± 0.8 and 47.7±0.8 respectively (P＜0.01 ).The metastatic ability of OVCAR3 cells was attenuated when TrkB was silenced,and the volume of the tumors developed by OVCAR3 adhesive cells and OVCAR3 adhesive cells with TrkB silenced were (16.3±4.7) mm3 and(6.0±1.4) mm3 respectively (P＜0.01).Conclusion As an anoikis-suppressur,TrkB may increase the invasion and metastasis of OVCAR3 ovarian cancer cells.

Objective To investigate the role of nuclear factor-kappa B(NF-κB) in preterm birth with subclinical chorioamnionitis.Methods From October 2005 to October 2006,111 cases including 36 cases of preterm birth in labor,37 cases of full term gravida with spontaneous labor and 38 cases of full term gravida without threatened labor in the Hunan Province People's Hospital,third Xiangya Hospital of Central South University and Changsha Maternal and Child Care Service Center were enrolled in the study.After delivery,by pathology results of fetal membrane they were divided into two groups:subclinical chorioamnionitis group(subclinical infectious group ) and non-infectious group. Immunohistochemical staining and RT-PCR were used to observe the change of the p65 subunit of NF-κB family in maternal blood and fetal membrane in subclinical infectious group and non-infectious group.Results (1) The incidence of subclinical chorioamnionitis:there were 24 cases of subclinical chofioamnionitis in the 36 cases of preterm birth in labor(67% ),7 cases in the 37 cases of full term gravida with spontaneous labor group (19%)and 3 cases in the 38 cases of full term gravida without threatened labor group(8%).There was a significant difference among the three groups (P＜0.01). In the totally 111 cases,34 eases were classified as subclinical infectious group and 77 cases as non-infectious group.(2) In fetal membrane,the median of the average staining intensity of NF-κB p65 protein was higher in the subclinical chorioamnionitis group (8.0)than those in non-infectious group(4.0).Similarly,the average staining intensity of NF-κB p65 mRNA was higher in the subclinical infectious group (47.5±17.2) than those in non-infectious group(31.3±13.6).There was a significant difference between the two groups (P＜0.01).(3)In maternal blood,the expression of NF-KB p65 protein and mRNA was higher in subclinical chorioamnionitis group (8.0 and 42.6) than these in non-infectious group(4.0 and 23.6).There was a significant difference between the two groups (P＜0.01).(4) The concentration of NF-κB p65 protein in fetal membrane was positively correlated with that of maternal blood(r=0.581,P＜0.01 ) and the concentration of NF-KB p65 mRNA in fetal membrane was positively correlated with that of maternal blood (r=0.571,P＜0.01 ).Condusion The expression of NF-KB in maternal blood and fetal membrane in preterm birth with subclinical chorioamnionitis is higher and the two are correlated with each other.NF-KB p65 could be an accurate biochemical marker in predicting subclinical chorioamnionitis in preterm birth. NF-KB p65 plays an important role in the pathogenesis of subclinical chorioamnionitis in preterm birth.

Objective To determine the effect of dihydroartiminisin on the proliferation and phosphorylation of mitogen-activated protein kinase (MAPK) in SKOV3 and OVCAR3 ovarian cancer cell lines.Methods Methyl thiazolyl tetrazolium assay was performed to evaluate the anti-proliferative effect of dihydroartiminisin in SKOV3 and OVCAR3 cells,and Western blot was used to determine its effect on phosphorylation level of MAPK,including extra-cell regulated kinase (ERK)1/2 and p38 protein kinase,in the two cell lines.Results Dihydroartiminisin inhibited the proliferation of ovarian cancer cells in vitro,with a mean of 50% inhibition concentration (IC50) at 72 h of (9.0 ±1.4) μmol/L for SKOV3 and (5.5 ±1.2)μmol/L for OVCAR3 respectively. Compared to cells without dihydroartiminisin treatment,phosphorylation level of ERK 1/2 in SKOV3 and OVCAR3 cells treated with dihydroartiminisin decreased by 64.2% and 75.3% respectively (P＜0.05),while phosphorylation of p38 protein kinase in SKOV3 and OVCAR3 only decreased by 8.5% and 6.4% respectively (P ＞0.05).Conclusion Dihydroartiminisin can inhibit the proliferation of ovarian cancer cell in vitro, probably through down-regulation of the phosphorylation of ERK 1/2 in ovarian cancer cells.

Objective To investigate genes involved in the mechanisms underlying the progression of severe preeclampsia.Methods We conducted a muhiregional gene expression analysis using peripheral leucocytes from patients with preeclampsia and normal controls.Total RNA was extracted from peripheral blood of six severe preeclampsia and five normotensive pregnancies.We performed genome-wide expression profiling using Affymetrix HG_U133 plus 2.0 chips to screen out differentially expressed genes of 2 fold or more and q_value ＜ 5.4%.Using Gene Ontology we identified the function of differentially expressed genes after cluster analysis.Results Among the 47 000 genes that were screened in the microarray,140 genes were found to be differentially expressed between normal and preeclamptic pregnancies. Eighty six up-regulated candidate genes were mainly involved in cysteine metabolism urea cycle and metabolism of amiogroups,proteasome,TGF-beta signaling pathway, and the ratio of calponin2 (CNN2), matrix metallopeptidase 8 (MMP8),V-set and immunoglobulin domain containing 4 (VSIG4),proteasome 26S subunit ATPase 5 (PSMC5) was evidently increased in preeclampsia patients.Among 54 down-regulatedcandidates,natural killer cell mediated cytotoxicity,antigen processing and presentation,metabolism of xenobiotics by cytochrome P450 were the main pathways.KIR3DL2,AKR1C3,CHURC1 and SLC25A13 were obviously decreased in preeclampsia patients. Conclusions The gene expression of peripheral leucocytes in preeclampsia patients is significantly different from that of uncomplicated pregnancies.CNN2,MMP8,VSIG4,PSMC5,KIR3DL2,AKR1C3,CHURC1 and SLC25A13 may be involved in the molecular mechanisms underlying the progression of severe preeclampsia.

Objective To investigate the incidence of glucose metabolism disorder during pregnancy and establish the diagnosis criteria for gestational diabetes mellitus (GDM) among Chinese patients.Method A prospective popolation-bused study of 16 286 pregnant women,who received 50 g glucose challenge test (GCT) for the first time between Apr 1,2006 and Sept 30,2006,was performed throughout 18 cities in China. Results According to national diabetes data group (NDDG)criteria,the overall incidence of GDM and glucose impaired glucose tolerance (GIGT) was 2.763% (450/16 286) and 3.862% (629/16 286),respectively; it changed to 5.078% (827/16 286)and 5.268% (858/16 286) when using American diabetes association (ADA) criteria.If the women who had 2 or more abnormal oral glucose tolerance test (OGTT) values meeting or exceeding ADA criteria was classified as group 1,and the women who had one or more meeting or exceeding NDDG criteria was group 2,the ratio of women who met both the criteria of ADA and NDDG in group 1 was 94.2%.The 95% CI of normal glucose when using ADA criteria were fasting glucose 5.3 mmol/L,1 hour 10.4 mmol/L,2 hour 8.7 mmol/L,3 hour 7.7 mmol/L,which is close to ADA criteria.Conclusions ADA criterion is more suitable for Chinese patients.According to NDDG criteria,it is reasonable to treat the patient with 1 or more abnormal OGTT values,and if choosing ADA criteria,2 or more abnormal OGrIT values is more reasonable.

Objective To investigate whether the proteasomes inhibitor MG262 exerts its anticancer function by inducing apoptosis in human ovarian cancer cells,and whether the extracellular signal regulated kinase (ERK) signaling pathway is involved in the regulation of apoptosis induction.Method Human ovarian cancer cell line SKOV3 was incubated with different concentrations of MG262 for 24 and 48 hours.Cell viability was evaluated with 3-(4,5-dimethyhhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points of culturing.Flow cytometry was used to detect cell apoptosis rate.The expression of vascular endothelial growth factor (VEGF) was evaluated with western blot and enzyme-linked immtmosorbent assay (ELISA).Western blot was used to detect the expression of phosphorylated ERK(pERK) .Results The viability of SKOV3 cells was decreased by MG262 in a concentration-dependent fashion(P＜0.05).After 24 h incubation with MG262 at 1,10,20,40,60 and 80 nmol/L,the viability rates of SKOV3 were (94.6±3.1)%,(92.7±3.7)%,(89.5±7.7)%,(84.2±5.1)%,(82.0±7.4)%and(76.8±11.0) % respectively,and after 48 h incubation,those figures were further decreased to (91.3±10.1)%,(86.8±4.5)%,(74.6±4.2)%,(56.8±2.1)%,(49.3±4.5)% and (37.4±5.4) %,respectively(P＜0.05).Apoptosis rate of SKOV3 cells induced by MG262,PD98059 or their combination was (30.7±4.3)%,(26.8±8.6)% and (50.3±10.6)%,respectively,which were significantly different compared with controls (P＜0.05).In contrast to SKOV3 cells,apoptosis rate of 293T ceils induced by MG262,PD98059 or their combination was (14.5±5.3) %,(16.2±7.5) % and (10.8±7.3)%,respectively,which were not significantly different compared with controls (P＞0.05).pERK expression decreased gradually in a time-dependent manner. And wild-type p53 expression was not significantly different.There was no significant difference between experimental and control 293T cells(P＜0.05).In addition,MG262 down-regulated VEGF secretion and expression in SKOV3 ceils (P＜0.05).Conclusions Proteasome inhibitors can induce apoptosis and inhibit cell proliferation and angiogenesis through ERK signal pathway in SKOV3 cells.

Objective To investigate expression of elastin,lysyl oxidase (LOX)and elafin in cardinal ligament of women with pelvic organ prolapse(POP) so as to determine their contributions to POP.Methods The cardinal ligament samples were obtained from 60 POP subjects and 60 non-POP control women undergoing hysterectomy.RT-PCR was used to verify the mRNA level of elastin,LOX and elafin.The protein concentration of the three genes was determined by western blotting technique,electrophoretic separation and quantification.Results The premenopansal and postmenopausal POP groups demonstrated significantly decreased expressions of elastin in cardinal ligament both in mRNA and protein levels than control group (mRNA 0.42±0.22,0.26±0.20 versus 0.79±0.30,0.63±0.23 ; protein 0.44±0.32,0.20±0.19 versus 0.89±0.27,0.78±0.25; P＜0.05).There was an identical tendency in the expression of LOX (mRNA 0.37±0.18,0.20±0.14 versus 0.65±0.22,0.53±0.20; protein 0.45±0.27,0.26±0.21 versus 0.85±0.39,0.69±0.31 ; P＜0.05).In POP group,the mRNA and protein levels of elastin and LOX in postmenopansal patients were significantly lower than premenopansal patients (P＜0.05).Inversely,POP group demonstrated an increased expression of elafin in cardinal ligament both in mRNA and protein levels than corresponding control group (mRNA 1.33±0.35,1.47±0.37 versus 0.62±0.25,0.55±0.24; protein 0.85±0.30,0.76±0.35 versus 0.21±0.15,0.29±0.22; P＜0.05). There was no significant difference in the expression of elafin between premenopansal and postmenopausal POP groups either in mRNA or protein levels(P＞0.05).There was a positive correlation between elastin and LOX both in mRNA and protein levels in POP group(r=0.9959,0.9708; P＜0.05),but there was no correlation between elastin and elafin(r=-0.0402,-0.0365;P＞0.05).Conclusions The results suggest that the decreased expression of elastin and LOX and the increased expression of elafin in the cardinal ligaments may contribute to POP.

Objective To evaluate the diagnostic value of MRI in the cases suspected of ventriculomegaly by prenatal uhrasonography.Methods 104 patients of suspected fetal ventriculomegaly (VM) diagnosed by uhrasonography were included from the Shengjing Hospital,China Medical University from March 2006 to October 2007.All cases were divided into 4 groups based on the standard of Gaglioti:10-12 mm(66 cases),13-15 mm(22 cases),16-20 mm(14 cases),and 21-25 mm(2 cases);they included 75 eases of single intracerebroventricular expansion and 29 eases of double intracerebroventricular expansion.All of them were subjected to MRI scan within 48 h of uhrasonographic examination to determine the prenatal diagnosis by MRI pregnancy outcomes.Results Among the 26 072 cases who received prenatal uhrasonography,104 cases (0.39%) were VM. (1) MRI detected 3 cases (5%) in 10-12 mm group:one ease of cerebellar hypoplasia,vascular malformation,chest and abdominal anomalies each; 5 cases(23%)in 13 -15 mm group:one case of agenesis of corpus callosum (ACC),cerebral hemorrhage,cerebral hemorrhage with cerebral meningocele,cerebral meningocele,intracranial mass meningocele each; 6 cases(43% )in 16 -20 mm group:4 cases of ACC,one case of intraventricular hemorrhage and ACC combined with ventricular hemorrhage each; 2 cases in 21-25 mm group:one case of ACC and intraventricular hemorrhage each.(2) MRI detected 4 eases(5%) among 75 unilateral VM cases and 12 eases (41%) among 29 bilateral VM eases.The differences were significant (P＜0.01).MRI diagnosis rate was 15.38% (16 cases).Follow-up of the outcomes of the pregnancy showed induction of 0labor in 15 cases (14%) all of which were the same as MRI results on autopsy,full-term delivery of 88 cases,of which all the neonates were healthy.Conclusions When the expansion width is above 16 mm or bilateral VM is suspected by ultrasonography,we suggest MRI examination to determine fetal central nervous system disease.

Objective To investigate the prevalence, clinical features, diagnosis, treatment and the influence on procreation of uterine malformation. Methods Totally 225 women with uterine malformation referred to our hospital from Mar 1990 to Jan 2005 were involved in this retrospective analysis.The constituent ratio, clinical feature, diagnosis, treatment and the influence on procreation were analyzed.Results (1) Among 225 cases of uterine malformation, 125 cases (55.6%) were septate uterus; 122(97.6%, 122/125 ) of these patients became pregnant, but always associated with a high risk of reproductive failure and obstetrical complications, such as abortion, abnormal fetal presentation, premature rupture of membrane. (2) Among 51 cases (22. 2%, 51/225 ) of didelphia, 50 cases (98%, 50/51 )became pregnant without any influence from this malformation. Five cases were combined with urinary system complication. (3) Among 26 cases (9.7%, 26/225 ) of rudimentary horn of uterus, 4 cases (15%, 4/26)became pregnant, and 1 case (4%, 1/26) presented with ipsilateral oviduct ectopic pregnancy; 2 cases (8%, 2/26) were combined with urinary deformities. (4) In 14 cases (6. 2%, 14/225) of bicornuate uterus, 1 case (7%, 1/14) presented with urinary tract abnormalities; gestations were more common, but due to the uterine structural abnormality, recurrent miscarriage and premature delivery were unavoidable.(5) Others: none came up with sterility in 4 cases (1. 8%, 4/225) of saddle shaped uterus. All the three cases ( 1.3% , 3/225) of ametria were with the chief complaint of primary amenorrhea. Two cases of unicornis uterus (0.9%, 2/225) were both complicated with sterility, while neither with urinary system deformities. Conclusions Among uterine malformations, septate uterine is the most common type. Septate uterus, didelphic uterus, bicornuate and rudimentary horn of uterus do not affect pregnancy, but can cause different complications. Unicornis uterus always causes sterility. Uterus malformations often come up with urinary system deformities.

Objective To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2)in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. Methods Spontaneous abortion embryo tissues were collected,including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos(35 cases)from the Department of Gynaecology and Obstetrics of the Affilisted Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007.FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein;primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis.Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAIY2 genes in embryonic cells which have normal karyotypes;the groups were defined as the first experimental group(transfeeted with pshRNA-hsMAD2-1),the second experimental group(transfected with pshRNA-hsMAD2-2),the third experimental group(transfected with pshRNA-hsMAD2-3),the first control group(transfected with nothing),the second control group(transfected with pTZU6+1)and the independent group(transfected with pshRNA-N1).Interference efficiency was demonstrated by FQ-PCR and western blot:cell prolireration was meagured by methyl thiazolyl tetrazolium(MTT)assay;cell-cycle was assessed by flow cytometry (FCM):the chromosome numbers were calculated to analyze the variation of chromosomes.Results(1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue,twice or more spontaneous abortion tissue and indueed abortion tissue were 0.00879±0.00035.0.00901±0.00033 and 0.00941±0.00026 respectively,and there Wag no significant ditierence(P＞0.05)compared with each other;however,the protein levels of hsMAD2 in three groups were 0.2791±0.0311.0.0431±0.0020 and 0.5790±0.0331 respectively,and there were significant difierences(P＜0.05)compared with each other.(2)Recombinant shRNA plagmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic cells.Compared with the first control group(4%)and the second control group(3%),the recombinant shRNA could inhibit embryonic cell proliferation to 54% at 48 h after transfection(P＜0.05):compared with the first control group(8.2%)and the second control group(8.0%),the ratios of G2/M phase cells in the experimental group(17.9%)was significantly increased(P＜0.05);compared with the first control group (4.8%),the ratios of abnormal chromosomes in the experimental group was increased to 30.0%(P＜0.05).Conclusions Down-expression of hsMAD2 gene may be one of the mechanisms inducing numerical chromosome aberration,abnormal embryo development and the occurrence of spontaneous abortion.