Corticotropin-releasing hormone binding to the syncytiotrophoblast membranes

The human placenta secretes large amounts of corticotropin-releasing hormone (CRH) which was thought to exert a paracrine action in the placenta. We have recently characterized high-affinity binding sites for CRH in the human placenta. However, our studies utilized whole placental membranes, which did not identify the site of binding of CRH in the plasma membrane. In this study we investigated the characteristics of CRH binding to purified mother-facing, brush border membranes (BBM) and fetus-facing, basal plasma membranes (BPM) of the syncytiotrophoblast. The two membranes were separated by a series of differential and density-gradient centrifugations. The purity of the membranes was determined by measuring alkaline phosphatase, as a marker of BBM and Na+/K+ATPase as a marker of BPM. Each membrane showed specific and high-affinity binding. Scatchard analysis revealed a high-affinity binding site for CRH with Kd of 1.0 +/- 0.15 and 1.3 +/- 0.176 for BBM and BPM, respectively. The maximal number of binding sites was significantly different (P < 0.01) in the two plasma membranes: Bmax of 79 +/- 6.4 fmol/mg protein for BBM and 23 +/- 3.9 fmol/mg protein for BPM. Both the mother-facing and fetus-facing membranes of the syncytiotrophoblast contain binding proteins for CRH, with significantly more binding sites on the mother-facing membranes. The functional consequences of CRH binding could be different for the two polar membranes due to differential localization of second messenger systems between the two membrane types. It is proposed that partial purification of BBM and BPM provides a better system to study CRH action in the placenta, than whole placental membrane preparations.     

脂质相关膜蛋白体外诱导hBD-2基因表达的研究

目的探讨解脲支原体(UU)的L AMPs对β-防御素表达分泌量与作用时间的研究。方法应用LAMPs体外诱导绒毛膜及胎盘组织hBD-2基因表达,RT-PCR检测hBD-2 mRMA表达。结果不同浓度的LAMPs均能明显诱导绒毛膜及胎盘组织产生hBD-2,相同浓度的LAMPS刺激绒毛膜及胎盘组织12 h,24 h绒毛膜及胎盘组织的HBD-2 mRNA表达量不同。结论绒毛膜及胎盘组织HBD-2 mRNA表达与LAMPs呈明显的剂量依赖性;24 h内绒毛膜及胎盘组织的HBD-2 mRNA表达呈现出时间依赖性。     

子痫前期胎盘中第10号染色体缺失性磷酸酶和张力蛋白同源物基因、Syncytin-1与母体内皮损伤、滋养细胞损伤的相关性

目的研究子痫前期胎盘中第10号染色体缺失性磷酸酶和张力蛋白同源物基因(phosphatase and tensin homologue-deleted chromosome ten gene,PTEN)、Syncytin-1与母体内皮损伤、滋养细胞损伤的相关性。方法选择2015年2月至2017年12月在南方医科大学附属小榄医院诊断为子痫前期并分娩的46例患者及同期正常分娩的的80名孕妇,分娩前留取母体外周血并测定内皮损伤标志物的含量,分娩后留取胎盘并测定PTEN、Syncytin-1、细胞凋亡基因的表达量以及氧化应激标志物的含量。结果子痫前期胎盘组织中PTEN的mRNA表达量显著高于正常胎盘组织(t=32.797、P<0.000),Syncytin-1的mRNA表达量显著低于正常胎盘组织(t=28.506、P<0.000);子痫前期患者母体血液循环中内皮素-1(endothelin-1,ET-1)、可溶性血管内皮生长因子受体-1(soluble fms-like tyrosine kinase receptor 1,sFlt-1)的含量显著高于正常妊娠孕妇(t值分别为39.884、40.243、P均<0.001),且与胎盘组织中PTEN的mRNA表达量呈正相关(相关系数分别为0.674、0.596,P分别为0.004、0.007),与Syncytin-1的mRNA表达量呈负相关(相关系数分别为-0.615、-0.637,P分别为0.009、0.008),一氧化氮(nitric oxide,NO)、前列腺素I 2(prostaglandin I2,PGI2)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、胎盘生长因子(Placental growth factor,PLGF)的含量显著低于正常妊娠孕妇(t值分别为30.944、27.404、32.827、26.644、P均<0.001),且与胎盘组织中PTEN的mRNA表达量呈负相关(相关系数分别为-0.648、-0.592、-0.537、-0.613,P分别为0.006、0.009、0.013、0.007),与Syncytin-1的mRNA表达量呈正相关(相关系数分别为0.632、0.572、0.537、0.654,P值分别为0.010、0.013、0.015、0.009);子痫前期胎盘组织中还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphate-reduced oxidase 4,NOX4)、丙二醛(malondialdehyde,MDA)、8-羟基脱氧鸟苷酸(8-hydroxydeoxyguanosine,8-OHdG)的含量以及自杀相关因子(factors associated suicide,Fas)、Fas配体(fas ligand,FasL)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)、含半胱氨酸的天冬氨酸蛋白水解酶(cysteine-containing aspartic acid proteolytic enzymes-3,Caspase-3)的mRNA表达量显著高于正常胎盘组织(t值分别为27.714、27.704、26.786、22.622、27.964、25.496、25.893、30.837、28.975、P均<0.001),且与胎盘组织中PTEN的mRNA表达量呈正相关(相关系数分别为0.703、0.651、0.593、0.628、0.449、0.552、0.612、0.569、0.502,P值分别为0.003、0.007、0.004、0.006、0.016、0.010、0.006、0.012、0.018),gn Syncytin-1的mRNA表达量呈负相关(相关系数分别为-0.615、-0.714、-0.577、-0.548、-0.512、-0.585、-0.619、-0.495、-0.662,P值分别为0.009、0.001、0.013、0.015、0.018、0.011、0.009、0.023、0.003),Peroxiredoxin-Ⅱ蛋白(PrxⅡ)、PrxⅢ、超氧化物歧化酶(superoxide dismutase,SOD)的含量以及B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)的mRNA表达量显著低于正常胎盘组织(t值分别为26.115、23.502、21.439、17.194、P均<0.001),且与胎盘组织中PTEN的mRNA表达量呈负相关(相关系数分别为-0.713、-0.657、-0.591、-0.642,P值分别为<0.001、0.006、0.009、0.007),与Syncytin-1的mRNA表达量呈正相关(相关系数分别为0.562、0.711、0.682、0.608,P值分别为0.013、<0.001、0.002、0.008)。结论子痫前期胎盘中PTEN的高表达以及Syncytin-1的低表达能够加重母体内皮损伤以及胎盘滋养细胞损伤。     

Local secretion of corticotropin-releasing hormone in the joints of Lewis rats with inflammatory arthritis.

Corticotropin-releasing hormone (CRH), the principal regulator of the hypothalamic-pituitary-adrenal axis, is also secreted in peripheral inflammatory sites, where it acts as a local proinflammatory agent. Arthritis-susceptible LEW/N rats have profoundly deficient hypothalamic CRH responses to inflammatory stimuli and other stressors. Arthritis-resistant F344/N rats, on the other hand, have a robust increase in hypothalamic CRH in response to the same stimuli. Contrasting with these hypothalamic CRH responses, we now show that CRH expression is markedly increased in the joints and surrounding tissues of LEW/N rats with streptococcal cell wall- and adjuvant-induced arthritis, whereas it is not increased in similarly treated F344/N rats and is only transiently increased in congenitally athymic nude LEW.rnu/rnu rats. Glucocorticoid treatment suppressed, but did not eliminate, CRH immunoreactivity in the joints of LEW/N rats. CRH mRNA was present in inflamed synovia, as well as in spinal cord, and inflamed synovia also expressed specific CRH-binding sites. We compared CRH expression in inflamed joints with another well-characterized proinflammatory neuropeptide, substance P (SP), and found that SP immunoreactivity paralleled that of CRH. In summary, although LEW/N rats have deficient hypothalamic CRH responses to inflammatory stimuli compared with F344/N rats, they express relatively high levels of CRH at the site of inflammation. Analogous to SP, CRH may be delivered to the inflammatory site by peripheral nerves and/or synthesized at the inflammatory site. These data provide further support for the concept that CRH not only triggers the pituitary-adrenal antiinflammatory cascade, but also functions as an antithetically active local mediator of acute and chronic inflammatory arthritis. These data also illustrate the complex interrelationships of the nervous, endocrine, immune, and inflammatory systems.     

Effect of a high-fat diet on food intake and hypothalamic neuropeptide gene expression in streptozotocin diabetes.

Insulin-deficient diabetic rats are markedly hyperphagic when fed a high-carbohydrate (HC) diet, but normophagic when fed a high-fat (HF) diet. When maintained on a HC diet, diabetic rats also exhibit increased gene expression of the orexigenic peptide neuropeptide Y (NPY) in the hypothalamic arcuate nucleus, and reduced expression of the anorectic peptide corticotropin-releasing hormone (CRH) in the paraventricular nucleus, and these changes are hypothesized to contribute to diabetic hyperphagia. In this experiment we assessed whether the normophagia displayed by HF-fed diabetic rats is associated with the opposite profile of NPY and CRH expression. Our results show that relative to diabetic rats on the HC diet, the diabetic rats on the HF diet exhibited significantly reduced caloric intake (-40%), NPY expression in the arcuate nucleus (-27%), and elevated CRH expression in the paraventricular nucleus (+37%). Insulin and corticosterone, which are known to affect hypothalamic NPY and CRH expression, were not different between these two groups, making it unlikely that they can account for the differences in either feeding behavior or hypothalamic peptide expression. There was a small but significant increase in plasma leptin levels in the diabetic animals maintained on the HF, and large differences in parameters associated with elevated fat oxidation. These observations support the hypothesis that the normalization of food intake observed in diabetic rats consuming a HF diet may in part be mediated by reductions in NPY expression and elevations in CRH expression.     

Placental glucose transporter gene expression and metabolism in the rat.

In situ hybridization was used to evaluate patterns of gene expression for glucose transporters 1-4 (GT1-4) in the rat uteroplacenta from the time of implantation through term, and in vivo regional placental glucose metabolism was measured by 14C-labeled 2-deoxyglucose uptake. GT1 mRNA was highly abundant and GT3 was barely detected in the postimplantation decidual reaction. GT1 and 3 mRNAs were colocalized in the labyrinthine syncitiotrophoblast layer of the chorioallantoic placenta, which forms the membranous barrier between maternal and fetal circulations. The level of labyrinthine GT3 mRNA showed no change from midgestation through term; however, the volume of the labyrinth and hence total GT 3 gene expression increased greatly during this period. Labyrinthine GT1 mRNA levels, in contrast, showed significant diminution near term. GT1 mRNA was also localized in the placental growth plate, or junctional zone, where it was most abundant during the period of rapid placental growth and was decreased at term. Placental glucose metabolism, as reflected by steady-state 2-deoxyglucose uptake, was highest in the junctional zone during the rapid growth phase during midgestation, and decreased significantly at term, in parallel with GT1 gene expression. These findings suggest that GT1 is responsible for supplying glucose for use as a placental fuel and that GT3 is important for glucose transfer to the embryo.     

17 beta-Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium.

The cDNAs for two separate human 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) have been isolated and sequenced. The well-studied human placental cytosolic 17 beta-HSD (also referred to as estradiol dehydrogenase) preferentially catalyzes the reduction of estrone to estradiol-17 beta and the reduction of the C-20-ketone of progesterone to 20 alpha-dihydroprogesterone. This isoform of the enzyme has been referred to as 17 beta-HSD type 1 and localized to chromosome 17. A second 17 beta-HSD isoform (referred to as type 2) is localized in the endoplasmic reticulum of human trophoblast and is characterized by the preferential oxidation of the C-17 beta-hydroxyl group of C18- and C19-steroids and the C-20 alpha-hydroxyl group of 20 alpha-dihydroprogesterone. In this study, we determined the chromosomal localization of human 17 beta-HSD type 2, the expression of this gene in human endometrium, and the tissue distribution of the mRNA. We found that the human 17 beta-HSD type 2 gene is localized on chromosome 16, 16q24. 17 beta-HSD type 2 mRNA (approximately 1.5 kb) was identified in human endometrial tissues by Northern analysis of total RNA (10 micrograms). The highest levels of 17 beta-HSD type 2 mRNA were found in endometrial tissues obtained during the mid- to late secretory phase of the ovarian cycle (i.e., during the time of high plasma levels of progesterone). 17 beta-HSD type 2 mRNA levels were much greater in glandular epithelium than in the stromal cells isolated from secretory phase endometrium. The levels of 17 beta-HSD type 2 mRNA in secretory phase endometrium were approximately one-tenth that in villous trophoblast tissue from human placenta. We did not detect 17 beta-HSD type 1 mRNA in endometrial tissue by Northern analysis of total (10 micrograms) RNA. These findings are consistent with the view that the progestin-regulated 17 beta-HSD of the glandular epithelium of the human endometrium is primarily, if not exclusively, the product of the 17 beta-HSD type 2 gene. 17 beta-HSD type 2 mRNA was present in human placenta, liver, and small intestine; much smaller amounts, barely detectable by Northern analysis of poly(A)+ RNA, were present in prostate, kidney, pancreas, and colon, but not in heart, brain, skeletal muscle, spleen, thymus, ovary, or testis.     

糖皮质激素对体外培养的胎盘组织促肾上腺皮质激素释放激素分泌水平的影响

目的观察糖皮质激素对胎盘组织促肾上腺皮质激素释放激素(CRH)的分泌水平的影响。方法收集2006年1月-3月住院分娩的正常妊娠妇女的胎盘组织与妊娠肝内胆汁淤积症(ICP)患者胎盘及其血清各10例。分3组进行胎盘组织培养,即正常胎盘组、ICP胎盘组,正常胎盘组织加ICP患者血清组,分别用放射免疫法测定各组加与不加地塞米松胎盘组织培养液中CRH的水平。结果正常组与正常胎盘加地塞米松组培养24、48、72、96 h其CRH分泌水平分别为(:74.81±27.92)、(63.71±24.72)、(91.87±41.64)、(98.90±42.52)pg/mL(;66.94±29.62)、(77.39±31.84)、(61.89±33.94)、(75.13±36.98)pg/mL,两组比较差异有统计学意义(P>0.05)。ICP组与ICP加地塞米松组培养上清液中CRH水平在24、48、72、96 h其CRH分泌水平分别为(:48.28±16.56)、(60.20±29.97)、(72.92±31.65)、(69.22±29.33)pg/mL(;41.81±25.00)、(57.36±39.75)、(57.72±23.29)、(61.43±20.77)pg/mL,两组比较差异有统计学意义(P>0.05);正常胎盘加ICP血清培养组与正常胎盘加ICP血清加地塞米松培养组上清液中CRH水平在24、48、72、96 h其CRH分泌水平分别为:(84.9±34.98)、(74.5±29.93)、(71.1±27.26)、(81.0±37.18)pg/mL;(76.29±33.11)、(63.70±24.20)、(64.85±28.39)、(67.65±33.20)pg/mL,两组比较差异有统计学意义(P>0.05)。3组加入地塞米松培养的胎盘组织,CRH分泌水平并无明显改变。结论地塞米松不影响体外培养胎盘组织CRH分泌。     

宫内暴露于高雄激素对小鼠胎盘营养转运体表达的影响

目的探讨宫内暴露于高雄激素对小鼠胎盘营养转运体表达的影响。方法选择2018年1-3月购置的6~8周龄ICR雌性小鼠,自然受精后6.5 d开始每天给予颈部皮下注射脱氢表雄酮(60 mg·kg^-1·d^-1)构建实验组,单纯玉米油注射构建对照组,第18.5天采用颈椎脱臼法处死小鼠后,收集胎鼠及胎盘标本,比较两组小鼠存活率、胎鼠体质量、胎盘质量、胎盘转运效率及目的基因mRNA表达水平。结果实验组与对照组胚胎总着床数、存活数差异均无统计学意义(P>0.05);实验组平均窝仔数、存活率低于对照组(P<0.05);实验组与对照组胎盘转运效率差异无统计学意义(P>0.05);实验组胎鼠体质量、胎盘质量均低于对照组(P<0.05);实时荧光定量PCR法检测两组胎盘的葡萄糖转运体mRNA表达水平,结果表明,与对照组相比,实验组胎盘Slc2a1、Slc2a2、Slc2a3、Slc2a9、Slc2a12 mRNA表达水平均出现不同程度下调(P<0.05);实验组Slc38a1、Slc38a2、Slc38a4、Slc43a2、Slc7a5、Slc7a8 mRNA表达水平下调(P<0.05)。结论宫内暴露于高雄激素中能降低胎盘质量与胎鼠体质量,能降低胎盘氨基酸转运体和葡萄糖转运体mRNA表达水平,提示宫内暴露于高雄激素会影响妊娠末期胎盘营养转运能力。     

Identification of targets of leptin action in rat hypothalamus.

The hypothesis that leptin (OB protein) acts in the hypothalamus to reduce food intake and body weight is based primarily on evidence from leptin-deficient, ob/ob mice. To investigate whether leptin exerts similar effects in normal animals, we administered leptin intracerebroventricularly (icv) to Long-Evans rats. Leptin administration (3.5 microg icv) at the onset of nocturnal feeding reduced food intake by 50% at 1 h and by 42% at 4 h, as compared with vehicle-treated controls (both P < 0.05). To investigate the basis for this effect, we used in situ hybridization (ISH) to determine whether leptin alters expression of hypothalamic neuropeptides involved in energy homeostasis. Two injections of leptin (3.5 microg icv) during a 40 h fast significantly decreased levels of mRNA for neuropeptide Y (NPY, which stimulates food intake) in the arcuate nucleus (-24%) and increased levels of mRNA for corticotrophin releasing hormone (CRH, an inhibitor of food intake) in the paraventricular nucleus (by 38%) (both P < 0.05 vs. vehicle-treated controls). To investigate the anatomic basis for these effects, we measured leptin receptor gene expression in rat brain by ISH using a probe complementary to mRNA for all leptin receptor splice variants. Leptin receptor mRNA was densely concentrated in the arcuate nucleus, with lower levels present in the ventromedial and dorsomedial hypothalamic nuclei and other brain areas involved in energy balance. These findings suggest that leptin action in rat hypothalamus involves altered expression of key neuropeptide genes, and implicate leptin in the hypothalamic response to fasting.     

妊娠期高血压疾病患者胎盘组织叶酸代谢基因MTR 的表达水平及其作用机制研究

目的 研究叶酸代谢相关基因甲硫氨酸合成酶(methionine synthase,MTR) 在妊娠期高血压疾病患者胎盘组织中的差异表达以及对于人绒毛滋养层细胞HTR8 迁移及侵袭能力调控的机制研究。方法 2018 年1 月～ 2020 年12 月在成都市龙泉驿区妇幼保健院分娩的30 例妊娠期高血压,28 例子痫前期和20 例重度子痫前期患者及25 例正常产妇作为研究对象,分娩时采集研究对象胎盘组织。使用实时荧光定量PCR(real-time quantitative PCR detecting system,qPCR)及蛋白质印迹法(western blotting,WB) 检测了各组胎盘组织中MTR,上皮型钙黏附蛋白(E-Cadherin,E-Cad),锌指E 盒结合蛋白-1(zinc finger E-box binding protein1,ZEB1) 基因转录及蛋白表达情况。进一步通过siRNA 敲减人绒毛滋养层细胞HTR8 中MTR 基因的表达,研究MTR 对于E-Cadherin 和ZEB1 基因的调控作用。通过Transwell 迁移及侵袭实验验证敲减MTR 后对于HTR8 细胞迁移和侵袭的影响。结果 与对照组相比,随着妊娠期高血压疾病分期的进展,子痫前期与重度子痫前期患者胎盘组织中E-Cadherin 的表达增加（1.14±0.35, 1.53±0.41 vs 1.00±0.30）,胎盘组织中MTRmRNA 的表达量逐渐增加（1.72±0.17, 2.58±0.13 vs 1.00±0.33）,妊娠期高血压疾病患者胎盘组织中ZEB1 的表达量下降（0.48±0.10, 0.13±0.06 vs 1.00±0.22）,差异具有统计学意义（F=13.28,67.1,65.41,均 P ＜ 0.01）。随着疾病分期进展,MTR 及E-Cadherin 的蛋白表达量明显增加,而ZEB1 的蛋白表达量明显降低。siRNA 可以显著抑制HTR8中MTR 基因mRNA 的表达,敲低MTR 基因后可显著抑制上皮相关标志物E-Cadherin 基因mRNA 的表达, 可以显著促进间充质相关标志物ZEB1 基因mRNA 的表达,差异具有统计学意义（t=5.906,6.715,9.777, 均 P ＜ 0.01）。敲减HTR8 细胞中MTR 基因可显著促进迁移能力,侵袭实验结果显示敲减MTR 基因可促进HTR8 细胞的侵袭能力,差异具有统计学意义（t=6.241,22.37, 均 P ＜ 0.01）。结论 妊娠期高血压疾病患者胎盘组织中MTR 基因表达上调, MTR可通过调节HTR8 细胞EMT 相关基因E-Cadherin,ZEB1 表达,从而抑制其迁移及侵袭能力。MTR 可能成为妊娠期高血压疾病潜在的治疗靶点之一。     

Placental corticotropin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans.

To clarify the physiological role of placental corticotropin-releasing hormone (CRH), we measured plasma CRH, ACTH, and cortisol throughout pregnancy. Cerebrospinal fluid (CSF) CRH levels and ACTH responsiveness to synthetic CRH were also quantified in pregnant and nonpregnant women. Maternal plasma CRH levels, which increased progressively during pregnancy, correlated well with both ACTH and cortisol in early labor, delivery, and postpartum samples, and also with cortisol levels in samples before labor. CSF CRH levels in term pregnant women did not differ from those of nonpregnant women. CRH infusion that attained similar plasma CRH levels to those found in late pregnancy elicited significant ACTH release in vivo and regular CRH test provoked normal ACTH response during early pregnancy but no response during late pregnancy. We concluded that: (a) maternal pituitary-adrenal axis correlates well with plasma CRH levels, which are high enough to provoke ACTH release from maternal pituitary; (b) hypothalamic CRH secretion in term pregnant women is not exaggerated; and (c) maternal pituitary is responsive to synthetic CRH in early but not late pregnancy, suggesting that maternal pituitary-adrenal axis is already activated by high circulating CRH. Placental CRH may be an important stimulator of the maternal pituitary-adrenal axis during pregnancy.     

Comparative in vivo approaches for selective adenovirus-mediated gene delivery to the placenta

Gene delivery to the placenta is one potential way of specifically modifying placental biological processes and fetal development. The aim of this study was to determine the most efficient and least invasive route of placental adenovirus delivery. The feasibility of adenovirus-mediated gene transfer to the rat placenta was addressed by maternal intravenous or direct intraplacental injection of adenoviral vectors expressing the glucose transporter GLUT3, a noncirculating integral membrane protein. Both routes led to transgene expression in the placenta. However, direct intraplacental delivery on day 14 of gestation yielded a higher transduction efficiency than maternal intravenous administration, and markedly reduced transgene expression in maternal liver. Most importantly, the amount of the GLUT3 transgene and the adenovirus itself in fetal tissues was only 1 to 3% of that found in the placenta. These results indicate that the nature of the transgene and the route of adenovirus administration are key parameters in selective placental somatic gene transfer. This novel strategy may prove useful for modifying a placental function without altering the fetal genome.     

妊娠肝内胆汁淤积症胎盘组织促肾上腺皮质激素释放激素分泌水平研究

目的观察妊娠肝内胆汁淤积症(ICP)胎盘组织促肾上腺皮质激素释放激素(CRH)的分泌水平。方法通过胎盘组织培养,放免法测定胎盘组织培养液中CRH的水平。结果ICP胎盘组织CRH基础分泌量低于正常胎盘(P<0.05),但48h后却高于正常胎盘组织CRH分泌量(P<0.05)。结论可能因ICP胎盘组织细胞暴露于某些因素而抑制其CRH分泌,导致其CRH分泌量低于正常胎盘。当这些暴露因素去除以后,ICP胎盘组织细胞CRH分泌水平呈升高趋势。     

Corticotropin-releasing hormone, proopiomelanocortin, and glucocorticoid receptor gene expression in adrenocorticotropin-producing tumors in vitro.

To differentiate between ectopic ACTH syndrome and Cushing's disease, gene expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and glucocorticoid receptor was examined in 10 pituitary adenomas (Cushing's disease) and in 10 ectopic ACTH-producing tumors. CRH increased plasma ACTH levels in all patients with Cushing's disease and in five patients with ectopic ACTH syndrome whose tumors contained CRH and CRH mRNA. In five CRH nonresponders, CRH was not detected in tumors that contained no CRH mRNA or that contained only long-size CRH mRNA. Dexamethasone (Dex) decreased plasma ACTH levels in all patients with Cushing's disease and in three patients with ectopic ACTH-producing bronchial carcinoid. These tumors contained glucocorticoid receptor mRNA. CRH increased and Dex decreased ACTH release and POMC mRNA levels in pituitary adenoma and bronchial carcinoid cells. PMA increased POMC mRNA levels only in carcinoid cells. These results reveal characteristics of ectopic ACTH-producing tumors: long-size CRH mRNA and PMA-induced POMC gene expression. In addition, there are two ectopic ACTH syndrome subtypes: tumors containing ACTH with CRH (CRH responder) and tumors without CRH. Dex decreases ACTH release and POMC mRNA levels in some bronchial carcinoids. Therefore, CRH and Dex tests have limited usefulness in differentiating between Cushing's disease and ectopic ACTH syndrome.     

Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.     

水通道蛋白8在人胎盘胎膜中的表达

背景:胎盘和胎膜在母胎液体平衡过程中起着重要作用,而水穿过胎盘和胎膜运输的分子和细胞机制目前尚不十分清楚,推测母胎液体交换可能通过胎盘和胎膜上的水通道进行.目的:观察人正常妊娠晚期胎盘与胎膜组织中水通道蛋白8蛋白质水平的表达及其分布.设计、时间及地点:对比观察,于2005-07/12在广州医学院实验中心完成.材料:收集正常足月妊娠剖宫分娩的胎盘与胎膜组织样本各5例,产妇年龄(27±5)岁,实验方法获医院伦理委员会批准.方法:胎盘胎膜组织在分娩30 min内取样,用无菌生理盐水漂洗干净去除血液,立即放入液氮中,转运至-80℃冰箱保存备用.主要观察指标:运用反转录-聚合酶链反应法、免疫组织化学法及Western印迹法检测水通道蛋白8在胎盘与胎膜组织中的表达与分布.结果:反转录-聚合酶链反应显示,水通道蛋白8 mRNA在胎盘和胎膜组织中均有表达.水通道蛋白8 Western印迹检测胎盘和胎膜组织显示,一特异性条带在Mr45 000左右.免疫组织化学结果显示,水通道蛋白8表达于胎盘合体滋养细胞、羊膜上皮细胞及平滑绒毛膜细胞滋养细胞.结论:在蛋白质水平显示,水通道蛋白8表达于胎盘的合体滋养细胞、羊膜上皮细胞及平滑绒毛膜细胞滋养细胞.     

Lactation as a model for naturally reversible hypercorticalism plasticity in the mechanisms governing hypothalamo-pituitary- adrenocortical activity in rats.

Steady state levels of hypothalamic expression of the genes encoding corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), arginine vasopressin (AVP), and oxytocin (OT) were studied in rats to investigate the mechanisms underlying the transitions between hypercorticalism during lactation and normocorticalism upon weaning. During lactation, CRH mRNA levels and blood titers of adrenocorticotropin (ACTH) were found to be significantly reduced, although POMC mRNA levels in the anterior pituitary were not significantly different from those found in cycling virgin (control) rats; during all phases of lactation, an inverse relationship was observed between the blood levels of ACTH and corticosterone (CORT). Plasma prolactin (PRL) concentrations were elevated approximately 30-fold during lactation. Whereas steady state levels of OT mRNA were markedly increased throughout lactation, those of AVP mRNA were only transiently (initially) elevated, and the blood levels of these hormones were not significantly altered in lactating as compared with cycling virgin and postlactating rats. CRH and POMC gene expression and blood levels of ACTH, CORT, and PRL were normalized within 1-3 d of removal of suckling pups. The temporal relationships between the biosynthetic profiles of the various peptide hormones and the patterns of ACTH and CORT secretion during the two physiological states suggest that lactation-associated hypercorticalism does not merely result from increased ACTH secretion; although still not well substantiated at this time, the evidence points to contributory roles of PRL, OT, and AVP in the hypercorticalismic state found during lactation.