【作者】 颜耀华；

【导师】 李力；

【作者基本信息】 第三军医大学 ， 妇产科学， 2013， 博士

【摘要】 背景与目的胎盘是人类胚胎发育成熟、妊娠顺利完成的基本要素。晚期囊胚着床之后，绒毛外滋养层细胞(extravillous trophoblast cell, EVT)向子宫蜕膜层和肌层血管的进步侵袭、迁移、分化是母-胎循环建立和胎盘锚定的关键环节，对胚胎发育、母胎间对话尤为重要。已证实，EVT侵袭性迁移不足可导致子宫螺旋动脉重铸障碍，是自然流产、子痫前期-子痫、胎儿生长受限等疾病的重要病理过程；同时，滋养细胞过度侵袭则是胎盘植入、胎盘附着部位异常的重要病理特征，滋养细胞恶性侵袭则最终可导致葡萄胎、绒癌等疾病。明确滋养细胞侵袭性的调节机制，是解决上述疾病的关键问题与希望所在。子痫前期及子痫是孕期以高血压为主要特征的多系统疾病，严重威胁母婴健康，对其病因和发病机制的研究直是产科的重要研究课题。多年来，通过几代学者对子痫前期的研究，虽仍未明确其发病的机制，但却形成了几种发病学说：子宫螺旋小动脉重铸不足、炎症免疫过度激活、血管内皮细胞损伤、遗传因素、营养缺乏、胰岛素抵抗等，其中子宫螺旋小动脉重铸不足是目前比较公认的观点。自1914年有学者根据子痫前期临床病征提出子宫缺血学说以来,现多数学者认为子宫缺血实质上是胎盘或滋养细胞缺血，其原因是子宫螺旋形小动脉生理重铸过程障碍,此病理现象也称之为“胎盘浅着床”,研究发现其与胚胎着床过程中滋养浸润能力受累有关。近来的大量研究表明，滋养细胞的生物学行为的改变可能与其相关基因表达异常从而导致些影响滋养细胞侵袭相关的蛋白酶或其它分子的分泌有关，另外可能还与子宫局部激素、母胎界面的免疫状况、细胞因子等内环境的变化和些控制胚胎和胎盘发育基因的表达有关。我们通过基因表达谱芯片对子痫前期患者胎盘组织中差异表达的基因进行筛选和分析，从中选出差异表达最明显的基因LAIR-2，研究其不同表达情况下滋养细胞生物学行为的改变，探讨子痫前期差异表达基因对滋养细胞侵袭功能的影响及机制。JEG-3细胞系来自人绒毛膜癌组织，其细胞形态、生化标志、激素合成分泌等特征与原代绒毛滋养细胞极其相似。本研究基于JEG-3体外培养等实验平台，应用构建cDNA过表达及RNAi慢病毒载体与细胞转染、Transwell、Western blot、RT-PCR等实验技术，研究并探讨子痫前期差异表达基因LAIR-2对人滋养细胞侵袭能力的影响及可能的机制。主要研究结果及结论如下：1．本研究通过基因表达谱芯片共筛选出1000余种在子痫前期患者与正常妊娠相比胎盘组织中表达有差异的基因，其中实验组中表达明显上调且表达量的差异超过3倍的基因共15种，表达下调且有明显表达量差异的基因共14种，差异均有显著性(P<0.05)。这些基因有与妊娠相关性疾病有关的、有与细胞侵袭有关的、有与胚胎和胎盘发育相关而影响妊娠结局的、有与生殖功能相关的，还有些基因目前鲜有报道。这些研究结果为子痫前期在分子水平的研究提供了理论依据。2．本实验研究中，我们通过Western blot和PCR技术对子痫前期患者晚孕胎盘组织与正常妊娠胎盘组织中LAIR-2的表达进行对照研究，结果显示在子痫前期患者的晚孕胎盘组织中，LAIR-2mRNA及蛋白的表达均高于正常妊娠组，此结果验证了第部分的实验结果，提示LAIR-2与子痫前期疾病密切相关。其在对子痫前期患者胎盘发育的影响及其因果关系尚待后续研究。3．(1)通过基因重组技术构建了LAIR-2cDNA过表达和RNA干扰重组慢病毒载体并成功稳定转染了滋养细胞株。(2)RNAi慢病毒载体转导滋养细胞降调了LAIR-2的表达，而cDNA过表达慢病毒载体转导滋养细胞同则上调了LAIR-2的表达。(3)LAIR-2过表达和基因沉默稳转细胞株的成功建立，为今后LAIR-2功能研究奠定了基础。4、(1)子痫前期差异表达基因LAIR-2参加了滋养细胞生物学行为的调节。(2)LAIR-2影响滋养细胞的增殖、迁移及侵袭能力，LAIR-2表达上调后细胞上述能力减弱，反之其表达下调后，上述能力增强。(3)LAIR-2影响滋养细胞MMPs的表达，LAIR-2表达上调后，其MMP-2、MMP-9、MMP-14的表达均下降，反之其表达下调后，MMP-2、MMP-9、MMP-14的表达升高。上述结果表明：LAIR-2可能在以滋养细胞侵袭不足为主要病理生理表现的子痫前期的发病中发挥重要的作用，而影响MMPs的表达则可能是其重要的中间环节和机制。对LAIR-2的深入研究有助于阐明子痫前期的发病机制，并为子痫前期的预测、早期诊断和早期治疗等临床应用研究提供方向。更多还原

【Abstract】 Background and ObjectivePlacenta is essential for the intrauterine development of the human embryo. After theembryonic implantation, Extravillous cytotrophoblasts (EVT) invade the underlyingdecidua, then surround and migrate into the wall of the uterine spiral arteries, which resultsin the remodeling of uterine vasculature. This process plays a pivotal role in mammalianplacentation and is stringently regulated to ensure a successful pregnancy. Poor invasion ofEVT is believed to be associated with insufficient remodeling of the spiral arteries, which istypical of the pathological changes of miscarriage, preeclampsia and intrauterine growthrestriction. Conversely, excessive invasion leads to placenta percreta, persistenttrophoblastic disease or invasive cancer, such as choriocarcinoma. Elucidating theregulatory mechanism of trophoblast invasion is critical to understand these diseases.Preeclampsia (PE) is a multi-system disorder of human pregnancy that is characterizedby hypertension and proteinuria, posing a serious threat to maternal and child health.Studies on its etiology and pathogenesis have become an important research topic inobstetrics. Several generations of scholars have been active in preeclampsia, and severalpathogenesis theories have been proposed despite of their unclear pathogenesis mechanisms,including insufficient remodeling of the spiral arteries, excessive activation ofinflammatory immunization, vascular endothelial cell injury, genetic factor, nutritionaldeficiency, insulin resistance, etc. Among the above factors, insufficient remodeling of thespiral arteries is more widely accepted. Since1914, some scholars had proposed the uterineischemia theory based on the clinical signs of preeclampsia, most scholars believe thatuterine ischemia is due to ischemic placenta or trophoblast, resulting from placental spiralarteries recast obstacles, known as "shallow placental implantation." Uterine ischemia hasbeen reported to be related with the involvement of trophoblast invasion in the process ofembryo implantation. Recently, a large number of studies have shown that the changes in biological behavior oftrophoblast cells may be associated with abnormal gene expression, thus affecting thesecretion of trophoblast invasion-related proteases or other molecules; on the other hand, itmay be associated with the internal environment changes in hormone, immune status ofmaternal-fetal interface or cytokine in womb and the expression of genes controllingembryonic and placental development.In this study, the differentially expressed genes in preeclampsia placenta tissues werescreened and analyzed through the microarray technique, and LAIR-2was identified as themost differentially expressed gene. Our experiment aims to study the biology behaviorchange in trophoblastic cells under different expression circumstances and to explore theeffect of the differentially expressed genes in preeclampsia on the invasion of trophoblastcells and its mechanism.JEG-3cell lines are derived from human choriocarcinoma tissues and share manyproperties with the villous trophoblast in terms of their morphology, biochemical markers,and hormone secretion. Based on the JEG-3cells culture, we applies cDNA overexpressionand RNAi lentiviral vectors and cell transfection, Transwell, Western blot, RT-PCR andother experimental techniques to study and explore the impact of the preeclampsiadifferentially expressed gene LAIR-2on the human trophoblast invasion and its possiblemechanism.Major findings and conclusions:1. In this study, microarray technique was applied to select more than1,000geneswhich were differentially expressed between normal pregnancy placenta tissues andpreeclampsia placenta tissues. There were15kinds of genes which were significantlyup-regulated and displayed more than3-fold expression level differences in theexperimental group, and14kinds of genes which were significantly down-regulated withremarkable differences in gene expression (P <0.05). These genes are associated withpregnancy-related diseases, cell invasion, embryo development or pregnancy outcomes.Some genes are currently rarely reported. These findings provide a theoretical basis for thestudy of the preeclampsia at the molecular level.2. We adopted Western blot and PCR techniques to conduct a comparative study ofLAIR-2expression between the placental tissue in late pregnancy and placental tissue in normal pregnancy in preeclampsia patients. LAIR-2mRNA and protein expression inpreeclampsia patients during late pregnancy was higher than that in normal pregnancy. Thisresult verifies the first part of the experiment, suggesting a close relationship betweenLAIR-2and preeclampsia diseases. The effect of LAIR-2on the placental development inpreeclampsia patients and their relationship need further study.3.(1) LAIR-2cDNA overexpression and RNA interference lentiviral vectors weresuccessfully constructed by gene recombination technology and trophoblastic cell lineswere stably transfected.(2) The transduction of trophoblast cells with RNA interference lentiviraldown-regulated the LAIR-2expression, whereas the transduction of trophoblast cells withthe overexpressed cDNA lentiviral up-regulated the AIR-2expression.(3)Successful construction of LAIR-2overexpression and cell lines with genesilencing laid the foundation for the future research about LAIR-2functions.4.(1) LAIR-2, as being a differentially expressed gene in preeclampsia, participated inregulating the biological behavior of trophoblastic cells.(2) LAIR-2affected the trophoblast proliferation, migration and invasion. The LAIR-2up-regulation decreased the trophoblast proliferation, migration and invasion, whereasLAIR-2down-regulation increased the above capabilities.(3) LAIR-2affected the expression of MMPs in trophoblast. LAIR-2up-regulationdecreased the expression of MMP-2, MMP-9and MMP-14, whereas its down-regulationincreased their expression.The above results indicate that: LAIR-2may play an important role in preeclampsiacharacterized with insufficient trophoblast invasion, and its role in affecting the expressionof MMPs is an important intermediate link and mechanism. In-depth study of LAIR-2helpsto clarify the pathogenesis of preeclampsia, and to provide new directions for the earlydiagnosis and treatment of preeclampsia in clinical practice.更多还原