沉默lncRNA-HOTAIR 对重症急性胰腺炎小鼠肠黏膜屏障保护作用的实验研究

Pancreatic secondary infection is the most serious complication of severe acute pancreatitis (SAP), intestinal tract is the main origin to cause secondary infection. Our previous study showed that the uncontrolled release of inflammatory cytokines is the main reason to induce SAP-associated intestinal mucosal barrier damage in vivo, recently study finds lncRNAs take part in inflammation response led by monocytes and macrophage. Our previous work has demonstrated the expression of lncRNAs is different markedly in SAP mouse model and the expression of lncRNA-HOTAIR is significantly increased in the intestinal mucosal of SAP mouse model .the present project intends to transfect lncRNA- HOTAIR siRNA to intestinal epithelial cells in vitro, identify the protein which can interact with lncRNA-HOTAIR by RNA-pulldown experiments and mass spectrometry , explore the function and mechanism how lncRNA-HOTAIR influences SAP-associated inflammation and intestinal barrier damage. On this basis, we inject lncRNA-HOTAIR SiRNA mediated by in vivo-jetPEITM to vein of SAP mouse model, investigate the mechanism lncRNA-HOTAIR affects inflammation and protects intestinal mucosal barrier from damage in SAP mouse model , thus provide the evidence for the feasibility that clinical gene intervenes the SAP-associated intestinal mucosal barrier damage .

胰腺继发感染是重症急性胰腺炎（SAP）最严重的并发症,肠道是引发胰腺继发感染的主要来源。本课题组前期研究表明,体内炎症因子失控性释放是SAP肠黏膜屏障损伤的主要原因，最新研究发现lncRNAs与单核巨噬细胞炎症反应密切相关。我们的前期工作已证实lncRNAs在小鼠SAP模型中差异表达，且lncRNA-HOTAIR在SAP小鼠肠上皮中的表达明显升高。本项目拟体外转染lncRNA-HOTAIR siRNA至肠上皮细胞，并通过RNA-pulldown实验联合质谱分析，鉴定与lncRNA-HOTAIR相互作用的蛋白，探讨其对SAP炎症反应及肠屏障作用及机制，并在此基础上，将in vivo-jetPEITM介导的lncRNA-HOTAIR SiRNA静脉注入SAP小鼠模型，探讨其对SAP小鼠炎症反应的影响及肠黏膜屏障保护作用及其机制，为临床上基因干预SAP肠黏膜屏障损伤的可行性提供依据。

重症急性胰腺炎是临床上常见的急腹症，起病急，进展快，病情重，死亡率高达20-40%，其典型特征为持续性器官衰竭。肠道是重症急性胰腺炎最常见的受累器官之一。然而，重症急性胰腺炎发生肠黏膜屏障功能障碍的具体机制目前仍未完全清楚。长链非编码RNA（lncRNA）具有重要的生物学功能，广泛参与机体几乎所有生理和病理过程。大量的研究证实lncRNA参与了免疫反应的调节。我们前期测序结果提示在重症急性胰腺炎小鼠肠上皮中lncRNA-HOTAIR具有显著差异表达，此外，lncRNA-HOTAIR的高表达与胰腺癌、肠癌密切相关。因此，lncRNA-HOTAIR与重症急性胰腺炎肠道炎症反应及肠道功能密切相关。为进一步探索重症急性胰腺炎肠道炎症及肠黏膜屏障损伤机制研究，明确lncRNA-HOTAIR在其中的作用以及参与机制。本研究通过干扰 lncRNA-HOTAIR的表达，观察了重症急性胰腺炎肠上皮细胞炎症反应变化以及对重症急性胰腺炎小鼠肠黏膜屏障损伤的影响，并进一步通过miRNA 测序检测出与lncRNA-HOTAIR相互作用的miRNA。结果表明，在体外实验中，lncRNA-HOTAIR过表达后可促进肠道炎症，加重肠黏膜屏障破坏，降低lncRNA-HOTAIR表达后可下调肠上皮细胞炎症进而减少细胞损伤。在体内实验中，降低lncRNA-HOTAIR后可缓解重症急性胰腺炎小鼠肠道炎症与肠黏膜屏障的损伤。本研究结果证明lncRNA-HOTAIR对重症急性胰腺炎肠黏膜屏障破坏的促进作用。 通过miRNA测序，检测出干扰肠上皮细胞内lncRNA-HOTAIR后有差异的miRNA，并与目前数据库进行比对，筛选出与lncRNA-HOTAIR可能的下游靶标miRNA-20a。综上所述，本研究发现了SAP小鼠肠道内lncRNA-HOTAIR的高表达，同时通过体内外实验证明lncRNA-HOTAIR对SAP小鼠中肠黏膜屏障破坏的促进作用，阐明lncRNA-HOTAIR通过靶向miRNA-20a调控肠屏障与炎症，为寻找SAP肠黏膜屏障破坏的有效治疗靶点提供理论依据。