米非司酮对人早孕绒毛及蜕膜中骨桥蛋白和绒毛膜促性腺激素-β表达的影响

目的:探讨米非司酮对早孕绒毛及蜕膜中骨桥蛋白(OPN)和绒毛膜促性腺激素-β(β-hCG)表达的影响。方法:采用免疫组化半定量法检测米非司酮药物流产(n=20)和人工流产(n=20)的早孕绒毛及蜕膜中的OPN(骨桥蛋白)和β-hCG的表达。结果:OPN和β-hCG在早孕妇女绒毛组织和蜕膜组织中的表达米非司酮组均显著低于手术流产组,差异有统计学意义(P<0.01)。结论:米非司酮可能抑制了早孕绒毛及蜕膜中OPN和β-hCG的合成,进而影响胚泡着床和胎盘的形成,使妊娠难以维持,最终流产。     

白血病抑制因子在早孕蜕膜组织中的表达

目的 探讨白血病抑制因子（ＬＩＦ）ｍＲＮＡ及其蛋白在人类早孕蜕膜组织中的表达及定位。方法 采用原位杂交及免疫组织化学方法对１６例正常早孕妇女蜕膜组织中的ＬＩＦ ｍＲＮＡ及其蛋白的表达进行研究。结果 全部早孕蜕膜组织标本中腺体及间质均有ＬＩＦｍＲＮＡ及其蛋白表达，腺体的中ＬＩＦ基因表达高于基质。结论 人类蜕膜组织中ＬＩＦ表达可能与胚泡着床、维持胎盘功能和促进胚胎发育有关。     

Galectin-1基因在人孕早期绒毛和蜕膜组织中表达的研究

目的：探讨早孕绒毛和蜕膜组织中Galectin-1mRNA及蛋白的表达与胚胎发育和胎盘形成的关系。方法：对70例非意愿妊娠的6-12周正常早孕行人工流产者,按孕周分为7组,每组10例。收集其绒毛和蜕膜组织,分别采用半定量RT-PCR及Western blot方法检测Galectin-1mRNA和蛋白的表达水平。结果：在整个孕早期绒毛组织中都有Galectin-1mRNA的表达,孕6周表达最低,随孕周增加而逐渐增高,孕12周最高,Galectin-1蛋白表达与mRNA表达趋势一致；其在蜕膜组织中的表达与绒毛组织中类似。结论：Galectin-1mRNA和蛋白在早孕绒毛和蜕膜组织中的表达均随孕周增加而增高,提示该基因在早孕期胎盘形成过程中起重要作用。     

人类白细胞抗原G、E在人胎盘组织中的表达及其意义

目的:探讨人类白细胞抗原G、E(HLA-G、E)在人胎盘组织中的表达及意义。方法:用原位杂交及免疫组化法分别检测HLA-G、E mRNA及蛋白质在人正常早孕绒毛组织、晚孕胎盘组织中的表达。结果:在人正常早孕绒毛组织中的绒毛细胞滋养细胞、合体滋养细胞及绒毛外滋养细胞(EVCT)内均有HLA-G、E mRNA及HLA-E蛋白质表达,而HLA-G蛋白质仅在EVCT内表达;晚孕胎盘组织中HLA-G、E mRNA及蛋白质表达于蜕膜板内的EVCT及羊膜上皮细胞。结论:HLA-E表达于人正常早孕绒毛组织中所有类型的滋养细胞及晚孕胎盘组织中的EVCT和羊膜上皮细胞。抗人HLA-G单克隆抗体4H84仅能检测出人胎盘组织中EVCT及羊膜上皮细胞内的HLA-G蛋白。     

凝集素受体WGA、RCA和ECL在米非司酮流产绒毛及蜕膜组织中表达的研究

目的:通过麦胚凝集素(WGA)受体、蓖麻凝集素(RCA)受体和鸡冠珊瑚树凝集素(ECL)受体在米非司酮流产的绒毛及蜕膜组织中的表达,探讨米非司酮的抗早孕机理。方法:以生物素标记的凝集素WGA、RCA和ECL为探针,应用亲和组织化学染色法检测3种凝集素受体在米非司酮流产组(实验组)和人工流产组(对照组)早孕绒毛及蜕膜组织上皮细胞及其间充质细胞的分布。结果:实验组WGA受体、RCA受体和ECL受体在蜕膜组织上皮细胞的表达比对照组均显著降低(P0.01)。实验组ECL受体在绒毛上皮的表达显著低于对照组(P0.01),实验组WGA和RCA受体在绒毛上皮细胞的表达与对照组无显著差异(P0.05)。实验组3种凝集素受体在绒毛及蜕膜的间充质细胞的表达,与对照组无显著差异(P0.05)。结论:米非司酮可能通过下调蜕膜上皮组织中WGA受体、RCA受体和ECL受体的表达,阻碍胚胎发育而导致流产。     

细胞外基质金属蛋白酶诱导因子在绒毛和胎盘组织中的表达

目的 探讨细胞外基质金属蛋白酶诱导因子(EMMPRIN)在绒毛及胎盘组织中的表达以及表达部位。方法 分别取36例妊娠6～9周妇女的早孕绒毛组织、7例因病理指征行中期引产妇女的胎盘组织和11例足月妊娠妇女的胎盘组织,免疫组化方法检测绒毛组织和胎盘组织中EMMPRIN的表达部位变化特点。结果 (1)表达部位：EMMPRIN在早孕绒毛组织、中期和足月妊娠的胎盘组织中均有高度表达。在早孕绒毛组织中的表达部位主要集中在绒毛内细胞滋养细胞、合体滋养细胞及细胞滋养层柱细胞;在中期和足月妊娠的胎盘组织中,主要表达于底蜕膜的绒毛外细胞滋养细胞。(2)表达特点：在早孕绒毛组织中细胞滋养细胞、合体滋养细胞和细胞滋养层柱细胞的。EMMPRIN阳性率随妊娠进展逐渐下降。在妊娠中期的胎盘组织中,细胞滋养细胞、合体滋养细胞和细胞滋养层柱细胞的EMMPRIN阳性率分别为5／7、3／7、5／7;在足月妊娠的胎盘组织中,细胞滋养细胞、合体滋养细胞和细胞滋养层柱细胞的EMMPRIN阳性率分别为73％、18％和82％。在中期和足月妊娠的底蜕膜中的EMMPRIN阳性率较弱,且趋于稳定。而早期妊娠阶段,侵入到底蜕膜的绒毛外细胞滋养细胞中。EMMPRIN阳性表达则随孕周进展逐渐增强。结论 EMMPRIN在妊娠早期与胚胎植入有关,在妊娠的中晚期则可能参与妊娠维持。     

自然流产蜕膜及绒毛组织中钙网蛋白的表达及意义

目的:检测早期自然流产蜕膜及绒毛组织中钙网蛋白(CRT)和凋亡蛋白酶Caspase-3的表达及定位,初步探讨其在早期自然流产过程中的作用及相关性。方法:正常早孕者和早期自然流产者(先兆流产组、稽留流产组)行负压吸引人工流产术,取蜕膜及绒毛组织,HE染色比较其形态学变化,免疫荧光法检测CRT及Caspase-3的表达,统计学分析其相关性。结果:(1)CRT及Caspase-3在早期自然流产蜕膜及绒毛组织中表达高于正常早孕组;(2)稽留流产组蜕膜及绒毛组织中CRT及Caspase-3的表达均高于先兆流产组;(3)CRT与Caspase-3呈正相关。结论:CRT及Caspase-3异常表达可能参与早期自然流产的发生机制。     

绒毛外滋养细胞浸润能力的调节

细胞滋养细胞起源于胚泡的滋养外胚层，是胎盘的主要细胞。妊娠早期，细胞滋养细胞代表着一个异源细胞群，在胚泡植入子宫壁后，其沿两条途经分化，即分化为绒毛滋养细胞(VCTs)和绒毛外滋养细胞(EVTs)^[1]。。其中EVTs是具有浸润能力的滋养细胞。Aplin^[2]将其分为浸润型和非浸润型两种亚型。定位在蜕膜组织的EVTs称间质滋养细胞。     

细胞因子信号抑制因子3(SOCS3)在先兆流产绒毛和蜕膜组织中的表达

目的:初步探讨早孕母胎界面细胞因子信号抑制因子3(SOCS3)的作用。方法:26例妊娠35-49d先兆流产患者,采用RT-PCR法及Western blotting法、免疫组化法检测绒毛、蜕膜组织SOCS3的表达,并以30例正常早孕者作对照。结果:①绒毛、蜕膜组织均表达SOCS3,但先兆流产组SOCS3表达明显低于对照组,差异有统计学意义(P<0.05)。②绒毛中的SOCS3表达于滋养细胞和绒毛间质,蜕膜中的SOCS3主要位于蜕膜腺体及间质细胞,二组SOCS3表达定位一致。结论:母胎界面SOCS3可能在早期妊娠的维持中起重要作用,其表达水平降低可能与先兆流产的发生、发展有关。     

血红素氧合酶在正常妊娠绒毛和胎盘组织中的表达及定位

目的　探讨血红素氧合酶 (HO)的两种同工酶基因及蛋白在正常早期妊娠绒毛和晚期妊娠胎盘组织中的表达。方法　选择正常妊娠 6～ 10周的孕妇 (早孕组 )和妊娠 3 7～ 41周的孕妇 (晚孕组 )各 2 0例 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测绒毛和胎盘组织中诱导型HO(HO 1)mRNA及结构型HO(HO 2 )mRNA的表达 ;并采用免疫组织化学 (免疫组化 )方法 ,进行HO蛋白定位和半定量分析。结果　两组HO 1mRNA的表达均较弱 ,早孕组绒毛HO 1mRNA表达水平为 ( 0 3 1±0 19) ,晚孕组胎盘HO 1mRNA表达水平为 ( 0 2 8± 0 14 ) ,两组比较 ,差异无显著性 (P >0 0 5) ;而两组HO 2mRNA的表达均较强 ,晚孕组胎盘表达水平为 ( 1 12± 0 58) ,早孕组绒毛表达水平为 ( 0 70±0 48) ,两组比较 ,差异有显著性 (P <0 0 5)。免疫组化定位结果显示 ,HO 1蛋白主要定位于绒毛间质细胞和胎盘滋养细胞 ,HO 2蛋白主要定位于胎盘滋养细胞及血管内皮细胞 ,绒毛间质也有染色。蛋白半定量结果显示 ,胎盘细胞染色分数为非正态分布 ,HO 1在早孕组绒毛滋养细胞、间质细胞和血管内皮细胞中的染色分数中位数分别为 9 0、2 6和 2 8,晚孕组分别为 8 7、2 0和 1 4,两组比较 ,差异均无显著性 (P >0 0 5)。HO 2在早孕组绒毛血管内皮细胞中     

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

Introduction

Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT).

Methods and results

Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast.

Discussion and conclusion

This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.     

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.     

Adriana and Luisa Castellucci award lecture 1999: role of oxygen in the regulation of trophoblast gene expression and invasion

Changes in oxygen levels characterize normal and pathological human placentation. For example, relatively low Po(2)values are present around the blastocyst during implantation and in the placenta of the first trimester of pregnancy, a time of maximal trophoblast invasion. Our studies have revealed that low oxygen levels stimulate the in vitro invasiveness of cultured first trimester trophoblasts. This increased invasive ability is linked to elevated expression of some components of the plasminogen activator system and requires the participation of a putative haem protein. As gestation proceeds beyond the first trimester, and the extent of trophoblast invasion decreases, placental oxygen levels rise with a corresponding increase in blood flow. However, during certain pathological conditions, such as pre-eclampsia/intrauterine growth restriction, impaired remodelling of the uterine spiral arterioles leads to vessels with reduced diameters and localized regions of placental ischaemia/hypoxia. Placental hypoxia in the second half of gestation, as a consequence of reduced uteroplacental blood flow, may result in aberrant expression of genes that contribute to the pathophysiology of pre-eclampsia. Some of these genes encode certain cytokines and vasoactive molecules. We have also identified other genes whose expression is regulated by oxygen. Expression of one of them is induced in trophoblast and other cell types cultured under low oxygen levels and the product of the gene is a 43-kDa protein which we have termed PROXY-1. Compared to placental tissues and membranes isolated from uncomplicated pregnancies, PROXY-1 expression is elevated in tissues from pre-eclamptic pregnancies such as chorionic villi of peri-infarct regions, basal plate and membrane decidua, as well as chorion. Overall, these observations suggest that oxygen levels play an important role in placentation and in the pathophysiology of certain complications of pregnancy.     

人类白细胞抗原-E在正常妊娠、妊娠期高血压及子(疒间)前期绒毛或蜕膜组织中的表达

目的 探讨人类白细胞抗原-E(human leukocyte antigen-E,HLA-E)在正常妊娠、妊娠期高血压和子(疒间)前期绒毛或蜕膜组织中的表达与意义. 方法 选择早、晚期正常妊娠及妊娠期高血压、轻度子(疒间)前期、重度子(疒间)前期患者各6例,应用免疫组化法分别检测HLA-E在绒毛及蜕膜的表达. 结果HLA-E在早、晚期正常妊娠细胞滋养细胞和合体滋养细胞中的表达均较弱分别为0.246±0.031和0.252±0.045,t=2.85,P>0.05);而在侵入蜕膜的绒毛外滋养细胞和腺上皮细胞中强表达,且在妊娠晚期的表达较早期增强(0.308±0.057和0.270±0.035,t=3.79,P<0.05).在正常足月妊娠、妊娠期高血压、轻度子(疒间)前期及重度子痢前期绒毛的平均吸光度分别为0.252±0.045、0.227±0.032、0.210±0.019和0.214±0.023(F=13.18,P<0.05);而在上述各组蜕膜中表达的平均吸光度分别为0.308±0.057、0.211±0.017、0.196±0.013和0.187±0.014(F=33.91,P<0.05).HLA-E在妊娠期高血压、轻度子(疒间)前期和重度子瘌前期绒毛和蜕膜的表达分别与正常足月妊娠比较,均显著减少(P均<0.05). 结论 HLA-E的表达在正常妊娠早期可能与胚胎植入有关,在晚期参与妊娠的维持,而在妊娠期高血压及子(疒间)前期中的低表达可能与该疾病的发生有关.     

Hepatocyte growth factor activator (HGF-A) and its zymogen in human placenta

HGF-activator (HGF-A) is a circulating serine protease known to be responsible for activation of hepatocyte growth factor (HGF). Active HGF is thought to be an important regulator of trophoblast growth. In vitro, HGF-A is produced via proteolytic cleavage of its zymogen by thrombin. Immunocytochemistry and Western immunoblotting were performed using human placental tissue from all three trimesters with an antibody that recognizes both HGF-A and its zymogen. Western immunoblotting revealed a 97 kDa band equivalent to the zymogen in placenta from all three trimesters. A smaller 34 kDa band equivalent to HGF-A was only seen in first and second trimester placenta. The anti-HGF-A/zymogen antibody demonstrated immunostaining in placental villi and membranes throughout gestation. Within first trimester villi immunostaining was strongest within the syncytio- and cytotrophoblast layers, but was also seen within stromal and endothelial cells. Likewise, in third trimester placenta the syncytio-cytotrophoblast layer showed the strongest immunoreactivity. In vitro, HGF can induce trophoblast DNA synthesis and the localization of HGF-A to the peri-villous trophoblast layer (which expresses c-met, the HGF receptor) suggests that it may be responsible for activation of pro-HGF at this site. This adds further weight to the hypothesis that HGF in vivo is an important regulator of trophoblast growth.     

Expression of Gadd45α in human early placenta and its role in trophoblast invasion

Objectives

Well-controlled trophoblast migration and invasion at the maternal–foetal interface are crucial events for normal placentation and successful pregnancy. Growing evidence has revealed that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) participates in tumour migration and invasion as a tumour suppressor. However, the expression and function of Gadd45α in trophoblasts is unknown. This study aimed to determine the Gadd45α expression and function in the human first trimester placenta and identify the underlying mechanisms.

Methods

The expression of Gadd45α in human first trimester placenta was determined using immunohistochemistry. HTR8/SVneo cell line was used to investigate the effects of Gadd45α on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP)2/9 activities, and tissue inhibitor of metalloproteinase (TIMP)1/2 expression using cell proliferation assays, flow cytometric analysis, transwell migration/invasion assays, gelatin gel zymography, and western blotting, respectively. Moreover, a placental villous explant model was employed to verify its functions in placentation.

Results

Gadd45α was strongly expressed in syncytiotrophoblasts and trophoblast columns of human placental villi, extravillous trophoblast cells and glandular epithelium within the maternal decidua. Gadd45α knockdown significantly promoted migration and invasion of HTR8/SVneo cells, whereas it did not affect cell proliferation or apoptosis. Silencing Gadd45α also enhanced trophoblast outgrowth and migration in placental explants. These effects were related to increased activities of MMP2/9 and the decreased expression of TIMP1/2.

Discussion and conclusion

Gadd45α may be involved in human trophoblast migration and invasion and may function as an important negative regulator at the foetal–maternal interface during early pregnancy by directly or indirectly regulating MMP2/9 activities.     

Glycodelin A and differentiation of first trimester trophoblast cells in vitro

Aim The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro.Methods Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry.Results Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid.Conclusions The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.