siRNA靶向诱导maspin启动子甲基化对子痫前期胎盘功能缺陷的调控机制研究

There is closely related between preeclampsia and placental dysfunction which is characterized by inadequate trophoblastic invasion and microvascular endothelial cell damage. Trophoblastic cell has invasive capacity and molecular mechanism similar to tumor cell. Research demonstrates that the change of maspin expression during pregnancy is consistent with self-limited invasiveness of trophoblastic cell, and maspin could inhibit tumor angiogenesis. Our previous study suggests that mRNA expression of maspin in placenta of preeclampsia and hypoxic trophoblastic cell is increased. In hypoxic environment, the invasiveness of trophoblastic cell is weak, the permeability of endothelial cell was increased, while CpG site in maspin promoter region is hypermethylated. So this subject focuses on maspin gene. The methylation status of CpG site in maspin promoter region of preeclampsia by methylation specific PCR will be detected to confirm methylated site for future interference. After screening siRNA which can effectively induce methylation, we will analyse the expression of maspin and invasion-associated gene, the capacity of proliferation and invasion in trophoblastic cell before and after transfection, furthermore, we will analyze apoptosis and injury of endothelial cell, so we will clarify the function of target siRNA, which induces methylation of CpG site in maspin promoter region and transcriptional gene silencing in placental dysfunction. Above all, it may provide new perspective for revealing the pathogenesis of preeclampsia by epigenetic method.

子痫前期与滋养细胞浸润不足和微血管内皮细胞损伤导致的胎盘功能缺陷密切相关。滋养细胞具有与肿瘤相似的浸润能力及分子基础，研究发现妊娠时maspin表达的动态变化与滋养细胞浸润自限性一致，并可抑制肿瘤血管生成。我们前期研究发现子痫前期胎盘及缺氧滋养细胞maspin mRNA表达增多，缺氧时滋养细胞浸润不足，内皮细胞通透性增加，maspin启动子非甲基化率增高。因此本课题将以maspin为切入点，利用甲基化PCR检测子痫前期maspin启动子CpG岛甲基化状态，并确定甲基化预干扰位点；针对不同甲基化位点筛选有效诱导启动子甲基化的siRNA，分析转染前后滋养细胞maspin表达、增殖、浸润能力和浸润相关基因表达谱的差异，并分析转染前后微血管内皮细胞凋亡及损伤情况，明确靶向siRNA诱导maspin启动子甲基化及转录沉默对胎盘功能缺陷的调控作用，为运用表观遗传学方法研究子痫前期发病机制提供全新视野。

子痫前期与滋养细胞浸润不足和微血管内皮细胞损伤导致的胎盘功能缺陷密切相关。滋养细胞具有与肿瘤相似的浸润能力及分子基础，研究发现妊娠时maspin表达的动态变化与滋养细胞浸润自限性一致，并可抑制肿瘤血管生成。因此本项目以maspin基因为切入点，从分子水平阐明了maspin基因在子痫前期中对滋养细胞浸润功能及血管内皮细胞血管形成能力的抑制作用，并通过BSP和MSP结果解析了子痫前期胎盘及缺氧培养的绒毛外滋养细胞株TEV－1中maspin基因启动子的低甲基化状态，并在此基础上设计和合成了针对maspin启动子区域的siRNA，建立靶向特异性转染体系，筛选出有效的诱导maspin启动子甲基化的siRNA，在细胞水平明确靶向siRNA诱导maspin启动子甲基化，降低maspin基因表达，促进了滋养细胞的侵袭和迁移能力，解释了maspin基因及其启动子甲基化对胎盘功能缺陷的调控作用。另外，在整体动物模型中，我们发现脱甲基化药物可以明显改善子痫前期孕鼠尿蛋白情况，这为进一步运用表观遗传学方法研究子痫前期发病机制提供了全新的视野，为改善子痫前期母儿预后提供了新的治疗思路。