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Objective To investigate the induction and regulatory mechanism of placental trophoblast cell autophagy in women with preeclampsia (PE).Methods Twenty gravidas with severe PE who underwent cesarean section in the Department of Obstetrics and Gynecology of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University from August 2016 to November 2016 were enrolled in PE group.An equal number of normotensive gravidas without proteinuria who also underwent cesarean section during the same period were randomly selected as control group.Placental tissue samples were collected from all gravidas.Ultrastructure of placental trophoblast cells and changes in autophagosome formation were observed by transmission electron microscope.Expressions ofmicrotubule associated protein 1 light chain 3B (MAP1LC3B,or LC3B) and Beclin 1 in placental tissue samples were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot.Activities of protein kinase B (PKB,also known as Akt)/mammalian target of rapamycin (mTOR) pathway in placental tissue samples were detected by Western blot.Two independent samples t-test or Mann-Whitney U test was used for statistical analysis.Results Sparse and disordered villi and many typical autophagosomes were observed in placental trophoblast cells from patients with severe PE.Significantly enhanced expression of LC3B at mRNA and protein levels and increased ratio of LC3-Ⅱ/LC3-Ⅰ were observed in the PE group as compared with the control group [3.37 (2.37-6.11) vs 0.62 (0.25-4.15),1.40±0.17 vs 1.00±0.13,1.57±0.25 vs 1.00±0.31,Zor t=--4.440,3.274 and 3.113,all P＜0.05].No significant difference in the expression ofBeclin 1 at mRNA or protein level in placental tissues was found between the two groups (both P＞0.05).Furthermore,Akt and mTOR phosphorylation in the PE group was significantly suppressed as compared with that in the control group (1.00±0.29 vs 0.64±0.21,1.00±0.32 vs 0.60±0.22,t=--3.672 and-2.895,both P＜0.05).However,the two groups showed no significant difference in the expression of Akt or mTOR protein (both P＞0.05).Conclusions Suppressed activity of Akt/mTOR pathway and enhanced induction of trophoblast cell autophagy are detected in placental tissues of patients with severe PE,indicating that excessive trophoblast cell autophagy,induced by decreased activity of Akt/mTOR pathway,may be the pathogenesis for PE.
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Objective To investigate the effects ofpravastatin on the expression ofmicroRNA-155 (miR-155) and the functions of lipopolysaccharide (LPS)-treated extravillous trophoblast cells.Methods In vitro cultured HTR-8/SVneo cells were divided into the following groups:control group,enhanced plasmid with green fluoscent protein (pEGFP)-miR-155 group (transfected with green fluorescent protein-tagged miR-155),LPS group (treated with 100 ng/mL of LPS),miR-155 inhibitor+LPS group,pravastatin+LPS group (treated with 100 ng/mL of LPS following pretreatment with 12.50,25.00,50.00 and 100.00 μ g/ml of pravastatin),and pravastatin+pEGFP-miR-155 group (transfected with pEGFP-miR-155 following pretreatment with 50 μ g/ml of pravastatin).Levels of miR-155 in HTR-8/SVneo cells treated with different strategies were measured by real-time polymerase chain reaction.Expression of phosphorylated JunB (p-JunB) and p-FosB proteins was analyzed by Western blotting.Migration,invasion and apoptosis of HTR-8/SVneo cells were also analyzed.All data were analyzed with t test.Results (1) Compared with the control group,HTR-8/SVneo cells in the pEGFP-miR-155 group were characterized by shorter migration distance [(274.70± 18.82) vs (181.00±8.62) μ m],less transmembrane cells [(123.00±4.36) vs (63.00±6.08)] and enhanced apoptosis [(5.40± 0.68)％ vs (9.27±0.68)％] (all P＜0.05).(2) Compared with the LPS group,the miR-155 inhibitor+LPS group showed longer migration distance of HTR-8/SVneo cells [(166.30±5.07) vs (242.00±18.07) μm,P＜0.05],more transmembrane cells [(71.67±6.12) vs (109.00±7.81),P＜0.05] and decreased cell apoptosis [(14.40±1.69)％ vs (6.23± 0.44)％,P＜0.05].(3) Expression of miR-155 at mRNA level in the LPS group was increased as compared with that of the control group (1.65 0.07 vs 0.79±0.12,P＜0.05).Compared with the LPS group,pretreatment with 12.50,25.00,50.00 and 100.00 μ g/ml of pravastatin decreased the expression of miR-155 at mRNA level [(1.14±0.10),(1.02±0.10),(0.74±0.15) and (1.140.02)],especially at the concentration of 50 μμ g/ml (all P＜0.05).(4) Expression ofp-JunB and p-FosB proteins in the control,LPS and pravastatin (50 μ g/ml)+LPS groups were (0.33 ±0.06) vs (0.37±0.07),(1.22±0.20) vs (0.80±-0.13),and (0.31 ±0.02) vs (0.21 ±0.05),respectively,showing higher expression level in both p-JunB and p-FosB proteins in the LPS group compared with that of the other two groups (all P＜0.05).(5) Compared with the LPS group,the pravastatin (50 μμ g/ml)+LPS group showed increased migration distance [(166.30±5.07) vs (246.80± 13.42) μ m,P＜0.05],increased numbers of transmembrane cells [(71.67 ± 6.12) vs (95.33 ± 2.73),P＜0.05] and decreased cell apoptosis [(14.40± 1.69) vs (6.05 ± 0.35)％,P＜0.05].(6) Compared with the pEGFP-miR-155 group,the pravastatin (50.00.00 μμ g/mL)+pEGFP-miR-155 group showed longer migration distance [(197.50± 13.86) vs (275.80± 13.63) μ m,P＜0.05],more transmembrane cells [(52.67±5.49) vs (125.00±6.66),P＜0.05] and lower rate of cell apoptosis [(8.90± 1.00) vs (5.05±0.35)％,P＜0.05].Conclusions Pretreatment of extravillous trophoblast cells with pravastatin can protect them from apoptosis and loss of migratory and invasive abilities through inhibiting the activation of AP-1 and down-regulating the expression of miR-155,which may be a mechanism that inhibits the development of preeclampsia.
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Objective During pregnancy , exosomes can be released from the placenta into maternal circulation and play im-portant roles in normal pregnancy or placenta-related diseases .We aimed to establish a simple and efficient method for isolating and i-dentifying placental exosomes from maternal serum and lay a foundation for the studies of pregnancy -related diseases . Methods Using sucrose gradient centrifugation with 8% PEG6000 precipitation twice , we isolated and purified placenta-derived exosomes from normal maternal serum and detected their molecular markers CD 63 , CD81 and PLAP by Western blot , followed by silver staining anal-ysis of the protein profile of the exosome pellet .We identified the morphology of the placenta-derived exosomes by transmission electron microscopy ( TEM) and measured the size and distribution of the particles by dynamic light scattering ( DLS) . Results Silver stai-ning of the protein profiles of the exosomes after sucrose gradient centrifugation clearly revealed the bands of the protein molecules . Western blot showed the expressions of CD 63, CD81, and PLAP in the 21-34%density layer, which demonstrated the presence of serum placental exosomes mainly in the 1.09-1.16 g/mL density layer.TEM exhibited that the placenta-derived exosomes were round or oval cup-shaped, specifically expressing PLAP, and the particles were uniform in size, with a mean diameter of (41.79 ±11.94) nm. Conclusion A simple, fast, and efficient method was successfully established for isolating placenta-derived exosomes from ma-ternal serum, which provides a basis for studying the roles of placental exosomes in normal pregnancy and placenta -related diseases.
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Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.
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Objective To establish an animal model of preeclampsia by injecting ultra-low-dose lipopolysaccharide (LPS) to rats in early pregnancy,and to lay the foundation for further study on mechanisms of preeclampsia.Methods Twenty-four pregnant rats were divided into six groups according to the random number table and were injected with LPS 0.3,0.5,0.7,1.0,2.0 μg/kg or saline 2 ml respectively through tail veins on day 5 of pregnancy.The differences in blood pressure,urinary protein and pathological changes in placenta among groups were compared to confirm the suitable dose of LPS for establishing preeclamptic model.Then another 19 pregnant rats were injected with the chosen dose of LPS slowly through tail veins on day 5 of pregnancy; 15 of which were chosen as model group; the other four were chosen as postpartum group.Three non-pregnant rats were as non-pregnant group.Besides,another 15 pregnant rats were injected with saline as pregnant control group.Systolic blood pressure,urinary protein excretion,placental weight,fetal weight,serum white blood cell counts,blood platelet counts,plasma anti-thrombin-Ⅲ content,D-dimer content were examined and compared among groups with one way analysis of variance; histopathologic studies were also done on the placentas,kidneys and aortas of the rats.Results (1) Placental weight of LPS 0.3 μg/kg group increased compared with control group.One pregnant rats(1/4) in LPS 1.0 μg/kg group and LPS 2.0 μg/kg group died on day 16 of pregnancy as a result of vaginal bleeding.Systolic blood pressure of LPS 0.5 μg/kg group rose steadily,while no significant changes were found in other groups.Urinary protein increased in all LPS groups,while urinary protein of LPS 0.7 μg/kg group and LPS 1.0 μg/kg group peaked on day 12 of pregnancy and then decreased; urinary protein of LPS 0.5 μg/kg group increased most significantly,and fetus in LPS 0.5,0.7 and 2.0 μg/kg groups had lighter body weight.So LPS 0.5 μg/kg was chosen as the suitable dose to establish preeclamptic model.(2)Compared with pregnant control group,model group had higher systolic blood pressure [(124.89±1.79) mm Hg vs (119.02±1.80) mm Hg,LSD test,P=0.03] from day 6 of pregnancy,more urinary protein [(2.02±0.29) mg vs (1.11±0.18) mg,LSD test,P=0.00] from day 9 of pregnancy,more absorbed embryos [3.6％ (7/194) vs 0.0％ (0/200),Fisher exact test,P=0.01] at day 20 of pregnancy,higher incidence of placenta bleeding [4.1％ (8/194) vs 0.0％ (0/200),Fisher exact test,P=0.00] and fetal growth restriction [13.9％ (27/194) vs 6.0％ (12/200),X2=6.92,Fisher exacttest,P=0.01].Model group showed more inflammatory cells infiltration in the placenta,more glomerular mesangial cells,swelling and desquamated of renal tubular epithelial cells compared to control group.Blood pressure and urinary protein of the model group recovered to the baseline at the sixth day of postpartum,and no changes in blood pressure and urinary protein were found in non-pregnant rats.Conclusions Injection of LPS 0.5 μg/kg on day 5 of pregnancy through tail veins could induce the clinical symptoms of preeclampsia in rats,which might be an ideal model for further preeclampsia research.
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Objective To investigate the different expression of microRNA-155 (miR-155) and cysteine-rich 61 (CYR61) in human placentas between severe preeclamptic and normal pregnancies.Methods Placentas were obtained from severe preeclamptic and healthy control pregnant women (n=18 for each group) at 36～40 gestational weeks. The expressions of miR-155 and CYR61 mRNA were assessed by real-time quantitative reverse transcription-polymerase chain reaction, and the levels of CYR61 protein were tested by Western blot. Results Compared with the control group, the miR-155 expression was increased in placentas from severe preeclampsia groups ( 165. 7 ± 16. 4 vs 527.9±49.1,t=7.00, P＜0.01), and the CYR61 mRNA expression (31.7±2.7 vs 16.4±1.2,t=5.10,P＜0. 01), as well as the CYR61 protein expression (36.4±1.5 vs 19.7±1.2,t=36.26, P＜0.01 ) were decreased. There was a significantly negative correlation between the expression of miR-155 and CYR61 mRNA within both groups (preeclamptic group: r=-0.52, P＜0.05;control:r=-0.57, P＜0.05). Conclusions Up-regulation of placental miR-155 in severe preeclampsia may be related to the decreased expression of CYR61. Both miR-155 and CYR61 may contribute to the disorders of placental angiogenesis in severe preeclampsia in human.
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Objectives: To express HBeAg in prokaryotic and eukaryotic cells and compare the two types of HBeAg in the anti-HBeAg testing. Methods: HBeAg was expressed both in E.coli cells and in silk worm cells, purified by Sephacryl S-200.HBeAg protein concentration and antigenic titer were determined respectively by ultraviolet-spectroscopy and EIA. Results: HBeAg produced by E.coli cells: Activation ratio was 10 000/mg, HBeAg/HBcAg = 50; The specificity in testing anti-HbeAg was 96%;HBeAg produced by silk worm cells: Activation ratio was 160 000/mg, HBeAg/HBcAg = 5 000, The specificity in testing anti-HbeAg was 100%. Conclusions: HBeAg produced by eukaryotic cells contained much lower proportion of HbcAg and higher activation ratio, which therefore bring about a possibility to improve the quality of the kit for testing Anti-HBe.
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Objectives:Using IE 1 gene promoter of Autograph californica nuclear polyhedrosis virus(AcNPV),a transfer vector with an immediately early gene promoter was constructed. Methods:Transfer vector pAcPIneo which contains neomycin resistance gene(neo)coding sequence downstream of IE 1-promoter was constructed and cotransfected with the wild type of AcNPV DNA into Sf9 insect cells.Recombinant virus was selected by G418 resistance since the neo gene can be expressed in Sf9 cells. Results:Northern blot hybridzation with 32 P labeled neo gene fragement as probe showed that the neogene was integrated in plogene of AcNPV genome. Conclusions:Transfer vector with an immediately early gene promoter of baculovirus was constructed successfully,the neo gene was transcribed from the immediately early phase to the very late phase in infected cells.