靶向阻断EPAS1基因调控TGF-β/Smad4信号通路在妊娠期SAP肠屏障损伤中的作用机制研究

Severe Acute Pancreatitis (SAP) in pregnancy is a disease of rapid progression, high mortality rate, and complicated pathogenesis. The incidence of SAP in China were increasing significantly with the adjustment of China's family planning policy as well as the amount of women of advanced maternal age. EPAS1 is a newly found cytokin with ODD domain as well as a most important component in the inflammatory/hypoxic microenvironment of the human body. Our previous study indicated that EPAS1 plays a pivotal role in the SAP-induced intestinal barrier dysfunction, while the mechanism remains to be illuminated. On this basis, the gene-nano composites was prepared by the combination of bio-nanotechnology and gene targeting technology. qRT-PCR, WB, FCM and other classical methods were applied to explore the role of EPAS1 in regulating Crosstalk between inflammatory/hypoxic microenvironment and intestinal mucosal barrier via TGF -β/Smad4 signal pathway. The results revealed that EPAS1 regulate proliferation and apoptosis of intestinal mucosa epithelial cells through down regulating the expression of Occludin, miR-130a and other downstream target genes of TGF-β/Smads pathway, so as to provide a new potential strategy for the treatment of SAP intestinal mucosal barrier dysfunction during pregnancy.

随着我国计划生育政策的调整及高龄孕妇的增加，妊娠期重症急性胰腺炎（SAP）的发病率也明显上升。EPAS1是新近发现的一个含有ODD结构域的细胞因子，是机体炎症/缺氧微环境中最重要的一员，我们的前期研究也发现：EPAS1在SAP肠粘膜屏障损伤中起重要作用，但其作用机制还有待进一步研究。基于此，本项目利用生物纳米技术和基因靶向技术相融合，构建靶向EPAS1基因的白蛋白纳米微球实现高效、缓释，采用qRT-PCR、ELISA、FCM等经典方法，从分子、细胞、动物模型多水平探讨EPAS1基因对妊娠期SAP肠粘膜上皮细胞增殖、凋亡的调控，揭示EPAS1通过抑制TGF-β表达，从而下调TGF-β/Smad4信号通路下游靶基因miR-130a、Occludin等表达的分子机制，明确靶向EPAS1基因的纳米微球对肠屏障损伤的治疗效果，本研究将为妊娠期SAP肠屏障损伤中靶基因选择和基因载体的优化提供新的策略。

妊娠期SAP是一种始于局部而累及全身的危急重症，其病程进展迅速，并发症多，常伴多器官功能衰竭。新近国外研究表明，肠道屏障包括机械屏障、化学屏障、免疫屏障和生物屏障，肠粘膜上皮细胞及紧密连接所组成的肠道机械屏障对维持正常肠粘膜功能尤为重要，是整个肠屏障功能完整的物质基础和支撑平台。在SAP时肠粘膜屏障功能受损，肠粘膜的完整性破坏，使肠粘膜通透性增加，导致肠道细菌及内毒素发生移位，加速脓毒症（sepsis）的进程，从而诱发全身炎症反应综合征（SIRS）和多器官功能衰竭，严重者导致病人死亡，因此在治疗原发病的同时对肠粘膜屏障的保护至关重要。.本科研团队在研究中发现，利用ELISA法对SAP大鼠血浆进行检测，发现EPAS1与致炎因子IL-6、TNF-α的表达增高，而抗炎因子 IL-4、IL-10的表达显著降低，提示EPAS1可能参与了 SAP 的炎症反应调控；然后我们利用Western b1ot对SAP大鼠胰腺和小肠组织检测发现EPAS1表达也显著增高，而TGF-β的表达则显著降低，二者呈显著负相关，而且EPAS1的表达程度与SAP大鼠的生存预后密切相关，通过本研究我们得出结论：胰腺和小肠在炎症/缺氧微环境下激活EPAS1高表达，从而通过TGF-β/Smad4信号通路上调下游含有TGF-β结合位点的相关炎症基因如TNF-α、IL-6等炎性细胞因子转录，同时下调相关靶基因Occludin、ZO-1等的表达，导致肠粘膜上皮细胞功能障碍、肠道通透性增加、肠道细菌及内毒素易位的分子机制。.本科学研究的结果和研究成果，将为妊娠期SAP致肠粘膜屏障损伤治疗中靶基因选择和基因载体的优化提供新的策略。

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