环磷酸腺苷对人羊膜上皮细胞水通道蛋白8表达及分布的影响

目的 探讨环磷酸腺苷(cAMP)对人羊膜上皮细胞水通道蛋白8(AQP8)表达及分布的影响.方法 培养人羊膜上皮细胞WISH细胞株,随机分为实验组和对照组.对照组给予PRMI 1640培养液常规培养.实验组将8-溴腺苷-3',5'环磷酸腺苷(8-Br-cAMP)加入培养液中,使其在细胞培养液中浓度分别为10、20、40、100、200、400 μmol/L,不同浓度分别培养2.4、8、16、24和48 h时取样.各浓度重复培养6瓶.采用蛋白印迹法定量检测WISH细胞AQP8的蛋白表达;采用RT-PCR技术半定量检测WISH细胞AQP8的mRNA表达.采用免疫荧光细胞化学法检测WISH细胞上AQP8的分布.结果 对照组WISH细胞AQP8蛋白表达水平为0.028±0.003,AQP8 mRNA表达水平为0.027±0.003.实验组WISH细胞随着培养液中8-Br-cAMP浓度的升高,AQP8蛋白表达水平明显升高.培养液中8-Br-cAMP浓度为20、40、100、200、400 μmoL/L时,其蛋白表达水平分别为0.062±0.007、0.063±0.006、0.125±0.008、0.145±0.010、0.100±0.008,培养液中8-Br-cAMP浓度为200 μmol/L,培养8 h时,AQP8蛋白水平开始升高,24 h时达到峰值,升高约5倍.随着8-Br-cAMP浓度的升高,AQP8 mRNA水平明显升高,培养液中8-Br-cAMP浓度为20、40、100、200、400 μmol/L时,其mRNA表达水平分别为0.065±0.007、0.069±0.006、0.130±0.008、0.150±0.010、0.110±0.009,同样在200 μmol/L时达到峰值;8-Br-cAMP浓度为200 μmol/L,培养2 h时AQP8 mRNA水平开始升高,培养8 h时达到峰值,16 h时开始下降,24 h时降到8-Br-cAMP培养前的水平.8-Br-cAMP浓度为200 μmol/L培养24 h时,细胞内的荧光减弱而细胞膜的荧光明显增强.结论 cAMP可能是调控羊膜上皮细胞AQP8表达及分布的重要介质.     

Comparison of magnetic resonance imaging to ultrasound in the estimation of birth weight at term

OBJECTIVE: This study was undertaken to compare magnetic resonance (MR) and ultrasound (US) fetal weight estimates obtained immediately before delivery with birth weight. STUDY DESIGN: Eighty women scheduled for a cesarean delivery underwent a fast acquisition MR and US for fetal weight estimation within 3 hours of delivery. Prospective MR calculation was based on the equation 0.12+1.031 g/mLxfetal volume (mL)=MR weight (g). US fetal weight estimation was calculated by the formula by Hadlock et al. Estimations were compared with birth weight. RESULTS: Correlation (95% CI) between birth weight and MR weight is 0.95 with a mean absolute error of 129 g (105-155) compared with the correlation between birth weight and US of 0.85 with a mean absolute error of 225 g (186-264). The correlation for birth weight and MR imaging is significantly greater than that of birth weight and US, P<.001. CONCLUSION: Birth weight estimation is more accurate by MR imaging than by US in term infants.     

Influence of oxytocin on prostaglandin E2, intracellular calcium, and cyclic adenosine monophosphate in human amnion-derived (WISH) cells

OBJECTIVE: This study was undertaken to investigate the regulation of prostaglandin release by oxytocin and the influence of oxytocin on intracellular calcium and on the cyclic adenosine monophosphate system in human amnion-derived WISH cells. STUDY DESIGN: We determined prostaglandin E(2) release from WISH cells treated with oxytocin, evaluated the cytosolic calcium concentration in single WISH cells by confocal microscopy, and measured both intracellular cyclic adenosine monophosphate levels and adenylyl cyclase activity after oxytocin treatment. RESULTS: Treatment of WISH cells with oxytocin resulted in a concentration-dependent release of prostaglandin E(2), which was increased by lithium chloride and inhibited by indomethacin and U-73122. In single WISH cells, oxytocin increased cytosolic calcium. Moreover, the hormone lowered levels of intracellular cyclic adenosine monophosphate but did not alter adenylyl cyclase activity. CONCLUSIONS: Our data demonstrate, for the first time, that WISH cells respond to oxytocin by increasing prostaglandin E(2) release. In addition to phospholipase C activation and cytosolic calcium increase, the hormone effect involves also a reduction of the cyclic adenosine monophosphate level.     

Effects of cyclic adenosine monophosphate on human chorionic gonadotropin and estradiol output by cultured human placental cells

We have previously shown that adenosine-3',5'-cyclic monophosphate (cAMP) inhibits basal estradiol output in human trophoblast cells. The objective of this study was to investigate further the effect of 8-bromo-cAMP on the conversion of C19 androgens to estradiol by placental cells. Trophoblast cells were prepared from human term placenta and maintained in monolayer culture. On days 3 and 4 of culture, these cells were treated with dehydroepiandrosterone sulfate, androstenedione, or testosterone with or without the concomitant presence of 8-Br-cAMP. 8-Br-cAMP markedly enhanced human chorionic gonadotropin secretion into the culture medium. On the other hand, the concomitant addition of 8-Br-cAMP with the androgen precursors led to an inhibition of estradiol output. The concentrations of androstenedione and dehydroepiandrosterone in the culture medium after treatment with dehydroepiandrosterone sulfate were elevated by the concomitant presence of 8-Br-cAMP. From these results we conclude that 8-Br-cAMP enhances human chorionic gonadotropin output in human term placental cells, whereas the presence of 8-Br-cAMP in cells given androgen precursors inhibits estradiol output, probably at the level of aromatization.     

Relation between cyclic adenosine monophosphate and prostaglandin output by dispersed cells from human amnion and decidua

We have examined the ability of activators of adenylate cyclase and cyclic adenosine monophosphate to affect the output of prostaglandins E and F by dispersed cells of amnion and decidua collected from women following spontaneous labor. Cyclic adenosine monophosphate production by amnion and decidua cells was stimulated in a dose-dependent fashion by cholera toxin and by forskolin in the absence or presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine. Forskolin and cholera toxin also stimulated prostaglandin E and F output from amnion and decidua cells. Similar effects were seen with cells incubated with dibutyryl cyclic adenosine monophosphate +/- 3-isobutyl-1-methyl xanthine. The beta-adrenergic receptor agonists salbutamol, isoproterenol, and epinephrine all stimulated prostaglandin E and F output from dispersed cells of both tissues. The stimulatory effect of 3-isobutyl-1-methyl xanthine was partially additive with the Ca2+ ionophore A23187. Basal outputs of prostaglandin and outputs stimulated by A23187 and by N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate were attenuated by the calmodulin antagonist trifluoperazine in a dose-dependent fashion. We conclude that mechanisms exist for stimulation of adenylate cyclase in human amnion and decidua resulting in enhanced prostaglandin output. This pathway requires basal interaction with Ca2+-calmodulin and may be additive with cyclic adenosine monophosphate-independent mechanisms for prostaglandin stimulation.     

Effect of the gonadotropin-releasing hormone antagonist ganirelix on cyclic adenosine monophosphate accumulation of human granulosa-lutein cells

OBJECTIVE: To evaluate whether the GnRH antagonist ganirelix exerts an effect on cyclic adenosine monophosphate (cAMP) production of human granulosa-lutein (GL) cells in vitro. DESIGN: In vitro cell culture study. SETTING: Research laboratory of a university hospital. PATIENT(S): Mural GL and cumulus cells were obtained from 15 patients on whom controlled ovarian hyperstimulation was being performed for intracytoplasmic sperm injection treatment. INTERVENTION(S): Mural GL and cumulus cells were cultured for 48 hours with and without 1 nM ganirelix or triptorelin. For the last 6 hours, the cells were either exposed to 1-5 IU hCG or left unstimulated. MAIN OUTCOME MEASURE(S): At the end of the culturing period, the intracellular and extracellular cAMP accumulations were measured by an (125)I-scintillation proximity assay. RESULT(S): hCG induced dose-dependent increases in total cAMP accumulation. Stimulation with 1 IU/mL hCG resulted in 9-fold and 13-fold increases, and 5 IU/mL hCG resulted in 19-fold and 14-fold increases in total cAMP release from cumulus and mural GL cells, respectively. On the other hand, treatments with 1 nM GnRH antagonist ganirelix and 1 nM GnRH agonist triptorelin did not exert any significant changes on the basal and hCG-stimulated cAMP accumulation of mural GL cells and cumulus cells as compared with controls. CONCLUSION(S): Ganirelix does not influence basal and hCG-stimulated cAMP accumulation of human GL cells in vitro. cAMP is apparently not involved in the mechanism of action of GnRH analogs in human ovary.     

Comparison of congenital pulmonary airway malformation volume ratios calculated by ultrasound and magnetic resonance imaging

Objective: To compare congenital pulmonary airway malformation (CPAM) volume to head circumference ratios (CVRs) determined by different imaging modalities and calculation techniques.

Methods: Fetal thoracic lesion images by ultrasound (US) and magnetic resonance imaging (MRI) were retrospectively reviewed and the CVRs were calculated. The CVR^US was determined by the standard method. The CVR^MRI was calculated from T2-weighted sequences (HASTE/SSH-TSE) in two ways, dimensional measurements analogous to US technique (MRI-D) and by using a MRI-software calculated volume (MRI-V). CVR values between methods were compared using Wilcoxon matched-pairs signed-rank testing, Bland–Altman analyses, and Spearman correlations.

Results: Appropriate images were available to compare CVR^US to CVR^MRI-D for 20 patients and CVR^US to CVR^MRI-V for 18 patients. There were no significant differences in CVR values between modalities. By Bland–Altman analyses, the CVR measurements were largely within the limits of agreement: 18 of 20 for CVR^MRI-D and 17 of 18 for CVR^MRI-V, with a slight bias towards larger measurements by MRI.

Conclusions: Though values varied between modalities for individual patients, there was no systematic difference in CVRs determined by US or MRI. Fetal prognostic category for CPAMs did not change based on MRI in any patient in this series.     

Basal and forskolin-stimulated cyclic adenosine monophosphate in intact human platelets during the menstrual cycle

In previous studies we observed modifications of cyclic adenosine monophosphate and adenylate cyclase activity in human endometrium during the menstrual cycle. In the present study our intension was to verify whether these modifications occur in isolated intact platelets. The results demonstrate that in nine normal women platelet cyclic adenosine monophosphate content varies during the menstrual cycle both in basal and in stimulated conditions (in vitro addition of forskolin). In fact, significantly higher levels of cyclic adenosine monophosphate were consistently observed during the proliferative phase. These findings provide evidence that platelet cyclic adenosine monophosphate metabolism normally varies during the menstrual cycle, which suggests a possible involvement of this system in some important clinical events.     

水通道蛋白3在人羊膜上皮细胞中的表达及调控

目的 研究丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)/细胞外信号调节激酶(extracellular signal regulated kinase,ERK)信号转导通路对人羊膜上皮细胞中水通道蛋白3(aquaporin,AQP3)表达的调控作用. 方法 选择2011年1月至11月,在温州医学院附属第二医院产科分娩的不明原因羊水过少和羊水量正常的足月单胎妊娠产妇各10例,均为剖宫产分娩.原代培养羊膜上皮细胞,并用ERK1/2抑制剂U0126进行处理.浓度为0、5、10、20和40 μmol/L的U0126分别作用12 h;再选取使磷酸化ERK1/2表达水平最低的浓度为最佳浓度,作用于细胞0、2、6、12和24 h.采用免疫细胞化学染色检测AQP3蛋白定位,Western印迹检测总ERK1/2、磷酸化ERK1/2和AQP3蛋白表达水平.统计学方法采用t检验和方差分析. 结果 (1)羊水过少组磷酸化ERK1/2和AQP3的表达均低于羊水量正常组(ERK1/2:2.46±0.25与3.46±0.33;AQP3:1.56±0.10与2.34±0.18,t=9.243和13.292,P＜0.01).(2)羊水过少组中U0126不同浓度组间比较,总ERK1/2表达差异无统计学意义(F=0.365,P＞0.05);5、10、20、40 μmol/L组磷酸化ERK1/2和AQP3表达均低于0μmol/L组(磷酸化ERK1/2:0.96±0.16、1.12±0.13、0.98±0.17、1.02±0.26与2.46±0.25; AQP3:1.10±0.09、1.12±0.08、1.13±0.06、1.11±0.06与1.56±0.10,P＜0.05);但5μmol/L组与10、20和40 μmol/L组比较,差异均无统计学意义(P＞0.05).羊水过少组U0126作用的最佳浓度为5 μmol/L.该浓度作用2h组磷酸化ERK1/2和AQP3表达均低于0h组(磷酸化ERK1/2:1.27±0.29与2.55±0.12;AQP3:1.44±0.12与2.15±0.09,P＜0.05),但2h组与6、12和24 h组比较,差异均无统计学意义(P＞0.05).最佳作用时间为2h.(3)羊水量正常组和羊水过少组产妇的羊膜上皮细胞中,胞浆及胞膜均有AQP3蛋白阳性染色,但主要位于胞浆.U0126作用后,AQP3蛋白在羊膜上皮细胞中定位无明显改变. 结论 U0126可抑制羊水过少者的羊膜上皮细胞ERK1/2磷酸化及AQP3蛋白表达,提示MAPK/ERK1/2信号转导通路可能对羊膜上皮细胞中AQP3蛋白表达起调控作用,从而影响羊水量的平衡.     

原发性羊水过多产妇胎膜和胎盘组织中水通道蛋白8的表达

目的 探讨水通道蛋白8(AQP8)在原发性羊水过多产妇胎膜和胎盘组织中的表达.方法 收集2005年10月-2007年5月重庆医科大学附属第一医院和重庆市妇幼保健院行足月剖宫产分娩(孕37～40周)的原发性羊水过多产妇12例为羊水过多组,同期因社会因素行剖宫产分娩的正常产妇12例为对照组.采用RT-PCR技术检测两组产妇胎膜和胎盘组织中的AQP8 mRNA表达水平;采用免疫组化技术检测两组产妇胎膜和胎盘组织中的AQP8蛋白表达水平.结果 (1)羊水过多组羊膜、绒毛膜和胎盘组织中的AQP8 mRNA表达水平分别为0.78±0.13、0.58±0.10、0.86±0.15,对照组分别为0.39±0.07、0.45±0.09、0.34±0.09,羊水过多组各组织中AQPS mRNA表达水平均高于对照组,两组分别比较,差异均有统计学意义(P<0.05).(2)羊水过多组羊膜、绒毛膜和胎盘组织中的AQP8蛋白表达水平分别为0.195±0.024、0.170±0.028、0.193±0.024,对照组分别为0.151±0.018、0.156±0.024、0.152±0.023,其中羊水过多组羊膜、胎盘组织中AQP8蛋白表达水平均高于对照组,差异有统计学意义(P<0.05),而羊水过多组绒毛膜组织中AQP8蛋白表达水平虽高于对照组,但两组比较,差异无统计学意义(P>0.05).结论 原发性羊水过多产妇羊膜和胎盘组织中AQP8mRNA及蛋白的表达水平均显著高于正常产妇,提示AQP8在产妇羊水量的调节中发挥重要作用.     

The antenatal diagnosis of placenta accreta

The antenatal diagnosis of placenta accreta is invaluable in planning an approach to delivery or to management of an early intrauterine demise. The women most at risk are those who have had uterine surgery and have placenta previa, but placenta accreta can occur in any pregnancy. In early pregnancy the most useful ultrasound finding is implantation of the sac over a uterine scar. Vascular sinuses, appearing as early as 15 weeks, are irregularly shaped, have obvious blood flow when evaluated with color Doppler, and have the highest sensitivity for placenta accreta. Loss of the usual retroplacental clear space as a sole finding will usually be false positive. Magnetic resonance imaging diagnosis is in its infancy and has not yet been proven to add information unless the placenta is posterior. In the future it will hopefully aid in distinguishing placenta accreta from percreta.     

Expression of aquaporin 9 in human chorioamniotic membranes and placenta

OBJECTIVE: Aquaporin 9 (AQP9) is one of the recently identified water channels that is also permeable to neutral solutes including urea. To investigate the molecular mechanism of intramembranous pathway of amniotic fluid regulation, we sought to determine whether AQP9 is expressed, and the cellular localization of AQP9 expression in human fetal membranes. STUDY DESIGN: Fetal membranes from 5 normal term human pregnancies were studied. Northern analysis was used to determine the tissue AQP9 messenger RNA (mRNA) expression. In situ hybridization and immunohistochemical staining with specific anti-AQP9 antibody was used for cellular AQP9 localization in the human fetal membranes. RESULTS: Northern analysis detected AQP9 mRNA expression in human amnion, chorion, and placenta. In situ hybridization revealed AQP9 mRNA expression in epithelial cells of the amnion, chorion cytotrophoblasts, and syncytiotrophoblasts and cytotrophoblasts of placenta. Further immunohistochemical study confirmed the AQP9 protein expression in these cell types of fetal membranes. CONCLUSION: This study demonstrated the expression of AQP9 mRNA and protein in human chorioamniotic membranes and placenta. The AQP9 expression in fetal membranes suggests that AQP9 may be an important water channel in intramembranous amniotic fluid water regulation.     

水通道蛋白9在人胎盘和胎膜中的表达

目的 检测正常人胎盘与胎膜中水通道蛋白9（aquaporin 9，AOP9）的表达。方法 收集5例足月剖宫分娩的胎盘和胎膜样本，运用RT—PCR方法从mRNA水平检测AQP9在胎盘与胎膜的表达；运用免疫组织化学和Western印迹方法检测AQP9蛋白在胎盘与胎膜中的表达。结果 RT—PCR结果显示AQP9mRNA在胎盘和胎膜均有表达；Western印迹显示两条带在相对分子量为30kD及45kD左右；免疫组织化学显示AQP9表达于胎盘的合体滋养细胞、羊膜上皮细胞及平滑绒毛膜滋养细胞。结论 AQP9在母胎液体交换、胎儿代谢物的排出及羊水平衡等的分子机制中可能发挥重要作用。