早期自然流产患者绒毛Notch受体mRNA和蛋白表达及与妊娠关系的初步探讨

目的:初步探讨早期自然流产患者绒毛Notch受体(Notch1、Notch2、Notch3、Notch4)mRNA及蛋白表达及对滋养细胞的调控作用。方法:用实时荧光定量逆转录聚合酶链反应技术和免疫印迹法检测6例自然难免流产患者和12例正常早期妊娠妇女绒毛Notch1、Notch2、Notch3、Notch4的mRNA和蛋白的表达水平。结果:(1)自然流产组绒毛Notch1和Notch3 mRNA表达水平高于正常早期妊娠组(P<0.05);(2)Western-blot检测显示早期自然流产患者绒毛中Notch1、Notch2、Notch3受体蛋白的表达水平高于正常早期妊娠组(P<0.05)。结论:Notch受体蛋白在早期自然流产患者绒毛中表达上调,可能与自然流产的发病原因有关。     

Syncytin在不同时期绒毛或胎盘组织中的表达及意义

目的:研究Syncytin在胎盘发育中的表达变化及意义。方法:采用荧光实时定量PCR(real-time FQ-PCR)方法检测正常早期妊娠绒毛组织(20例)、中期妊娠胎盘组织(15例)及晚期妊娠胎盘组织(20例)中Syncytin mRNA表达。结果:正常妊娠早、中、晚期胎盘组织Syncytin mRNA表达随孕周增加而升高,差异有统计学意义(P<0.05)。结论:Syncytin在胎盘发育过程中表达不同,推测可能参与胎盘发育之调节。     

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。     

cFLIP在早期自然流产绒毛组织的表达及意义

目的:检测早期正常妊娠及自然流产绒毛组织中细胞凋亡抑制蛋白cFLIPmRNA及其蛋白产物的表达,分析并探讨cFLIP与自然流产的关系。方法:应用RT-PCR法及免疫组化SP法检测cFLIP mRNA及其蛋白产物在20例自然流产患者(研究组)绒毛组织的表达,以同期正常妊娠要求人工流产20例为对照组。结果:研究组绒毛组织中cFLIP mRNA及蛋白表达明显低于对照组,差异有统计学意义(P<0.01)。结论:细胞凋亡抑制蛋白cFLIP参与维持正常妊娠,绒毛组织中其表达下降可能在自然流产的发生中起重要作用。     

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。     

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     

长链脂肪酸氧化酶mRNA在正常妊娠及重度子痫前期胎盘中的表达

目的研究长链羟酰基辅酶A脱氢酶(long-chain3-hydroxyacyl-CoAdehydrogenase,LCHAD)在正常妊娠不同孕期绒毛或胎盘组织的表达情况以及在伴有肝脏损害和不伴有肝脏损害重度子痫前期胎盘的表达差异。方法应用原位杂交和RT-PCR方法对早孕绒毛组织(10例)、妊娠中期胎盘组织(10例)、正常妊娠晚期胎盘组织(10例)及32例重度子痫前期胎盘组织进行LCHAD基因的定位表达及半定量测定。结果原位杂交实验显示正常妊娠早、中、晚期绒毛或胎盘组织及重度子痫前期胎盘组织滋养细胞中存在LCHAD阳性表达。RT-PCR实验显示①妊娠早期绒毛LCHAD表达与妊娠中期比较,P=0.844;妊娠早期绒毛LCHAD表达高于晚期胎盘,P=0.020;妊娠中期胎盘LCHAD表达也高于晚期胎盘P=0.026;②发病孕周≤34周早发型重度子痫前期伴肝损害胎盘组织中LCHAD表达均值为(0.449±0.038),不伴肝损害LCHAD表达均值为(0.482±0.042),伴肝损害较不伴肝损害者表达有减弱,但两组比较,P=0.084;发病孕周≤34周早发型重度子痫前期伴肝损害LCHAD表达量与正常晚期比较,P=0.05,而不伴肝损害重度子痫前期LCHAD表达量与正常晚期比较,P=0.775。结论本研究显示在妊娠的早中晚期滋养细胞中均存在长链脂肪酸氧化代谢,妊娠早中期LCHAD的mRNA表达高于妊娠晚期;早发型重度子痫前期伴肝损害胎盘组织中LCHAD表达均值与不伴有肝损害者比较虽无统计学差异,但是有明显降低趋势。提示长链脂肪酸氧化代谢对子痫前期伴发肝脏损害的影响还有待酶活性和蛋白水平以及代谢调节方面的深入研究。     

早期自然流产患者绒毛KiSS-1、MMP-9和MMP-2基因及其蛋白表达

目的研究早期自然流产患者绒毛肿瘤转移抑制基因KiSS-1与基质金属蛋白酶(MMP-9、MMP-2)mRNA及蛋白的表达,探讨其与自然流产的关系。方法采用半定量RT-PCR、免疫印迹法检测30例正常早期妊娠和30例早期自然流产患者绒毛KiSS-1、MMP-9和MMP-2的mRNA及蛋白水平,用明胶酶谱分析法检测上述样本孵育液MMP-9、MMP-2的酶活性。结果(1)自然流产患者绒毛组织中MMP-9及MMP-2mRNA表达水平低于正常早期妊娠组(P<0.05),KiSS-1mRNA的表达高于正常早期妊娠组差异有统计学意义(P<0.05);(2)Westernblotting分析显示自然流产患者绒毛MMP-9、MMP-2蛋白表达低于正常早期妊娠差异有统计学意义(P<0.05),KiSS-1蛋白表达高于正常早期妊娠差异有统计学意义(P<0.05);(3)明胶酶谱分析结果显示,自然流产患者绒毛MMP-9、MMP-2酶活性低于正常早期妊娠者差异有统计学意义(P<0.05)。结论自然流产患者绒毛促浸润基因MMP-9、MMP-2mRNA及其蛋白表达水平降低,同时抑制浸润基因KiSS-1mRNA及其蛋白表达增加,造成滋养细胞浸润能力下降,可能与自然流产的发生有关。     

人类白细胞抗原F在妊娠期肝内胆汁淤积症患者胎盘中的表达及意义

目的:研究人类白细胞抗原F(HLA-F)在妊娠期肝内胆汁淤积症(ICP)患者胎盘组织中的表达,并从免疫学角度探讨其与ICP发病的关系。方法:采用免疫组织化学技术对妊娠晚期(ICP组40例和正常对照组40例)胎盘组织中表达的HLA-F蛋白进行定位;再通过PCR及蛋白质免疫印迹技术,研究ICP患者(轻度20例,重度20例)胎盘组织中HLA-F在核酸分子(mRNA)及蛋白中的表达情况。结果:1妊娠晚期胎盘组织中仅蜕膜板绒毛外滋养细胞(EVCT)膜上有HLA-F蛋白表达。2正常对照组和ICP组间胎盘组织中HLA-F mRNA的表达差异无统计学意义(P0.05);正常对照组和轻度ICP、重度ICP3组间胎盘组织中HLA-F mRNA的表达差异均无统计学意义(P0.05);ICP组胎盘组织中HLA-F蛋白的表达水平低于正常对照组(0.6092±0.3370vs1.3451±0.5540),差异有统计学意义(P0.05);重度ICP胎盘组织中HLA-F蛋白的表达水平显著低于正常对照组(0.5328±0.2762vs 1.3451±0.5540)差异有统计学意义(P0.05);而轻度ICP和正常对照组间胎盘组织中HAL-F蛋白的表达量差异无统计学意义(P0.05)。3ICP非地塞米松组和ICP地塞米松组间胎盘组织中HLA-F mRNA及蛋白的表达差异均无统计学意义(P0.05)。结论:HLA-F蛋白表达于妊娠晚期胎盘组织蜕膜板EVCT细胞膜上;ICP患者胎盘组织中HLA-F蛋白表达水平降低可能是导致ICP患者妊娠免疫耐受平衡紊乱的重要机制之一。     

细胞因子IL-6mRNA与IL-10mRNA在早产合并绒毛膜羊膜炎胎盘组织中的表达水平及意义

目的:研究白细胞介素-6 mRNA(IL-6 mRNA)和白细胞介素-10 mRNA(IL-10 mRNA)在早产合并绒毛膜羊膜炎孕妇胎盘组织中的表达水平,探讨IL-6及IL-10在妊娠中的作用,以及其在早产合并绒毛膜羊膜炎中的变化及意义。方法:取足月分娩及早产孕妇部分胎盘胎膜组织做病理检查,根据病检结果分为3组:早产感染组20例、早产非感染组20例和足月组20例。采用二步法RT-PCR法测定3组胎盘组织中IL-6 mRNA和IL-10 mRNA的表达。结果:早产感染组胎盘组织中IL-6 mRNA表达水平明显高于早产非感染组和足月组,差异有显著性(P<0.05);而IL-10 mRNA表达水平明显低于另外两组,差异有显著性(P<0.05)。结论:感染可引起细胞因子IL-6 mRNA、IL-10 mRNA在胎盘组织中的表达发生改变,推测IL-6可促进分娩发动,使妊娠提前终止,而IL-10在维持正常妊娠中可能起重要作用。     

TGF-β和IGF表达异常与滋养层相关疾病关系的研究

目的:探讨转化生长因子(TGF-β)和胰岛素样生长因子(IGF)等与滋养层侵入调节密切相关的细胞因子在胎盘绒毛组织中的表达,及与葡萄胎、先兆子痫等滋养层细胞相关妊娠疾病病理机制的关系。方法:采用半定量逆转录多聚酶链反应(RT-PCR)检测方法,分别以正常早孕绒毛和正常足月(自然分娩或剖宫产)胎盘绒毛组织为对照,观察葡萄胎组织与先兆子痫足月胎盘绒毛组织中TGF-β和IGF的表达。结果:5种胎盘绒毛组织中均可检测到IGF-I mRNA的表达,且先兆子痫足月胎盘绒毛组织中IGF-I的表达量明显低于正常足月胎盘绒毛组织,而葡萄胎组织中IGF-I的表达略高于正常早孕绒毛。在3种足月胎盘绒毛组织中可检测到IGF-ⅡmRNA的表达,但相互之间无明显差异。葡萄胎组织中IGF-Ⅱ的表达低于早孕绒毛。5种胎盘绒毛组织中均有TGF-β3的表达,而TGF-β1和TGF-β2的表达并未检测到。在表达量上,葡萄胎组织中TGF-β3的表达明显高于正常早孕绒毛组织,先兆子痫胎盘绒毛组织中TGF-β3的表达强于足月自然分娩的正常胎盘绒毛组织,而剖宫产胎盘绒毛组织中,TGF-β3的表达亦相对较强。结论:TGF-β3和IGF-I在胎盘绒毛组织中的表达变化,可能和葡萄胎、先兆子痫等与滋养层密切相关的妊娠疾病的发生有关。     

The determination of basic fibroblast growth factor (bFGF) in human placental tissue and the changes in bFGF during pregnancy]

The purpose of this study is to develop an assay system for quantification of bFGF in human tissue and to investigate the changes in bFGF content in the human placenta during pregnancy. Sixty-two placental tissue samples from various stages of normal pregnancies were collected. Approximately 28 micrograms bFGF was obtained per 1 kg of placental tissue. The recovery rates were 17.1 +/- 7.4%. The purified samples were confirmed as bFGF by SDS-PAGE and enzyme-linked immunoelectrotransfer blot (EITB) with anti-human bFGF monoclonal antibody. The bFGF readings in the human placenta determined by RIA were 11.81 +/- 2.11 fmol/mg protein (first trimester), 20.45 +/- 4.85 (early second trimester), 9.52 +/- 5.02 (late second trimester), 7.41 +/- 2.07 (third trimester), and 7.75 +/- 1.86 (post trimester). The placental bFGF were significantly high in the early stage of second trimester and declined gradually during the remainder of the pregnancy. The RIA values were correlated closely with the values obtained by bioassay. These results demonstrate that our assay system provides a tool for the quantification of bFGF in biological samples and suggest that bFGF, the active mitogen and angiogenic factor, participates in the formation of the human placenta.     

Expression patterns of Notch receptors and their ligands Jagged and Delta in human placenta

The establishment of an appropriate fetomaternal vessel system is a prerequisite for prevention of pregnancy associated pathologies. Notch receptors and ligands are manifoldly involved in vascular development and angiogenesis. To further characterize the process of human placental vasculo- and angiogenesis we investigated the expression pattern of Notch receptors and their ligands during pregnancy.Real time RT-PCR, immunohistochemistry and flow cytometry analysis were performed in early (6-12) weeks of gestation (w.o.g.) and late placenta (37-41 w.o.g.). To specify the exact cellular localization immunofluorescent labelling of epithelial and endothelial cells (EC), respectively, with cytokeratin-7 and vonWillebrand factor (vWF) was done. One placenta from a patient with Alagille syndrome (AGS) was examined with real time RT-PCR and immunohistochemistry.The receptors Notch2, -3, -4 and their ligands Jagged1, -2 and Delta1, -4 were detected at both the mRNA and protein level in early and late placenta. Notch1 was only detected at protein level. The expression was found mainly in the stromal compartment: placental EC expressed Notch1, Delta4, Jagged1 and Delta1. A strong Jagged1 expression was found in the endothelium of arteries and veins supporting a role in differentiation of capillaries. Hofbauer cells (HC) primarily displayed the receptors Notch2, -3 and -4. Placental stromal cells (SC) were positive for Jagged2. The syncytiotrophoblast (ST) and cytotrophoblast (CT) cells revealed a weak but detectable co-localization with cytokeratin-7 and Notch1, -3 and Delta1. These results were verified by flow cytometry of freshly isolated placental cells of placental tissue. Interestingly Jagged1 expression was absent in endothelial cells from an AGS placenta.The Notch receptors and their ligands are expressed in human placental ST, CT, EC, SC and HC. The distribution pattern of Notch receptors and their ligands suggests their involvement in the process of placental vasculo- and angiogenesis via cell-cell communication between trophoblast, -stroma and endothelial cells.     

A comparison in concentration of heat shock proteins (HSP) 70 and 90 on chorionic villi of human placenta in normal pregnancies and in missed miscarriages

PURPOSE: To investigate the role of heat shock protein (HSP) on the chorionic villi of human placental cells and to compare the concentration of placental HSP70 & 90 in term deliveries and in missed miscarriages. MATERIALS AND METHODS: Fifty products of conception from women who experienced first trimester missed miscarriage and 50 placentas from women who gave birth at term were studied. An immunohistochemical investigation was carried out with which we marked the localization of heat shock proteins 70 and 90 on the syncytiotrophoblastic, cytotrophoblastic, stromal and blood vessel cells, using specific antibodies which can detect the presence of those proteins on light microscopy. We compared their expression with the normal placental tissue of term pregnancies and with material acquired from first trimester missed miscarriages. An indirect immunoperoxidase method was applied using polyclonal antibodies against HSP70 and HSP90 on formalin-fixed paraffin-embedded tissues. RESULTS: Expression of HSP90B was increased in chorionic villi of first trimester missed miscarriages concerning syncytiotrophoblasts, cytotrophoblasts, vessel and stroma cells compared to full-term placentas. There was a statistically significant increase of HSP90A expression in chorionic villi of first trimester missed miscarriages, concerning only the cytotrophoblast cells, compared to full-term placentas. Expression of HSP70 cognate protein was significantly increased in chorionic villi of first trimester missed miscarriages, concerning syncytiotrophoblastic cells only, compared to full-term placentas. Finally, HSP70 inducible protein was significantly increased in chorionic villi of first trimester missed miscarriages concerning syncytiotrophoblasts, cytotrophoblasts, vessel and stroma cells compared to full-term placentas. CONCLUSIONS: The results of the present study have sufficiently shown that there is an increase of HSP70 & 90 expression in chorionic villi of first trimester missed miscarriages compared to full-term placentas and this increase may have an important implication on the miscarriage process.     

Expression and distribution of notch protein members in human placenta throughout pregnancy

Notch signaling is an evolutionarily conserved mechanism used by invertebrates and vertebrates to control cell fates through close-range cell interactions. Four Notch receptors have been identified in vertebrates and different ligands, divided into Delta-like and Serrate-like (Jagged). Several studies have demonstrated that Notch signaling is involved in different branches of the cell fate decision tree: differentiation, proliferation and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and a correct fetal growth. Moreover, Notch and its ligands participate in the vascular remodelling and stabilization, other two processes much important and ticklish in human placenta. So, we decided to investigate the pattern of expression of Notch-1, Notch-4 and Jagged-1, together with two members related to Notch pathway and involved in angiogenesis: VEGF and p21, in human placenta during gestation by immunoblotting and immunohistochemistry. We showed a modulation of Notch proteins throughout the pregnancy; in particular we showed a slight decrease of Notch-1 throughout pregnancy, with a decreased cytoplasmic staining from the first to the third trimester of gestation in cytotrophoblast and syncytiotrophoblast. In contrast Jagged-1 showed an increase throughout pregnancy especially in syncytiotrophoblast and stroma during the third trimester of gestation. In addition, we found by immunoblotting an increase of VEGF expression from the first to the third trimester and an intense VEGF expression inside endothelial cells throughout the gestation as also confirmed by immunohistochemistry. We also showed a decrease of p21 expression during the pregnancy both through immunoblotting and immunohistochemistry assays. Moreover, we observed Notch localization in extravillous trophoblast cells that are able to invade the decidualized endometrium. Our results suggest an involvement of Notch signaling in regulation of placental cell fate decision and in angiogenesis that are dramatically important to maintain a normal physiology of this organ during pregnancy.     

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.