snail与E-box结合调控maspin启动子区甲基化对子痫前期绒毛外滋养细胞浸润能力的影响及机制研究

Placental dysfunction often leads to human pregnancy complications including preeclampsia which is characterized by inadequate trophoblastic invasion. Maspin inhibits cell growth, migration and invasion and promotes cell adhesion and apoptosis. There are 8 E-boxes (which the zinc finger motifs bind to) in the promoter region of maspin. Several cancer studies found that snail inhibited the invasion and the migration of tumor cells and down-regulated the expression of maspin. Our previous study found that maspin expression was significantly increased in placenta from preeclamptic pregnant women, the methylation level of the maspin promoter region was lower and the expression of snail was decreased. Thus, this study takes the regulation of maspin promoter methylation by snail binding E-box as the pointcut to explore the mechanism of snail regulating the aggressive ability of extravillous trophoblast through maspin in preeclampsia for providing the novel targeted cure of preeclampsia.

绒毛外滋养细胞浸润能力下降导致胎盘浅着床引起胎盘功能缺陷性疾病的发生，如子痫前期等。Maspin抑制肿瘤细胞的生长、迁移与侵袭和促进粘附与凋亡。而maspin基因启动子区含有8个E-box（snail锌指结构的结合位点）。多项肿瘤研究发现snail与肿瘤细胞的侵袭和转移相关，且负向调控maspin的表达。本课题组前期研究发现与正常孕妇胎盘组织相比，子痫前期患者胎盘组织中maspin表达明显增加，maspin启动子区呈低甲基化和snail表达下降。本研究以snail与maspin基因启动子区的E-box结合对maspin启动子区甲基化的调控为切入点探索snail通过maspin调控绒毛外滋养细胞浸润能力在子痫前期中的分子机制，为子痫前期提供新的靶向治疗方法。

绒毛外滋养细胞浸润能力下降导致胎盘浅着床引起胎盘功能缺陷性疾病的发生，如子痫前.期等。Maspin抑制肿瘤细胞的生长、迁移与侵袭和促进粘附与凋亡。而maspin基因启动子区含有8个E-box（snail锌指结构的结合位点）。本研究从以下四个部分进行了相关研究：①组织水平：通过BSP法发现子痫前期胎盘中maspin基因启动子区的甲基化程度明显下降；与血压正常组比较，子痫前期组孕妇胎盘中Snail基因mRNA和蛋白的表达量明显降低；②构建snail-siRNA质粒和人滋养细胞实验：构建的质粒转染效率达80%以上，siRNA转染后，snail基因的mRNA和蛋白表达明显下降，其中siRNA2的沉默效率最高；沉默snail基因和缺氧均可以显著提高人滋养细胞株maspin基因的mRNA和蛋白表达水平，降低MMP2的mRNA和蛋白表达水平；使细胞的增殖能力减弱，凋亡增加，迁移和侵袭能力减弱。③动物实验：与正常对照组比较，L-NAME腹腔注射可以使大鼠血压明显升高，24h尿蛋白明显增加，脱甲基化药物5-Aza干预可以改善大鼠的血压升高和24h尿蛋白；与正常对照组比较，L-NAME腹腔注射对大鼠胎盘组织的maspin表达水平无明显影响；但脱甲基化药物5-Aza干预可以使大鼠胎盘组织maspin表达（mRNA和蛋白水平）明显升高；④脐静脉内皮细胞实验：沉默snail基因可以显著提高人脐静脉内皮细胞株maspin基因的mRNA和蛋白表达水平，降低MMP2的mRNA和蛋白表达水平；可以使细胞的增殖能力减弱，凋亡增加，迁移和血管形成能力减弱。本研究从组织、细胞和动物实验三个水平发现snail可通过与maspin基因启动子区的E-box结合影响maspin基因启动子区的甲基化水平从而调控绒毛外滋养细胞和血管内皮细胞浸润行为在子痫前期中的分子机制，为子痫前期提供新的靶向治疗方法。