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。     

低分子肝素对不同氧浓度下早孕绒毛滋养层细胞侵袭力的影响作用

目的:研究低分子肝素(LMWH)对不同氧浓度下早孕绒毛滋养层细胞侵袭力的影响。方法:取妊娠8~10周正常绒毛组织,酶消化法分离人早孕绒毛滋养层细胞,分别置2%和20%O_2分压的细胞培养箱培养。用不同浓度LMWH(0、0.1、5、10IU/ml)处理滋养细胞,观察滋养层细胞侵袭力的改变,检测细胞中HIF-1α、MMP-2、MMP-9、TIMP-2、TIMP-3表达。结果:低氧浓度下,早孕绒毛滋养层细胞的侵袭能力随着LMWH浓度(0,0.1,5IU/ml)升高而增强,但高浓度的LMWH(10IU/ml)对其有抑制作用;随着LMWH浓度增加,HIF-1α和MMP-2表达呈先上升后下降的趋势;TIMP-2、TIMP-3表达改变的趋势与MMP-2相同。正常氧浓度下的滋养层细胞,其侵袭能力未见显著增强,各细胞因子的表达无上调。结论:低氧环境下,一定浓度的LMWH可提高早孕绒毛滋养层细胞的侵袭能力,其发生机制可能是通过诱导、增强滋养层细胞HIF-1α和MMP-2基因表达。     

早期自然流产患者TNF-α及TGF-β1的异常表达

目的:探讨自然流产患者外周血单个核细胞和胎盘组织肿瘤坏死因子-α(TNF-α)以及转化生长因子-β(TGF-β1)的异常表达。方法:采用RT-PCR技术,检测30例早期自然流产患者(自1然流产组)、25例正常妊娠妇女(正常妊娠组)、25例正常未妊娠妇女(未孕对照组)外周血单个核细胞内TNF-αmRNA和TGF-β1mRNA表达水平;采用免疫组织化学技术检测绒毛和蜕膜组织内TNF-α及TGF-β1表达强度。结果:三组研究对象外周血单个核细胞表达TNF-αmRNA相对含量分别为21.1±9.4%、68.6±14.1%、97.0±11.6%,组间比较有显著性差异(P<0.05);TGF-β1mRNA相对含量分别为21.5±9.4%、95.7±12.9%、87.0±13.8%,组间比较也均有显著性差异(P<0.05)。自然流产组细胞滋养层细胞及合体滋养层细胞表达TNF-a均显著强于人工流产组(P<0.01);自然流产组合体滋养层细胞表达TGF-β1显著低于人工流产组(P<0.01)。结论:①早期自然流产患者外周血单个核细胞TNF-αmRNA表达水平显著下降,而细胞滋养层细胞及合体滋养层细胞表达TNF-α均显著强于人工流产组,提示TNF-α可能在母胎界面局部发挥作用并导致流产。②正常早期妊娠妇女外周血单个核细胞TGF-β1mRNA表达水平较正常未妊娠妇女显著升高,提示TGF-β1对妊娠维护起重要作用。早期自然流产患者外周血单个?     

晚发型胎儿生长受限胎盘组织碱性成纤维细胞生长因子表达及超微结构的观察

目的:观察晚发型胎儿生长受限(fetal growth restriction,FGR)患者胎盘组织中碱性成纤维细胞生长因子(basic fibroblast growthfactor,bFGF)的表达及其超微结构的变化。方法:FGR组和足月正常妊娠组的胎盘组织标本各15例,应用免疫组织化学法检测bFGF的表达,2组标本各选取3例应用透射电镜观察其超微结构的变化。结果:2组均见bFGF表达于绒毛合体滋养层细胞、绒毛微血管内皮细胞和绒毛间质细胞的胞浆。FGR组胎盘滋养层bFGF的表达显著高于正常妊娠组(P<0.01)。FGR组胎盘合体结节增多,胎盘绒毛变性,基底膜增厚,微血管内皮可见突起及钙化砂粒体,自体胞质脱落。结论:FGR胎盘组织有超微结构病理改变,bFGF的表达可能是继发性增高。     

白血病抑制因子在正常早孕、先兆流产及难免流产患者绒毛组织中的表达

目的测定白血病抑制因子(LIF)在正常早孕、先兆流产及难免流产患者绒毛组织中表达的差异,探讨LIF在先兆流产及难免流产发病中的作用。方法采用放射免疫法检测正常早孕妇女(正常早孕组,30例)、先兆流产患者(先兆流产组,30例)及难免流产患者(难免流产组,30例)血清孕酮及人绒毛膜促性腺激素(hCG)水平;采用免疫组化技术———链霉菌抗生物素蛋白-过氧化酶连接(SP)法对LIF在3组妇女绒毛组织中的表达进行组织学定位和半定量分析;采用蛋白印迹法对3组妇女绒毛组织中LIF的相对表达量进行测定。结果(1)血清孕酮及hCG水平在正常早孕组、先兆流产组及难免流产组分别为(95±26)、(90±26)、(36±17)nmol/L及(75±14)、(68±13)、(13±3)kU/L。正常早孕组与先兆流产组血清孕酮及hCG水平分别比较,差异均无统计学意义(P>0·05);而难免流产组与其他两组妇女血清孕酮及hCG水平分别比较,差异均有统计学意义(P<0·01)。(2)3组妇女绒毛组织中LIF均呈现阳性表达,阳性染色主要位于滋养细胞胞质中,胞核无明显着色。(3)正常早孕组、先兆流产组及难免流产组LIF相对表达量分别为1·17±0·13、1·06±0·10、0·30±0·02,难免流产组与其他两组分别比较,差异均有统计学意义(P<0·01),而正常早孕组与先兆流产组比较,差异无统计学意义(P>0·05)。结论LIF对妊娠的正常维持有一定的保护作用,LIF在早孕绒毛组织中的低表达,可能与hCG及孕酮水平下降有关,是导致难免流产的原因之一。     

正常妊娠滋养层组织中Lin28B表达变化的研究

目的:探讨Lin28B在正常妊娠不同阶段滋养层细胞中的表达变化及其临床意义。方法:收集40例正常早期妊娠者(孕早期组),包括20例妊娠5~7+6周(孕早A组)和20例妊娠8~12+2周(孕早B组),15例孕17~27周(孕中期组),以及20例孕37~41周(孕晚期组),共75例正常孕妇的绒毛和胎盘组织。采用RT-q PCR、Western blotting和免疫组织化学方法检测胎盘组织Lin28B基因和蛋白的表达差异及细胞定位。结果:1在正常妊娠不同时期的滋养层组织中细胞滋养细胞和合体滋养细胞中均可检测到Lin28B蛋白的表达。2 Lin28B m RNA表达量各组间有统计学差异(P0.05),早孕A组最高,随着孕周进展逐渐下降,至孕晚期达最低水平。3正常妊娠不同时期的滋养层组织中Lin28B蛋白表达呈现为随着孕周的增加而逐渐下降趋势。结论:Lin28B在正常妊娠进展过程中表达下调,提示其可能参与了滋养细胞的增殖和浸润及胎盘的发育。     

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。     

孕酮对早孕人滋养层HLA-G mRNA转录水平的调控

目的:探讨孕酮对人早孕滋养层人类白细胞主要组织相容性抗原(HLA)-G基因转录的调控作用。方法:选取正常妊娠6-9周的人绒毛标本,进行组织块贴壁培养、纯化获得滋养层细胞后,采用半定量逆转录-聚合酶链式反应(RT-PCR)检测孕酮对体外培养的人滋养层细胞HLA-G基因转录的影响。结果:添加孕酮的滋养层细胞HLA-GmRNA水平显著增高(P<0.05)。结论:孕酮对滋养层HLA-G的转录具有明显的上调作用。     

妊娠滋养细胞疾病间隙连接蛋白CX43的表达

目的 :研究间隙连接蛋白 (CX43)在妊娠滋养细胞疾病中的组织定位和表达 ,探讨CX43与妊娠滋养细胞疾病的关系。方法 :采用单克隆抗体SP免疫组化技术和核酸原位杂交技术测定 1 1 0例妊娠滋养细胞疾病中CX43的表达情况。结果 :正常早孕绒毛中CX43阳性颗粒定位于细胞滋养层细胞的胞浆和胞膜中 ,胞核也有表达 ;合体滋养细胞和间质细胞几乎不表达。GTD中CX43表达明显减弱 ,有表达也仅局限于细胞膜上 ,胞核和胞浆中几乎不表达。正常早孕绒毛与妊娠滋养细胞疾病中CX43的表达有显著性意义 (P <0 .0 1 ) ,而滋养细胞疾病间则无显著性意义 (P >0 .0 5)。结论 :CX43表达降低及GJIC功能减弱 ,可能是导致滋养细胞恶变的因素之一     

先兆子痫患者母胎界面上细胞凋亡的研究

目的:研究先兆子痫患者母胎界面上细胞凋亡水平及凋亡相关分子的表达变化。方法:分别收集妊娠25、27、29、30和31周及足月分娩的正常妊娠者和先兆子痫患者的胎盘组织,采用脱氧核糖核酸末端转移酶介导的dUTP刻痕末端标记法(TUNEL)检测母胎界面上细胞凋亡情况, 并行凋亡细胞定位;RT-PCR检测正常妊娠和先兆子痫胎盘组织中P53,Bcl-2和Bax的mRNA表达水平的差异。结果:正常妊娠和先兆子痫患者胎盘中发生凋亡的细胞数目都随孕周延长而逐渐增高,但先兆子痫的胎盘中细胞凋亡率明显高于正常妊娠者;正常妊娠胎盘中凋亡细胞主要为合体滋养层细胞,足月胎盘绒毛内血管内皮细胞亦有少量细胞凋亡,而先兆子痫胎盘中细胞滋养层细胞、合体滋养层细胞和绒毛内血管内皮细胞均发生明显的凋亡;RT-PCR显示先兆子痫胎盘组织中P53和Bax的mRNA水平明显高于同期妊娠的正常胎盘,而Bcl-2水平显著降低。结论:正常妊娠过程中胎盘组织中只在有限部位出现少量细胞凋亡,而先兆子痫胎盘组织中细胞过度凋亡, 且凋亡细胞分布广泛,并伴有凋亡相关分子的表达上调。表明先兆子痫的发生与胎盘组织中滋养层细胞大量凋亡密切相关。     

Localisation of indoleamine 2,3-dioxygenase and kynurenine hydroxylase in the human placenta and decidua: implications for role of the kynurenine pathway in pregnancy

Indoleamine 2,3-dioxygenase (IDO) has been implicated in contributing to immunotolerance in early pregnancy, but the presence in the term placenta of mRNAs for enzymes that produce other biologically active kynurenine end-products suggests other functions for kynurenine pathway metabolites. The aim of this study was to investigate the localisation of two key enzymes - IDO and kynurenine hydroxylase (KYN-OHase) - in first trimester decidua and in the human placenta across pregnancy. Using immunocytochemistry, it was shown that there was strong expression of IDO and KYN-OHase in stromal and glandular epithelial cells of first trimester decidua. In first and second trimester placenta, IDO and KYN-OHase were localised to the syncytiotrophoblast, stroma and macrophages. IDO and KYN-OHase mRNAs were also identified, and the enzymes appear to be functional because kynurenine and 3-hydroxy-anthranilic acid (respective products of the activity of these enzyme) were released into the medium when first trimester placental explants were maintained in culture for 48h. In term placenta, both IDO and KYN-OHase immunoreactivities were confined mainly to vascular endothelial cells of villous blood vessels, and to macrophages within the fetal villus, whereas syncytial staining was very weak or absent. The shift of expression of these enzymes away from the syncytiotrophoblast to fetal endothelial cells in terminal villi suggests that the function of the enzymes may change from a role in immunosuppression at the maternal-fetal interface in early pregnancy, to one associated with regulation of fetoplacental blood flow or placental metabolism in late gestation.     

自然流产患者绒毛中ADAM19、MMP-9和KISS-1基因的表达及其意义

目的:探讨解聚素金属蛋白酶19(ADAM19)、基质金属蛋白酶9(MMP-9)和肿瘤转移抑制因子(即亲吻素-1,KISS-1)与自然流产的关系及其在妊娠中的作用。方法:正常妊娠(对照组)和自然流产(实验组)的绒毛组织各30例,应用免疫组织化学SP法和RT-PCR检测ADAM19、MMP-9及KISS-1的表达情况。结果:①ADAM19、MMP-9蛋白主要在滋养细胞的细胞膜上表达,在细胞质中仅有少量表达。其表达对照组高于实验组(P<0.05)。KISS-1蛋白主要在细胞质中表达,细胞膜上也有少量表达。其在对照组中的表达低于实验组(P<0.05)。②对照组ADAM19 mRNA和MMP-9 mRNA表达高于实验组(P<0.05),KISS-1 mRNA的表达低于实验组(P<0.05)。结论:患者ADAM19、MMP-9和KISS-1 mRNA和蛋白的表达变化影响滋养细胞的浸润能力,可能与自然流产的发生有关。     

自然流产患者绒毛组织PAI-14G/5G多态性和uPA基因的表达及其意义

目的:探讨纤溶酶原激活物抑制剂-1(PAI-1)4G/5G多态性和纤溶酶原激活剂(uPA)与自然流产的关系及其在妊娠中的作用。方法:正常早孕(对照组)人工流产和自然流产(实验组)稽留流产的绒毛组织各30例,应用免疫组织化学PV法检测绒毛组织中PAI-1和uPA蛋白表达情况,等位基因特异性-PCR(AS-PCR)法检测PAI-1 4G/5G多态性的表达情况,RT-PCR法检测uPA mRNA表达情况。结果:①PAI-1蛋白主要在绒毛组织的滋养细胞质中表达,其表达对照组低于实验组(P<0.05)。uPA与PAI-1蛋白在细胞中分布一致,其表达水平对照组高于实验组(P<0.05)。②PAI-1 4G/4G基因型和4G等位基因表达水平对照组低于实验组(P<0.05),uPA mRNA表达水平对照组高于实验组(P<0.05)。③PAI-1和uPA蛋白在自然流产患者绒毛组织中的表达呈负相关(r=-0.639)。结论:PAI-1 4G/5G多态性和uPA异常表达可能与自然流产的发生有关。     

GLUT12 expression in human placenta in first trimester and term

The aim of this study was to characterize the expression of a novel glucose transporter protein GLUT12 in human placenta. GLUT12 mRNA expression was identified by RT-PCR in extracts from five normal term placentae and in extracts from cultured cells of the JAR, JEG-3 and HTR-8Svneo cell lines. In further studies, paraffin sections of first trimester tissue from chorionic villus sampling and term tissue obtained after delivery were analysed by immunohistology with a GLUT12 specific polyclonal antibody. GLUT12 immunoreactivity was expressed predominantly in the syncytiotrophoblast and in extra-villous trophoblast cells in first trimester tissues at 10, 11 and 12 weeks' gestation. In term tissue, however, GLUT12 staining was not detected in syncytiotrophoblast and was found predominantly in villous vascular smooth muscle cells and villous stromal cells. These results suggest that there is a dynamic spatial and temporal expression pattern for the novel glucose transporter GLUT12 in human placenta.     

半乳糖凝集素-3在早期绒毛和蜕膜中的表达

目的:了解半乳糖凝集素-3(galectin-3)在早孕期绒毛与蜕膜中的表达,为进一步探讨稽留流产发病机制提供理论依据。方法:分别获取手术流产组(n=20)、药物流产组(n=20)及稽留流产组(n=20)患者的流产绒毛及蜕膜,用免疫组织化学法及实时荧光相对定量PCR法检测各组绒毛及蜕膜组织中半乳糖凝集素-3的表达。结果:免疫组织化学显示稽留流产组绒毛半乳糖凝集素-3强阳性表达率(30.00%)明显低于手术流产组(80.00%)(P<0.01)。RT-PCR结果进一步证实稽留流产组绒毛半乳糖凝集素-3 mRNA的相对表达量(2-△△Ct=0.31±0.70)明显低于手术流产组(2-△△Ct=1.00±0.00)(P<0.01),药物流产组蜕膜半乳糖凝集素-3 mRNA的相对表达量(2-△△Ct=0.74±2.20)明显低于手术流产组(2-△△Ct=1.00±0.00)(P<0.01)。结论:半乳糖凝集素-3与妊娠早期绒毛及蜕膜发育有关,其表达降低可能促进了稽留流产的发生。     

Expression of pregnancy-associated plasma protein-A (PAPP-A) during human villous trophoblast differentiation in vitro

Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae. In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independently of the age of gestation.     

人类组织相容性抗原-G mRNA表达与不明原因习惯性流产关系的初步研究

目的　探讨人类组织相容性抗原Ｇ（ＨＬＡＧ） ｍＲＮＡ 的表达与不明原因习惯性流产（ＲＳＡ） 的关系。方法　用原位杂交的方法检测正常妊娠早期、晚期孕妇胎盘和ＲＳＡ患者经免疫治疗并成功分娩的胎盘母胎界面处的ＨＬＡＧｍＲＮＡ的表达。结果　正常妊娠早期、晚期孕妇胎盘母胎界面均有ＨＬＡＧｍＲＮＡ的阳性表达,阳性表达位于绒毛内（ 或） 外细胞滋养细胞,以及合体滋养细胞的胞浆内。ＲＳＡ患者免疫治疗后的胎盘均不表达ＨＬＡＧｍＲＮＡ,免疫治疗没有调节ＨＬＡＧ ｍＲＮＡ 表达的作用。结论　ＨＬＡＧ 可能与ＲＳＡ的发病原因没有直接的关系;与胎儿存活也无直接关系。     

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal–fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal–fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal–fetal immune dialogue.