褪黑素调控GATA6在子痫前期胎盘滋养层细胞中的作用及机制研究

Preeclampsia (PE) is a serious pregnancy complication. The mechanism is unclear and there is no effective treatment for PE. Previous studies have found that melatonin (MT) and GATA6 are negatively correlated in placenta of PE patients. GATA6 inhibits the invasion of primary extravillous trophoblasts (EVT), and high expression of GATA6 induces eclampsia-like hypertension and proteinuria in pregnant mice. The expression of GATA6 can be regulated by MT. MT has been reported as an effective drug for the treatment of pre-eclampsia. However, it is unknown that which genes and what mechanisms is involved in PE. We propose that insufficient of MT synthesis/secretion in placental trophoblast cells leads to high expression of Gata6 involved in the pathogenesis of PE. We will: 1: Which receptor and signaling pathway are involved in the mechanism of GATA6 regulated by MT. How does GATA6 regulate the MT membrane receptor-encoding gene to form a circular regulatory pattern? 2: To evaluate the mechanism of MT regulating GATA6 affects trophoblast invasion, migration and integration, primary EVT, explants, co-culture of EVT and decidual cells, hypoxia/reoxygenation and other models was used.3: Can MT improve the eclampsia-like symptoms of over-GATA6-expression pregnant mice? Elucidate the molecular mechanism of MT and GATA6 involved in PE. Provide theoretical basis for disease prediction, diagnosis and clinical application of MT.

子痫前期（PE）是严重的妊娠并发症，机制不明尚无有效药物。前期发现：褪黑素（MT）和GATA6在PE患者胎盘中表达负相关，GATA6可抑制原代绒毛外滋养细胞（EVT）侵袭，GATA6高表达诱发孕鼠子痫样高血压和蛋白尿，并证实GATA6表达受MT调控。MT可作为治疗PE的有效药物已被报道，但其通过那些基因或何种机制调控PE的发生尚不清楚。因此，我们提出胎盘滋养层细胞MT合成/分泌不足引发Gata6高表达参与PE发病机制。本项目将1：研究MT通过那种受体和信号通路调控GATA6，GATA6又如何调控MT膜受体编码基因从而构成环状调节模式。2:通过在原代EVT、外植体、EVT和蜕膜细胞共培养、缺氧/复氧等模型中探索MT调控GATA6影响滋养层细胞侵袭、迁移和合体化。3：MT能否改善GATA6高表达孕鼠的子痫样症状。阐明MT、GATA6参与PE的分子机制，为疾病的预测、诊断及MT的临床应用提供依据

子痫前期（PE）是严重的妊娠并发症，机制不明尚无有效药物。褪黑素（MT）可作为治疗PE的有效药物已被报道，但其通过那些基因或何种机制调控PE的发生尚不清楚。我们通过尾静脉注射过表达的AAV-sFlt1和L-NAME两种方法构建了子痫前期模型大鼠，并用褪黑素对子痫前期模型鼠进行治疗并对胎盘进行了转录组测序。我们发现MT可以改善的子痫前期孕鼠的症状，并找到了一系列对子痫前期的发病机制有作用并可被褪黑素调控的基因，包括GATA2、GATA6和CD34。随后，对这3个基因进行细致的研究发现：GATA6主要在滋养层细胞的侵袭和糖代谢中发挥作用，GATA2主要在子宫内膜细胞中发挥作用。CD34主要参与内皮细胞炎症的氧化应激。.同时证实：PE孕妇血中MT和雌激素水平明显低于正常孕妇，孕激素和hCG在两者中未见明显差异。PE胎盘中的GLUT1、糖酵解关键酶PGK1的表达降低。GATA6和 GATA2在PE胎盘中低表达。干扰GATA2/6后，细胞的迁移、侵袭、增殖能力下降。干扰GATA6后GLUT1、PGK1、LDHA的蛋白和mRNA表达降低，乳酸生成减少；过表达GATA6后得到相反的结果。干扰GLUT1后，细胞降低的迁移、侵袭能力可通过表达GATA6后部分恢复。我们在孕鼠中转染了GATA6低表达的慢病毒（Ad-GATA6），发现与对照组（Ad-CTL）比孕鼠血清中GATA6表达降低，Ad-GATA6孕鼠D18胎盘中GATA6、PLGF、GLUT1、PGK1低表达，肾脏并出现轻微的病理改变和糖原累积。并且我们发现MT可以缓解Ad-GATA6大鼠的子痫样症状。对于在子痫前期孕鼠子宫内膜上另一个低表达并受MT调控的另一个GATA2也做了细胞水平的研究发现，MT可以通过MT1受体调控GATA2的表达，同时GATA2作为转录因子可以调控编码MT1蛋白的基因MTNR1A的上游启动子区域。对参与子痫前期内皮细胞氧化应激的CD34基因我没发现CD34在内皮中的低表达可以影响内皮细胞的单层细胞通透性，可以引起内皮损伤。损伤的内皮细胞引起了焦亡的发生。.因此，本研究阐明了MT作为子痫前期的潜在治疗药物可以通过在滋养层细胞，子宫内膜细胞和内皮细胞中调控GATA6、GATA2和CD34参与PE的分子机制，为疾病的预测、诊断及MT的临床应用提供依据。