Receptor expression by JEG-3 trophoblast cells in the presence of placenta secreted factors

Abstract

Preeclampsia still remains one of the most severe pregnancy complications and is an actual problem in the obstetrics practice. At present, the joint impact of cytokines and other placenta secreted factors on trophoblast cell functional activity during preeclampsia complicated pregnancy remains unclear. The aim of the study is to estimate the surface receptors expression by trophoblast cells in the presence of placenta secreted factors during physiological pregnancy and at preeclampsia. Trophoblast cells of the JEG-3?line were incubated in the presence of supernatants obtained by cultivation of placentas from women with physiological pregnancy and with preeclampsia. Surface receptors expression by trophoblast cells was estimated by FACS Canto II flow cytometer. It was established that in the third trimester both under normal and pathological conditions, the placenta secreted factors impact on the cytokine receptor expression by trophoblast differs while the trophoblast response capacity to the migration and proliferation stimulating and inhibiting signals remains stable. JEG-3?line cells enhanced the expression of CD186, CD140a, Integrin β6, VE-cadherin, CD29, and CD140a in the case of incubation in the presence of placenta supernatants from the third-trimester pregnancy complicated with preeclampsia compared to incubation in the presence of placenta supernatants form the third trimester of physiological pregnancy.     

同源异型盒基因DLX4在妊娠期高血压疾病中的表达研究

目的:检测妊娠期高血压疾病胎盘组织中同源异型盒基因DLX4的表达,探讨其在妊娠期高血压疾病发生、发展中的意义。方法:严格设置病例组和对照组,采用半定量逆转录聚合酶链反应法(RT-PCR)和免疫组化SABC法检测40例胎盘组织中DLX4mRNA和蛋白的表达情况。结果:DLX4 mRNA在子痫前期胎盘组织中的表达(轻度为0.17±0.05、重度为0.09±0.03)明显低于对照组(0.49±0.07,P<0.001)。DLX4蛋白在子痫前期胎盘中表达与对照组相比也显著降低(P<0.001)。结论:随着妊娠期高血压疾病临床症状的加重,胎盘组织中DLX4的表达呈明显下降趋势,同源异型盒基因DLX4参与了妊娠期高血压疾病胎盘的发育障碍及功能缺陷。     

过表达NDRG1通过调控Wnt/β-catenin信号通路促进滋养层细胞上皮-间质转化

目的:探讨N-myc下游调节基因1(NDRG1)过表达对滋养层细胞上皮-间质转化(EMT)的影响以及对Wnt/β-catenin信号通路的作用。方法:采用携带NDRG1基因的慢病毒感染人胎盘滋养层细胞系HTR-8/SVneo,建立NDRG1基因稳定过表达的HTR-8/SVneo细胞株。qRT-PCR和Western blot法检测慢病毒感染后细胞中NDRG1基因mRNA和蛋白的表达水平,Transwell小室法观察细胞侵袭和迁移能力,免疫荧光实验检测β-catenin蛋白核转位情况,Western blot法分析EMT相关蛋白、Wnt/β-catenin信号通路相关蛋白及基质金属蛋白酶-2(MMP-2)和MMP-9等蛋白表达。结果:慢病毒介导NDRG1过表达后,HTR-8/SVneo细胞中NDRG1 mRNA和蛋白表达水平显著上调。NDRG1过表达可显著增强细胞的侵袭和迁移能力,并上调MMP-2、MMP-9、波形蛋白(Vimentin)、神经性钙黏附蛋白(N-cadherin)、β-catenin、Snail1和环氧化酶-2(COX-2)等蛋白的表达水平,下调上皮钙黏蛋白(E-cadherin)和糖原合成酶激酶3β(GSK3β)磷酸化水平,同时促进β-catenin蛋白由细胞质向细胞核转位。结论:NDRG1过表达可促进HTR-8/SVneo细胞EMT过程,提高细胞侵袭和迁移能力,其机制可能与激活Wnt/β-catenin信号通路转导相关。     

STOX1在子痫前期滋养细胞炎症反应中的作用研究#br#

Objective?To investigate the STOX1 (Storkhead box1) in the inflammatory response of trophoblast cells simulated by human choriocarcinoma cell line JEG3 cells. Methods?The human choriocarcinoma cell line JEG3 cell line was used to mimic trophoblast cells, and a stable transitional cell line model with overexpression/knockdown of STOX1 gene was constructed. 0.05 mmol/L aspirin was added to the experimental and control groups, respectively. Cell migration ability was measured by Transwell, and apoptosis was detected in each group by flow cytometry, and real-time quantitative PCR and protein immunoblotting were used to detect the expression of cell-associated inflammatory, hypoxic and apoptotic factors, biological behavior, and the relative expression of mRNA and protein of molecular biology. Results?Hypoxia-inducible factor α(HIF-1α), endothelial-type nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), and apoptosis B lymphocytoma-2 (Bcl-2) were significantly reduced in the overexpression STOX1 trophoblast group (P<0.01); meanwhile, inflammatory response-related factor nuclear factor κB (NF-κB), tumor necrosis factor α (TNF-α), interleukin 8 (IL-8) and cysteine protease-3 (Caspase-3) were significantly increased at both gene and protein levels (P<0.05). The expression of hypoxia-inducible genes HIF-1α (P<0.01), eNOS (P<0.05), and iNOS (P<0.001) and anti-apoptotic genes Bcl-2 (P<0.01) was elevated after knockdown of STOX1, while inflammatory responses NF-κB (P<0.05), TNF-α (P<0.01), IL-8 (P<0.01), and Caspase-3 (P>0.05) levels were reduced. Conclusion?STOX1 induces inflammatory response, promotes hypoxia and apoptosis-related gene expression in JEG3 cell line, aspirin may inhibit the effect of STOX1, reduce apoptosis in JEG3 cells, decrease inflammatory response and improve hypoxia in trophoblast cells, and knockdown of STOX1 has synergistic effect with the use of aspirin.     

RNA干扰技术抑制HLX1基因表达对滋养细胞生物学性能的影响

目的:研究小分子干扰RNA(siRNA)技术抑制人类同源异型盒基因HLX1的表达对胎盘滋养细胞株HTR-8/SVneo细胞生物学性能的影响。方法:常规体外培养HTR-8/SVneo细胞,设实验组(转染HLX1基因的特异性siRNA)、阴性对照组(转染阴性对照siRNA)及空白对照组(除不含siRNA片段,余试剂与另两组相同)3组分别进行瞬时转染。用流式细胞术测定siRNA转染效率,应用实时定量PCR技术和蛋白印迹法测定转染后各组HTR-8/SVneo细胞中HLX1 mRNA和蛋白的表达;应用MTT比色实验、Matrigel侵袭实验分别检测转染后各组细胞的增殖能力与侵袭能力的变化。结果:(1)转染24h后测得siRNA转染效率达(86.3±2.6)%。(2)转染48h后HLX1 mRNA的表达水平下调(77.0±1.2)%(P<0.01),转染72h后HLX1蛋白的表达水平下调(82.6±1.2)%(P<0.01),与HLX1 mRNA的表达水平下调相一致。(3)转染HLX1 siRNA 24h后,HTR-8/SVneo细胞的增殖活性已受到抑制,转染72h后细胞增殖抑制最显著,抑制率达到(58.1±4.4)%(P<0.01)。(4)转染HLX1 siRNA后,HTR-8/SVneo细胞侵袭Matrigel基质胶的能力受到显著抑制,其穿膜细胞数为(29±3)个,与空白对照组(穿膜细胞数为[53±8]个)相比,差异有统计学意义(P<0.01)。结论:HLX1基因表达水平的下降可以显著抑制滋养细胞的增殖活性与侵袭能力;HLX1基因表达异常可能通过影响滋养细胞增殖、侵袭等过程参与IFGR、子痫前期等疾病的发生。     

DLX4在子痫前期胎盘组织的表达及与上皮细胞间质化相关性的研究

目的:探讨DLX4在妊娠期高血压疾病中的作用及其对滋养细胞EMT的影响。方法:采用Western blot法分别检测子痫前期胎盘组织及缺氧培养的滋养细胞系JEG-3中DLX4蛋白表达的变化;通过实时PCR技术,比较缺氧条件下滋养细胞中DLX4、HIF-1α和E-cad表达变化的差异。结果:滋养细胞缺氧培养后DLX4蛋白表达明显下降(P0.05),与其在子痫前期胎盘组织的表达变化一致;缺氧抑制滋养细胞中DLX4 mR-NA表达,同时伴有HIF-1α和E-cad mRNA表达增加。结论:缺氧引起滋养细胞DLX4表达下调,伴随EMT相关因子表达的改变。子痫前期滋养细胞侵袭力异常可能与缺氧引起DLX4表达下调及滋养细胞EMT异常有关。     

Altered protease expression by periarterial trophoblast cells in severe early-onset preeclampsia with IUGR

Adaptation of uteroplacental arteries in patients with early-onset preeclampsia combined with IUGR is compromised due to insufficient invasion of extravillous trophoblast cells (EVT) into the spiral artery wall. The underlying molecular mechanisms are widely unknown. We investigated expression and possible mechanisms of regulation of different matrix-metalloproteases (MMPs) by EVT in placental bed biopsies from patients with early onset preeclampsia combined with IUGR and healthy pregnant women. Expression of MMP-3 and MMP-7 by EVT was markedly reduced in preeclamptic patients, especially close to spiral arteries. In contrast to healthy pregnancies these cells strongly expressed the receptor for leukemia inhibitory factor (LIF). LIF is known to suppress MMP-expression and is produced by uterine natural killer (uNK) cells which we found to be present in higher concentrations in the placental bed of preeclamptic patients, and accumulating aside the spiral arteries. We speculate that in preeclampsia a maternal immune cell network accumulating and interfering in the placental bed leads to an altered cytokine environment, resulting in disturbed trophoblast cell function such as impaired MMP expression and reduced invasiveness.     

Upregulation of HLA-G in JEG-3 cells by dexamethasone and hydrocortisone

Background  

Various reports suggest that HLA-G molecule plays an important role in feto-maternal interface, protecting the allogenic fetus from maternal immune attack. It is shown that steroid hormones may upregulate the HLA-G gene expression. In the present study, we have made an attempt to upregulate the HLA-G gene expression in a HLA-G^+ve cell line (JEG-3) by using two glucocorticoids drugs, i.e., dexamethasone and hydrocortisone.     

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-μn pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of a5 and a6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly (P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG (P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl CAMP (db cAMP) and the CAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell (JEG-3) invasion. The collagenolytic activity of trophoblastic cells (IEG-3) was increased by hCG stimulation. No changes were shown in the expression of α5 and α6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.     

Regulation by hypoxia of adrenomedullin output and expression in human trophoblast cells

Objectives

Plasma adrenomedullin concentrations are increased in the fetal circulation in acute and chronic hypoxic conditions. The effect of hypoxia in regulating adrenomedullin synthesis and secretion was investigated in human placental trophoblast cells.

Study design

Human trophoblast cells obtained from term placentas (n = 7) were cultured in hypoxic condition (3% oxygen). Cytotrophoblast cells were cultured for up to 48 h and syncytiotrophoblasts for 2, 8 and 24 h. Changes in adrenomedullin output compared to normoxic conditions were measured by radioimmunoassay. Protein expression was evaluated with Western blot and immunocytochemistry.

Results

Hypoxia induced a time-dependent increase in adrenomedullin output and protein expression by placental trophoblast cells.

Conclusions

Hypoxia regulates adrenomedullin secretion and expression by human placenta, thereby promoting increased adrenomedullin concentration in the fetal circulation in clinical circumstances characterized by reduced oxygen levels.     

Expression of homeobox gene transcripts in trophoblastic cells

OBJECTIVE: This study was conducted to examine the dynamics of homeobox gene expression in the differentiation of trophoblasts as a key to the understanding of the regulatory mechanisms that are involved in placental development. STUDY DESIGN: Expression of homeobox genes was examined in primary trophoblastic cells and in the BeWo choriocarcinoma model cell lines by molecular and immunocytochemistry techniques. RESULTS: We demonstrated the expression of 3 homeobox genes (HOX B6, HOX C6, and HOX A11) in primary trophoblastic cells. BeWo cells showed an expression pattern similar to that of the primary cell lines. In both primary trophoblasts and BeWo cells, the HOX A11 gene, but not the HOX B6 or HOX C6 genes, were found to down-regulate with differentiation from single- to multinucleate giant cells. CONCLUSION: This study demonstrates a novel expression pattern for HOX A11 gene in trophoblastic differentiation and suggests that the down-regulation of HOX A11 may be necessary for the differentiation of cytotrophoblasts into syncytiotrophoblasts.     

Accelerated expression of Ca antigen by placental villous trophoblast in preeclampsia

The Ca antigen is expressed by a range of normal tissues, as well as by many malignant neoplasms. The former includes placental villous syncytiotrophoblast where it appears first on syncytial sprouts at 20 weeks gestation, and progressively becomes more intensely and widely expressed on the surface of the trophoblast of villi of all sizes. We report here the prematurely intense expression of the antigen in pre-eclampsia (22 cases), by comparison with control sections from uncomplicated pregnancies. The expression of this antigen may have value as a relatively objective marker of accelerated maturation of the placental villous tree. It is suggested that the premature expression of Ca antigen in villous trophoblast in pre-eclampsia is due to external modification of a temporally related gene activation.     

The role of trophoblast binucleate cells in implantation in the goat: a quantitative study

The number of goat trophoblastic binucleate cells, the incidence of their migration and the formation of trinucleate and syncytial cells in the maternal uterine epithelial layer was estimated quantitatively using transmission electron microscopy between 14 and 23 days postcoitum (dpc). Binucleate cells were first observed at 18 dpc and their proportions increased rapidly from less than 1 per cent to 16 per cent by 19 dpc and 22 per cent by 23 dpc. The appearance of trinucleate cells within the maternal uterine epithelial layer coincided with evidence of migration and fusion of binucleate cells with individual uterine epithelial cells, and an increased death rate among the other uninucleate uterine epithelial cells. There was also a slight increase in the incidence of intraepithelial lymphocytes close to the trinucleate cells. The quantitative studies uphold the hypothesis that at implantation in the goat, placental trinucleate cells and their subsequent multinucleate syncytial plaque derivatives are fetomaternal hybrid tissue formed by fusion of a binucleate cell(s) with a single uterine epithelial cell.