【作者】 俞丽丽；

【导师】 李力；

【作者基本信息】 第三军医大学 ， 妇产科学， 2009， 博士

【摘要】 背景与目的滋养细胞来源于形成中胚泡的滋养内胚层,是胚泡中最先分化的细胞。滋养细胞的增殖、分化、凋亡和程序性侵入是胚胎发育、胎盘形成及胎儿生长所必需的。滋养细胞的功能异常可导致子痫前期/子痫、不明原因的流产、胎儿生长受限等妊娠相关性疾病以及葡萄胎、侵袭性葡萄胎、绒癌等妊娠滋养细胞疾病(Gestational trophoblastic diseases,GTD)的发生。因而,对滋养细胞功能的调控机制的深入研究,有利于阐明妊娠相关性疾病以及妊娠滋养细胞疾病的发病机制,并对这些疾病的防治有重要的意义。研究滋养细胞增殖、分化、凋亡和程序性侵袭行为的调控因素已成为目前妇产科学研究热点,但确切的调控机制至今尚未阐明。调控因素很多,也很复杂。如何将这些错综复杂的调控因素有机地统一起来一直是困扰我们的难题。但不管通过何种因素起作用,其最初都是先出现某个基因或某组基因的改变,最终通过细胞信号传导途径来引起滋养细胞功能的变化。基因印迹(gene imprinting)是指体细胞来源于不同亲代的一对等位基因发生的差异性表达,它是一种不遵从经典的孟德尔遗传规律的依靠单亲传递某些遗传学性状的现象。正常的基因印迹在功能上与胎儿及胎盘的发育、细胞分化和增殖等有关。基因印迹异常(如印迹丢失、杂合性丢失、单亲二倍体等现象)与某些特殊的遗传性疾病及肿瘤的发生密切相关。其中印迹基因H19作为最早发现的印迹基因之一,是一种母系表达,父系沉默的印迹基因,又是一种非编码RNA,其转录产物不编码蛋白,而是在RNA水平发挥作用。但H19确切的作用机制目前仍未能完全阐明。有学者认为它是原癌基因,也有学者认为它是抑癌基因。因此,H19可能在细胞的增殖、分化和侵袭方面具有双重作用。印迹基因H19的不同作用取决于不同的组织类型以及不同的发育阶段,即H19基因具有组织特异性及发育阶段特异性。然而,目前的文献报道仍然无法明确在滋养细胞中H19到底是抑癌基因还是原癌基因。我们既往的研究结果发现H19的印迹状态在胎盘滋养细胞的发育过程中存在着动态变化,而这个动态变化的节点是在孕10周,恰好和滋养细胞的第一次侵袭高峰的时间非常一致。因此,我们有理由推测:印迹基因H19在滋养细胞节制性侵袭行为(或程序性侵袭行为)的调控中可能发挥着关键性的作用。我们既往的研究发现H19在45%的子痫前期胎盘组织中呈印迹丢失(Loss of imprinting,LOI),在正常晚孕胎盘组织中均呈印迹状态;然而在子痫前期胎盘组织中H19 RNA表达水平与正常晚孕胎盘组织却无差异。H19的印迹状态与H19 RNA表达水平的不一致也在很多肿瘤组织的研究中被认识到。H19的表达水平除了印迹或甲基化的调控方式外,可能还存在着其他的调控方式或者负反馈的保护作用。那么,H19到底是通过印迹状态的改变起作用还是通过RNA表达水平的变化起决定性作用,或者是两种方式均产生作用,目前仍不清楚。我们之前采用DNA甲基化转移酶抑制剂5-杂氮脱氧胞苷作用于人滋养细胞株JEG-3细胞后发现,H19启动子区域呈现低甲基化状态,H19 RNA表达明显上调,滋养细胞增殖、迁移和侵袭能力下降。虽然前期的实验可以证明H19启动子区域甲基化水平的高低可直接影响H19的表达水平,但是由于5-杂氮脱氧胞苷的甲基化抑制效应是广谱的,而缺乏特异性,因此,药物作用不仅引起H19基因的表达改变,还可能影响其它基因的印迹状态和表达水平。目前尚没有针对H19的甲基化转移酶抑制剂的药物。因此,为了进一步阐明或明确H19的表达水平对滋养细胞功能的影响,本研究拟通过人全长H19cDNA重组真核表达载体和H19-RNA干扰慢病毒载体的构建来深入探讨H19过表达和低表达对滋养细胞功能的影响;同时探讨H19通过哪些相关的信号传导分子来影响滋养细胞的功能以及相关信号传导分子在重度子痫前期胎盘组织中的表达状况,为印迹基因H19调控滋养细胞功能改变的分子机制的阐明奠定基础,为子痫前期的发病机制的阐明提供新的线索。本研究的主要结果和结论如下:1.滋养细胞株JEG-3细胞和JAR细胞以及正常胎盘滋养细胞H19的表达水平非常丰富,而且H19-RNA干扰慢病毒载体感染后不影响细胞形态学的改变,因此,采用H19-RNA干扰慢病毒载体构建H19低表达模型的方法优于含人全长H19cDNA重组真核表达质粒pRc/CMV的H19过表达模型,更适合用于H19在滋养细胞中的功能研究。H19-RNA干扰慢病毒载体的成功构建,为后续的H19的功能研究奠定了基础。2.H19干扰后可抑制JAR细胞的凋亡,使JAR细胞周期阻滞在G1期,而不影响JAR细胞侵袭力以及细胞基质金属蛋白酶9 (matrix metalloproteinase-9,MMP-9)mRNA的表达,提示H19不是直接通过影响滋养细胞的侵袭力,而是通过参与调节滋养细胞的增殖和凋亡过程来影响滋养细胞的生物学功能。3.分别采用含人全长H19cDNA重组真核表达质粒pRc/CMV转染的方法和H19 RNA干扰的方法均证实了在人绒癌滋养细胞株中H19表达水平的改变不能直接调控胰岛素生长因子2(insulin-like growth factor 2,IGF2)的表达水平。H19的过表达可下调JEG-3细胞HES1和双特异性磷酸酶5(dual specificity phosphatase 5,DUSP5)mRNA的表达,H19低表达可上调JAR细胞的HES1和DUSP5 mRNA的表达,提示HES1和DUSP5在H19调控滋养细胞的生物学功能中可能起着重要的信号传导作用。4.结合子痫前期H19呈印迹丢失状态以及重度子痫前期胎盘组织IGF2、HES1mRNA表达水平均较正常晚孕胎盘组织明显降低,H19可调节HES1和DUSP5的表达的研究结果推测:在妊娠早期H19印迹丢失导致H19过表达,从而使HES1和DUSP5表达受阻,引起滋养细胞的分化障碍、促进滋养细胞的凋亡,最终导致滋养细胞功能异常,从而参与子痫前期的发病。5.不管H19是过表达还是低表达都不影响内吞蛋白(EGFR pathway substrate15,EPS15)mRNA的表达变化,提示EPS15在H19调控滋养细胞的生物学功能中并不起重要作用;重度子痫前期胎盘组织EGFR及EPS15表达水平与正常晚孕胎盘组织比较也无明显改变,提示EGFR及EPS15不是参与调节表皮生长因子(epidermal growth factor, EGF)水平以及重度子痫前期发病的重要信号分子。本研究工作是探索H19在滋养细胞中的生物学功能以及对下游信号传导分子的影响的一次尝试,为进一步的表观遗传学研究奠定了良好基础,为开拓H19的新功能的研究提供了有益的信息,为阐明滋养细胞相关性疾病的发病机制和防治策略提供了新的视角。更多还原

【Abstract】 Background and Objective:Pre-eclampsia is a life-threatening, pregnancy-related disease and leading cause of global maternal and neonatal mortality, affecting 2-7% of pregnancies worldwide. Many risk factors contribute to the development and severity of pre-eclampsia, which include primiparity, exposure to sperm, multifetal gestation, chronic hypertension, previous pre-eclampsia, pregestational diabetes mellitus, maternal low birth weight, and others. Clinical symptoms and disease severities vary from mild to life-threatening, including seizure, hemolysis, liver dysfunction, low platelet count, fetal hypoxia, and growth retardation, even with disseminated intravascular coagulation, placental abruption, cerebral hemorrhage, acute renal failure, circulation collapse and death. Although pre-eclampsia is commonly associated with hypertension and proteinuria, the etiology and pathogenic process of pre-eclampsia remain unclear. Several maternal genetic factors and their trophoblast effects are important to the susceptibility of individual pregnant women to develop pre-eclampsia. Continuous approaches to identify genetic factors and their function will be of great significance in understanding the pathogenesis of pre-eclampsia.Genomic imprinting, unlike the majority of genes where expression is from both inherited alleles of a gene, leads to paternal and maternal alleles of some genes having different levels of activity, one of the paternal alleles silenced, another expressed. The silencing mechanism is known to allel specific DNA methylation and/or histone modifications. The imprinted H19 gene, as one of the first genes showed to be paternal imprinted and maternal expressed, is expressed abundantly in the human placenta and in several embryonic tissues,and is only marginally expressed in nearly all normal adult tissues. In adulthood a basal H19 gene expression has been detected only in mammary gland, cardiac and skeletal muscles and to a lesser extent in kidney, adrenal gland and lung.Previous studies have shown that the H19 gene is transcripted into a non-coding RNA, and acts mainly or exclusively at the RNA level. However, the function of this non-coding RNA remains to be elucidated. The H19 gene is highly expressed in human embryonic tissue, regulating the growth and development of the embryo and the differentiation of placental cytotrophoblasts. However, its precise role in the regulation of reproduction and fetal development is poorly understood. It has been suggested that H19 functions as a tumor suppressor, on the basis of loss of heterozygosity at 11p15.5 with the retention of the paternal chromosome in some Wilms’tumor cases, embryonal rhabdomyosarcoma, and Beckwith-Wiedemann cancer predisposing syndrome. Studies published by others show that H19 was an oncogene. Interestingly, the development of pre-eclampsia is associated with a decreased invasive capacity of trophoblasts. Given that a remarkable reduction in the expression of the H19 gene is associated with the development of choriocarcinoma and its highly invasive capacity, we reasonably hypothesized that the imprinting status of the H19 gene may contribute to the pathogenesis of pre-eclampsia. Our study also showed that the biallelic expression of H19 gene existed in some cases at the early stage of normal pregnancy and changed into monoallelic expression near at 10 weeks of gestation. The dynamic alternations in the patterns of H19 gene imprinting may regulate the maintenance of normal pregnancy and be related with the trophoblast regulative invasion.To investigate the possible function of H19 on human trophoblast and study the effect of H19 on the corresponding signal conduction gene, we transiently transfected human choriocarcinomal cell line JEG-3 cells with pRC/CMV–H19 containing a sense full-length H19 cDNA under the control of the cytomegalovirus promoter and pRC/CMV and a lentiviral expression vector containing H19 small interfering RNA was constructed. It provides the experimental basis for the regulation of the trophoblast cellular function with H19 gene.The study elucidate the pathogenesis of trophoblastic correlated diseases (gestational trophoblastic diseases, preeclampsia, abortion and fetal growth retardation) from the point of the gene imprinting and offer a new way for prevention and cure of preeclampsia.The results and conclusions were as follow:1. A lentiviral expression vector containing H19 small interfering RNA didn’t influence the cellular morphology, and was better than pRC/CMV–H19 containing a sense full-length H19 cDNA. It is better for study that adopt the method of RNA interfering because of the high expression of H19 in the placental trophoblast, human choriocarcinomal cell line JEG-3 cells and JAR cells. The successful construction of a lentiviral expression vector containing H19 small interfering RNA establish the basis for the continued function study.2. After the knockdown of H19 expression, the invasion ability and the expression of MMP-9 gene had never changed. The apoptosis of JAR cell were repressed and the cell cycle was holded up at the stage of G1 after the interferece of H19 RNA expression of JAR cells. It is suggested that H19 could regulate the proliferation and apoptosis of trophoblast cell and couldn’t direct influence the invasion of trophoblast cell.3. It was confirmed that H19 couldn’t regulate directly the expression of IGF2 in the human choriocarcinomal cell line by the two ways of RNA interfering and the transfection of pRC/CMV–H19 containing a sense full-length H19 cDNA . The excessive expression of H19 inhibited the expression of HES1 and DUSP5 in JEG-3 cells, and after the knockdown of H19 expression, the expression of HES1 and DUSP5 in JAR cells were up-regulated.It is suggested that HES1 and DUSP5 could have the important role in signal transduction in the regulation of biological function of trophoblast.4. The expression of IGF2 mRNA and HES1 mRNA decreased in the patients with severe pre-eclampsia than that in the normal pregnancy and pre-eclampsia patients showed biallelic expression of H19 gene in the previous experiment. It is suggested that the loss of H19 gene imprinting in the early stage of pregnancy could induce the excessive expression of H19, and impede the expression of HES1 and DUSP5, contributing to trophoblast differentiation obstruction, apoptosis of trophoblast cell and the pathogenic process of pre-eclampsia.5. The expression of H19 didn’t influence the expression of EPS15 and there was no difference in the expression of EGFR and EPS15 between the patients with severe pre-eclampsia and normal pregnancy. It is suggested that EGFR and EPS15 could not be important signaling molecules to regulate the expression of EGF and take part in the pathogenesis of pre-eclampsia.更多还原