【作者】 韩健；

【导师】 李力；

【作者基本信息】 第三军医大学 ， 妇产科学， 2010， 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一步侵袭、迁移、分化是母-胎循环建立和胎盘锚着固定的关键环节,对胎盘的形成尤为重要。已证实,EVT侵袭性迁移不足可导致子宫螺旋动脉重铸不足,是流产、子痫前期/子痫、胎儿宫内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、葡萄胎、绒癌等疾病的重要病理特征。明确滋养细胞侵袭性迁移的调节机制,是解决上述疾病的关键问题与希望所在。EVT的侵袭性迁移是由细胞粘附、胞外基质降解、运动迁移、穿越基底膜等多个环节构成多元化过程,受到体内微环境的精确调节。蜕膜/胎盘等局部器官、组织和/或机体其它器官、组织分泌的内源性因子如内分泌激素、细胞因子等能够作用于EVT的侵袭性迁移过程,是调节该过程的重要因素。但各种因素调节EVT侵袭性迁移的确切分子机制至今尚未阐明。表皮生长因子(epidermal growth factor,EGF)是一种对机体发育、代谢等多种生物学过程具有调节作用的重要的生长因子。已有证据表明,EGF及EGFR在人类妊娠组织的蜕膜层和滋养层均有表达,且EGF在滋养层的表达水平随妊娠进展而逐渐下降,提示其在妊娠早期滋养层的发育过程中扮演重要角色。研究发现,EGF对滋养层细胞的增殖、凋亡、分化、能量代谢、激素分泌、侵袭和迁移等多种生物学行为均存在调控,妊娠动物母体EGF的缺乏亦可造成明确的胎盘发育不良、子代宫内生长受限。因此,EGF对滋养细胞功能的调控研究成为本领域近年来的重要方向。EGF通过与细胞表面表皮生长因子受体(epidermal growth factor receptor,EGFR)的高亲和力结合而使EGFR内在的酪氨酸激酶活性激活,启动胞内信号传导级联反应。已发现受EGF/EGFR激活的信号通路包括:Ras/MAPK,PI3K/Akt,PLC/PKC和STAT等。Rho亚家族蛋白(Rho subfamily)属于Ras超家族小分子G蛋白范畴,主要包括RhoA、RhoB、RhoC等3个成员,它们在细胞的信号转导通路中作为分子开关作用于其下游靶效应分子调节细胞骨架的聚合与重建,进而参与调控细胞的粘附、细胞的极性和细胞迁移。Rho亚家族成员具有高度同源性,RhoA、RhoB、RhoC蛋白的氨基序列结构约有85%相同,但在胞内信号传导通路中的分工有所不同。早已发现,EGFR的激活能够通过Rho信号途径调节胞膜皱缩、骨架重组、局部粘附等对迁移、运动至关重要的细胞行为特性,但Rho蛋白各亚型在EGF介导的人滋养细胞迁移中的作用及机制至今未见报道。JEG-3和JAR细胞系分别来自人绒毛膜癌和人胎盘部位滋养细胞肿瘤组织,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,且细胞表面均表达EGFR。本研究基于JEG-3、JAR等两种滋养细胞系和人早孕绒毛体外培养等实验平台,应用Transwell、Western blot、RNAi等实验技术,研究并探讨Rho亚家族蛋白在EGF介导的人滋养细胞迁移中的作用及其机制。主要结果和结论如下:1.EGF以剂量依赖模式作用于人滋养细胞的迁移行为。在测试的浓度中,1ng/ml的EGF最大程度的上调JEG-3、JAR细胞的迁移行为(P < 0.01);浓度升高至10ng/ml时其上调效应有所下降;浓度升高至100ng/ml时EGF的迁移调节作用转为抑制(P < 0.01)。相同的实验条件下,JEG-3、JAR细胞的数目、增殖、凋亡等无显著变化。2.Rho蛋白活性抑制剂-C3胞外酶(5mg/ml)显著抑制JEG-3、JAR细胞的迁移(P < 0.01);C3预处理细胞后,EGF对细胞迁移的激活作用也被抑制(P < 0.01);相同实验条件下,细胞的数目、增殖、凋亡等无显著变化;同时,EGF还显著上调早孕绒毛组织EVT细胞的外生性迁移(P < 0.01),而C3可将此作用阻断(P < 0.01)。3.EGF处理JEG-3、JAR细胞后,RhoA和RhoC的表达显著上调(P < 0.01),而RhoB的表达轻微下调(P < 0.01),EGF处理绒毛组织后Rho表达谱与细胞结果相近;在3种实验平台,EGF处理均使处于活性状态的RhoA和RhoC水平升高(P < 0.01),而RhoB水平无显著变化。4.分别沉默RhoA和RhoC均能显著的抑制JEG-3、JAR细胞的迁移行为(P < 0.05),EGF对细胞迁移的刺激作用也同时被抑制(P < 0.01);相同实验条件,沉默RhoA和RhoC对细胞的数目、增殖、凋亡等行为均无显著影响。5.C3处理(5mg/ml, 2h)的JEG-3、JAR细胞出现细胞皱缩、细胞微丝骨架解聚、不规则等表现,C3处理之后应用EGF刺激不能使细胞形态和骨架复原。沉默RhoA (100nM, 24h)后细胞出现与C3处理相似的细胞皱缩、骨架解聚等表现,不能被EGF复原。而RhoC沉默(100nM, 24h)后细胞的形态、微丝骨架等未发生明显可见的变化。上述结果表明:低浓度的EGF(1ng/ml)能够刺激JEG-3、JAR迁移上调,促进EVT细胞自培养绒毛组织的外生性迁移,而高浓度EGF(100ng/ml)显著抑制JEG-3、JAR迁移;EGF对滋养细胞迁移行为的上调依赖于RhoA和RhoC的表达、激活水平上调,而非RhoB。同时,RhoA通过细胞微丝骨架的重组来参与调节细胞的迁移行为,而RhoC对细胞迁移的调节不依赖于微丝骨架。更多还原

【Abstract】 Background and Objective:Formation of a functional placenta is essential for intrauterine development of the human embryo. Extravillous cytotrophoblasts (EVT) invade the underlying decidua, then surround and migrate into the wall of the uterine spiral arteries, which results in the remodeling of uterine vasculature. This process plays a pivotal role in mammalian placentation and is stringently regulated to ensure a successful pregnancy. Poor invasion of EVT was believed to be associated with insufficient remodeling of the spiral arteries, which was typical of the pathological changes of miscarriage, preeclampsia and intrauterine growth restriction; conversely, excessive invasion leads to placenta percreta, persistent trophoblastic disease or invasive cancer, such as choriocarcinoma. Elucidating the key events of trophoblast invasion is critical to understand these diseases, as well as properly managing them. EVT invasion is a multi-step process involving attachment, degradation through matrix metalloproteinases (MMPs) and subsequent migration through the extracellular matrix. Endocrine factors produced locally or distally play an important role in regulating this process. A collection of endogenous growth factors and cytokines have been implicated in trophoblast migration and/or invasion, however, the mechanisms that link the endocrinal signals to migratory processes are still only partially understood.Epidermal growth factor (EGF) is a multifunctional growth factor that regulates a variety of fundamental cell properties. Maternal EGF deficiency causes fetal hypoglycemia and intrauterine growth retardation in mice. In humans, both the decidua and trophoblast express EGF and its receptor, and the expression of EGF in the trophoblast decreases as the pregnancy progresses, which hints at its important role in early pregnancy. There is evidence that EGF affects the trophoblast cell in many ways, including proliferation, survival, apoptosis, differentiation, secretion, motility, and invasion and/or migration. In recent years, more attention has been paid to the effect and mechanism of EGF on trophoblast cell migration and the majority of these results show that EGF is stimulatory. Binding of EGF to its receptor (EGFR) at the plasma membrane induces dimerization of EGFR, which results in the activation of EGFR tyrosine kinase and trans-autophosphorylation, thus launching a variety of intracellular pathways, including Ras/MAPK, PI(3)K/Akt, PLCg/PKC, and STAT. Rho proteins are part of the extensive Ras superfamily that shuttle between an inactive GDP-bound and an active GTP-bound form and exhibit intrinsic GTPase activities. Rho proteins consist of highly conserved RhoA, RhoB and RhoC isoforms that have been regarded as the master regulators of cytoskeletal reorganization required for cellular migration. Although EGFR activation has been claimed to lead to membrane ruffling and the reorganization of cytoskeleton and focal adhesion through activation of Rho proteins, the details of Rho isoforms involved in the regulation of EGF-induced trophoblast cell migration remain unclear.JEG-3 and JAR cell lines are derived spontaneously from choriocarcinoma and share many properties with the villous trophoblast in terms of their morphology, biochemical markers, and hormone secretion. In addition, EGFR has been reported to be expressed in both JEG-3 and JAR cells. To investigate the roles of Rho protein in epidermal growth factor (EGF)-induced trophoblast cell migration and its mechanism, we examined EGF-mediated stimulation of trophoblast migration, using JEG-3 and JAR and first-trimester human chorionic villus explant cultures on matrigel.Results and conclusions:1. EGF regulates trophoblast cell migration in a dose-dependent manner. Both JEG-3 and JAR cell migration peaked with EGF concentrations of 1ng/ml (P < 0.01); but then decreased with higher concentrations and was down-regulated by the highest concentration (100ng/ml; P < 0.01). Survival, proliferation and apoptosis appear to have no effect on the varied number of cells attributed to cell migration as a result of EGF treatment.2. C3 exoenzyme (5mg/ml), an inhibitor of Rho activity significantly reduced the basal migration rate in both JEG-3 and JAR cells (P < 0.01). Pretreatment with C3 exoenzyme plus EGF also significantly reduced the extent of cell migration (P < 0.01). C3 exoenzyme had no significant effect on survival, proliferation, or apoptosis for JEG-3 or JAR cells (P > 0.05). More over, EGF could significantly stimulate EVT cell outgrowth from explants of human villous tissue (6-9 wk gestation) cultured on matrigel (P < 0.01), and that C3 exoenzyme decreased the outgrowth in the absence or presence of EGF (P < 0.01). 3. Following EGF treatment, in JEG-3 and JAR cells, level of RhoA and RhoC had increased significantly by more than two fold by 12 and 24 h (P < 0.01), whereas the level of RhoB had decreased slightly (P < 0.01). In cultured villous explants, EGF treatment showed a similar pattern to JEG-3 cells. Moreover, EGF significantly increased the amount of the active form of RhoA (P < 0.01) as well as RhoC (P < 0.01) in JEG-3, JAR or cultured villous explants, whereas the level of GTP-bound RhoB was unaffected. These results suggest that RhoA and RhoC may play an important role in trophoblast cell response to EGF stimulation.5. Both RhoA and RhoC siRNA significantly reduced the migration of JEG-3 and JAR cells, and treatment with EGF following siRNA did not restore the migratory capacity of these cells, indicating that both RhoA and C are essential to EGF-stimulated trophoblast cell migration. Neither RhoA nor RhoC siRNA had significant effect on the survival, proliferation, and apoptosis of cells after 24 h in culture.6. Treatment with C3 exoenzyme (5mg/ml, 2h) led to cell shrinkage and disorganization of F-actin, which was not reversed by treatment with EGF after C3 exoenzyme treatment. RhoA siRNA (100nM, 24h) disrupted the F-actin and led to marked cell shrinkage, which was consistent with a decreased migratory ability and looked similar to results after C3 exoenzyme treatment and EGF did not restore the original cell morphology. RhoC siRNA (100nM, 24h) did not destroy the organization of F-actin in JEG-3 and JAR cells.In conclusion, our data confirm that EGF (1ng/ml) not only stimulates JEG-3 and JAR cell migration in serum reduced medium (0.5% FBS), but also stimulates EVT migration from cultured human first trimester villous tissue. We also show that trophoblast migration induced by EGF depends on the increased expression and activation of RhoA and RhoC, but not RhoB. Furthermore, RhoA was found to stimulate the migration by F-actin redistribution, while RhoC was not critical for F-actin redistribution.更多还原