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。     

早孕期绒毛和蜕膜的组织化学观察

111例正常早孕人工流产的胎盘组织作绒毛蜕膜组织化学观察。蜕膜细胞,富于糖元,碱性磷酸酶多呈阳性或弱阳性,酸性磷酸酶多数阴性或弱阳性,少数阳性。3β-HSD弱阳性,蜕膜腺体弯曲呈锯齿状,腺体壁呈扇形,有时细胞向内突出如鞋钉状,腔内充满分泌物,糖元强阳性,对维持早期妊娠是一个重要营养条件。早期妊娠绒毛具有完整两层滋养层,即合体滋养层及细胞滋养层,60天后则细胞滋养层逐步消失,绒毛大小不等,胎儿血管扩大并移向绒毛表面,从而使胎儿-母体胎盘屏障变薄,有利于胎儿-母体的物质交换,是适应胚胎发育的一个重要条件。早孕期绒毛组织化学检查显示绒毛两层细胞糖元和碱性磷酸酶活性都很强,反映代谢机能活跃,细胞滋养层胞浆酸性磷酸酶活性60—90天逐渐加强,这应与此时期细胞滋养层退化有关。3β-HSD 在甾体生物合成过程中起重要作用,绒毛滋养层细胞3β-HSD皆为阳性,表明具有甾体生物合成机能。着床、早孕过程中,绒毛、蜕膜是母体-胚胎直接接触和相互联系的两种组织,从计划生育角度,在发展抗着床、抗早孕药物研究工作中,对于蜕膜、绒毛形态和机能的研究应当受到重视。     

干扰素-r和干扰素-α对人蜕膜LAK细胞杀伤滋养层细胞能力的影响

人类正常妊娠过程中,胎儿绒毛外滋养层细胞必须侵入到子宫蜕膜中。滋养层细胞表达非典型I类HLA分子,如HLA-G等。这些滋养层细胞如何不被母亲效应细胞攻击,目前还未了解。蜕膜中存在一大群CD_3~-的大颗粒淋巴细胞(LGL),其与     

早期妊娠妇女胎盘绒毛滋养层组织蛋白质的生化分析

本研究对不同周数(6、7、8、9、10周)妊娠妇女胎盘绒毛滋养层组织水溶性蛋白质组分、分子量、等电点及其特性进行了分析和比较。用SDS-聚丙烯酰胺凝胶电泳分析结果指明,妊娠早期绒毛组织,除分泌绒毛膜促性腺激素(hCG)这一含量较多的蛋白以外,尚含有其他四种含量较高的蛋白质,这四种蛋白质的分子量分别为:140,000,128,000,94,000和83,000左右。用高压液相色谱层析和等电聚焦电泳分析结果指明,在不同周龄的组织中其蛋白量和组分间的比例稍有变化,较多的蛋白质为酸性蛋白,与蜕膜组织蛋白质组分作比较,绒毛组织具有蜕膜组织内不存在的蛋白组分,其等电点分别为:8.4、6.4、5.8、4.8。由此可知早期妊娠胎盘绒毛滋养层组织,存在一些含量较高的特殊蛋白,深入研究这些蛋白的特性和生理功能,将对阐明人类妊娠生理和寻找控制生育新途径有重要意义。     

孕酮和雌二醇对人单核细胞毒活性的抑制作用

妊娠时,在抗原不同的组织间存在着自然免疫监视。虽然对这种与免疫规律不完全符合的机制还不清楚,但有一概念似乎已被肯定,即在绒毛和蜕膜界面的局部免疫调节因素对胎儿的存活是必需的。机体单核细胞对外来细胞的细胞毒作用是重要的免疫防御手段,但胎儿滋养层细胞对细胞溶解作用有抵抗力,这是由于在绒毛-蜕膜界面间存在的胎盘甾类化合物对单核细胞活性有抑制作用。已证实     

体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。     

hCG及人胎盘泌乳素与妊娠并发症

人绒毛膜促性腺激素(hCG)、人胎盘泌乳素(hPL)水平异常与妊娠并发症有关,尤其是妊娠中、晚期hCG水平升高与妊娠高血压综合征(PIH)危险性的升高有关;还可能与早产、胎儿发育有关;与死产、脐带异常附着及妊娠甲状腺毒症的相关性还需研究;与胎膜早破、胎盘早剥、羊水过少、前置胎盘等无关.hPL与胎儿发育密切相关,与其他妊娠并发症无关.hCG、hPL联合测定的价值尚不肯定.异常的hCG和hPL可能作为妊娠并发症的预测因子而具有新的临床价值.     

蛋白酶体亚基LMP 2在稽留流产的蜕膜和绒毛组织中的表达

目的探讨蛋白酶体亚基LMP-2在稽留流产中的作用。方法 用免疫组化方法研究LMP-2在201例稽留流产中的48例绒毛和45例脱膜与114例正常早孕人工流产中的56例绒毛和58例蜕膜中的表达。结果LMP-2在稽留流产的绒毛和蜕膜中的表达均低于正常早孕人工流产的绒毛和蜕膜,二者间的表达强度差异有统计学意义（P〈0.05）。结论 LMP-2在稽留流产中绒毛与蜕膜的表达减弱提示泛素-蛋白酶体通路介导的细胞蛋白降解参与调控滋养层细胞的生长过程。     

炔诺酮对人早孕蜕膜和绒毛超微结构的影响

用炔诺酮（５ｍｇｑ８ｈ×５天）抗早孕后作人工流产，对蜕膜和绒毛的超微结构进行观察。结果显示绝大部分蜕膜和绒毛滋养细胞均发生不同程度的退变和坏死。蜕膜组织中大蜕膜细胞和绒毛合体滋养细胞变性坏死较重，而蜕膜颗粒细胞和细胞滋养细胞仅轻度变性。细胞退变的超微结构特征皆以线粒体肿胀和粗面内质网扩张为先导，继之，线粒体固缩伴高电子密度颗粒沉积，内质网不规则扩张，直至细胞全面崩解。本文就炔诺酮通过引起子宫局部缺血而导致蜕膜和绒毛滋养层细胞变性坏死的可能性进行了讨论。     

对米非司酮合并前列腺素终止早孕监护措施的探讨

我们对129例早孕患者应用米非司酮合并前列腺素终止妊娠并采用尿hCG 半定量测定,B 型超声检查及绒毛、蜕膜组织病理检查作为监护方法。结果表明在药流d15,出血在15d 内完全流产者中有70%尿hCG 滴度呈正常水平(<312mIU/ml)。而药流d15尿hCG 滴度≥1250mIU/ml 者,可能为不完全流产或完全流产但出血时间延长。B 型超声检查对诊断子宫出血时间长短有一定价值。绒毛蜕膜组织坏死程度则并非确定出血时间长短的唯一因素。     

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1 in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1) in villous and extravillous trophoblast at various stages of normal human gestation were studied, using hysterectomy specimens. In the chorionic villi, the capacity for synthesizing placental proteins seemed to develop in parallel with the morphological change from mononuclear cells to multinucleated syncytiotrophoblast and no villous trophoblast expressed HLA antigens. In contrast, extravillous trophoblast, including the multinucleated trophoblastic cells at the deciduomuscular junction, expressed HLA-A, -B, and -C, and their capacity for synthesizing placental proteins did not seem to correspond with the degree of morphological change: the location of alpha hCG, beta hCG CTP and SP1 was restricted to mononuclear trophoblast in the superficial decidua, while hPL was present extensively in extravillous trophoblast. These findings strongly suggest that extravillous trophoblast possesses many distinctive biological features and differentiates in an independent manner. Mononuclear trophoblast forming the cell columns was also positive for HLA-A, -B, and -C, and no placental protein was demonstrated in these cells; this, together with previous morphological observations, may indicate the germinative nature of these cells.     

Immunohistochemical localization of hCG alpha, hCG beta CTP, hPL and SP1 on villous and extravillous trophoblasts in normal human pregnancy

Trophoblasts are divided into villous trophoblasts and extravillous trophoblasts, depending on whether they constitute a villous structure, and cell columns intervene between them. We conducted an immunohistochemical localization of human chorionic gonadotropin (hCG) alpha, hCG beta C-terminal peptide (CTP), human placental lactogen (hPL) and pregnancy-specific beta 1-glycoprotein (SP1) on the trophoblasts of normal human pregnancy, using forty-one hysterectomized pregnant uteri (6-22 weeks). In villous trophoblasts, the capacity to synthesize hCG alpha, hCG beta CTP, hPL and SP1 seemed to develop according to the morphological change from mononuclear cells to multinuclear cells. In contrast, the synthetic capacity of these proteins seemed not to correspond with the morphological change in extravillous trophoblasts: The location of hCG alpha, hCG beta CTP and SP1 was restricted to the mononuclear trophoblasts in the superficial decidua, while hPL was present extensively in extravillous trophoblasts, including multinuclear trophoblasts in the deciduomuscular junction. Therefore, it may be reasonably said that extravillous trophoblasts have many biological features distinct from villous trophoblasts and differentiate in an independent manner. Mononuclear trophoblasts in the cell column were negative for these proteins, which, together with morphological observations, strongly suggest the germinative nature of these cells.     

Morphological and immunohistochemical study on specimens of trophoblastic diseases in which the presence of a villous formation could not be established

The histopathological discrimination between malignant trophoblastic diseases and benign trophoblastic diseases depends on the presence or absence of a villous structure. However, molar extravillous trophoblasts and cells in some placental site trophoblastic tumors (PSTT) of a benign nature, lack a villous structure. We therefore observed the morphology of trophoblastic cells which do not constitute a villous structure, including choriocarcinoma cells, and analyzed the location of placental proteins in these cells immunohistochemically. The results were as follows: 1. Molar extravillous trophoblasts were composed of large mononuclear cells and multinuclear cells. Most of them were positive for hPL and negative for hCG and SP1. 2. Choriocarcinoma consisted of cytotrophoblast-like cells, syncytiotrophoblast-like cells, large mononuclear cells and multinuclear cells resembling large mononuclear cells. HCG was noted in syncytiotrophoblast-like cells and large mononuclear cells, while hPL and SP1 were found only in syncytiotrophoblast-like cells. 3. PSTT was made up of large mononuclear cells and multinuclear cells which contained abundant hPL and very little hCG and SP1 or none at all. Molar extravillous trophoblasts were clearly distinguishable from choriocarcinoma cells in terms of their morphology and the location of placental proteins. In contrast, it seemed difficult to distinguish cells of PSTT from molar extravillous trophoblasts on a cell level.     

Oxygen tension directs the differentiation pathway of human cytotrophoblast cells

During placental development, human cytotrophoblast cells can differentiate to either villous syncytiotrophoblast cells or invasive extravillous trophoblast cells. We hypothesize that oxygen tension plays a critical role in determining the pathway of cytotrophoblast differentiation. A highly purified preparation of cytotrophoblast cells from human third trimester placenta was cultured for 5 days in either 20% or 1% oxygen tension. The cells incubated at 20% oxygen formed a syncytium as determined by immunohistochemistry using an anti-desmosomal protein antibody that identifies cell membranes. In addition, the mRNA was markedly induced for syncytin, a glycoprotein shown to be essential for syncytiotrophoblast formation, and for human placental lactogen (hPL), which is a specific marker for syncytiotrophoblast cells. In contrast, the cell incubated at 1% oxygen tension did not fuse by morphologic analysis and did not express syncytin or hPL mRNA. However, these cells expressed abundant amounts of HLA-G, a specific marker for extravillous trophoblast cells, which was not seen in cells incubated at 20% oxygen tension. These results suggest that low oxygen tension directs differentiation along the extravillous trophoblast cell pathway while greater oxygen tension directs differentiation along the villous trophoblast cell pathway.     

Effects of various growth factors on the proliferation and the differentiation of trophoblastic cells in vitro

EGF, PDGF, IL-6, TGF-beta 1 and supernatants from decidual cell cultures were added in various concentrations to cultures of normal trophoblastic cells in vitro. Effects on cell proliferation and differentiation were assessed by using 3H-thymidine uptake test and measuring hPL, hCG production in culture media, respectively. EGF stimulated only hPL, hCG production and in contrast, PDGF stimulated 3H-thymidine uptake only. IL-6, TGF-beta 1 and decidual supernatants stimulated both hPL, hCG production and 3H-thymidine uptake, especially TGF-beta 1 and decidual supernatants. These results suggest that EGF is related to the differentiation, PDGF is to the proliferation, and IL-6, TGF-beta 1 and decidual supernatants are to both the differentiation and proliferation of trophoblastic cells. Further understanding of the effects of these products on trophoblastic growth may provide important insights into mechanisms of early pregnancy development.     

The localization of pregnancy proteins (hPL, SP1 and PAPP-A) in intra- and extrauterine pregnancies

The localization of human placental lactogen (hPL), pregnancy-specific beta-1 glycoprotein (Schwangerschaftsprotein 1, SP1) and pregnancy-associated protein A (PAPP-A) was examined in intrauterine and tubal ectopic gestation (n = 5) by the immunoperoxidase technique. The distribution of hPL and SP1 was identical in placental tissues obtained from intra- and extrauterine pregnancies, being uniformly seen throughout the syncytiotrophoblast. hPL and SP1 were not demonstrated in uterine decidual tissue from ectopic pregnancies. During early (week 8) intrauterine pregnancy, PAPP-A was not restricted to the mature syncytiotrophoblast, being observed also in some trophoblast-like cells adjacent to islands of syncytiotrophoblast. In contrast, in ectopic gestation, PAPP-A was observed in these cells at six weeks' gestation only. We were unable to detect PAPP-A in trophoblastic tissue of chorionic villi and uterine decidual tissue from ectopic gestation.     

Villous culture of first trimester human placenta--model to study extravillous trophoblast (EVT) differentiation.

During implantation and subsequent placentation the human extravillous trophoblast (EVT) cells invade the endometrium and maternal vasculature within the uterus. The origin of the EVT and signals triggering its differentiation, migration and invasion are poorly understood. First and second trimester human chorionic villi explants were used as a source of EVT and a variety of substrates which resemble extracellular matrix (ECM) in vivo have been tested to induce EVT differentiation and migration. The obtained results demonstrate that villous explants from both 5-7 and 8-10 weeks of gestation give rise to EVT cells in vitro if maintained on the surface of Matrigel or decidual extract supplemented collagen gel. Fetal calf serum (FCS) supplemented media was essential for EVT differentiation and villous trophoblast viability. Immunostaining of both EVT cells and cells from the cytotrophoblastic column with monoclonal antibody Ki67 (cell proliferation marker) indicate that EVT cells differentiate in vitro by proliferation from the tip of anchoring villi. These mononucleated, round-shaped, migrating cells are HLA-A,B,C class I antigen (W6/32) antibody and low molecular weight cytokeratin positive, and do not immunostain with PAI-1 (plasminogen activator inhibitor) and HPL antibodies. Differentiation of EVT was restricted to first trimester villous tissue; explants from second trimester placentae did not give rise to EVT. Tissue viability as monitored by glucose utilization, lactate, progesterone and hCG production rates correlated with EVT differentiation. The production rates for hCG demonstrated significant variation among individual placentae and was maintained constant for 10 days consistently only in explants cultured on decidual extract supplemented collagen matrix. The described villous tissue culture system may be, therefore, a unique in vitro model to study proliferation and differentiation of EVT from cytotrophoblastic columns, the regulation of EVT proliferation and differentiation, the role of ECM in the induction of the migration and the interaction of extravillous and villous trophoblast at the level of the cytotrophoblastic column.     

In vitro secretory patterns of human chorionic gonadotrophin, placental lactogen and pregnancy-specific beta 1-glycoprotein

The control of secretion of the placental hormones human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein pregnancy-specific beta-glycoprotein (SP1), is not well understood. During pregnancy, the hCG concentrations peak in the first trimester then decrease, while hPL and SP1 increase steadily throughout gestation. In order to determine whether the discordance between hCG secretion and that of hPL and SP1 observed in vivo also occur in vitro, we cultured placental explants with and without dibutyryl cyclic AMP (dbcAMP) and theophylline. Between 5 and 12 explants were used for each treatment in each experiment. The concentration of the proteins secreted into the media each day was measured by specific radioimmunoassays. The quantities of hPL and SP1 secreted per day declined in a parallel fashion after 24 hours under both basal and dbcAMP-stimulated conditions. The hCG output progressively decreased in the unstimulated cultures until 48 hours, at which time an increase in hCG secretion was observed. The dbcAMP-stimulated placentae significantly increased their hCG output at both 48 and 72 hours. These data show that hCG secretion is regulated differently from that of hPL and SP1. The results do not negate the possibility that term placental tissue may contain an inhibitor of hCG release that is removed by experimental manipulation in vitro.     

Linkage of human chorionic gonadotrophin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotrophin (hCG) and placental lactogen (hPL). Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their mRNAs in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha/beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues which contain cytotrophoblastic elements.