miR-217介导BMP信号通路在运输应激导致鸭小肠黏膜损伤中的作用机制研究

Transportation stress is a common problem in the scaled raising of duck which damages the small intestinal mucosal and reduces the production performance. Transportation stress induces oxidative stress and causes the damage of small intestinal mucosa. It has been demonstrated that miR-217 and BMP signaling pathway play an important regulatory roles in stress damage of small intestinal mucosa. CHRDL1 is an inhibitor of BMP signaling pathway. Our previous results showed that miR-217 was highly expressed in duck intestine under transportation stress condition and could regulate the expression of CHRDL1. Compositing the previous results and relevant literature reports, we hypothesized that highly expressed mir-217 in intestine under transportation stress condition could induce BMP signaling pathway activation to boost antioxidant power by inhibiting the expression of CHRDL1 gene. Therefore, this study will confirm the expression and histological localization of miR-217, CHRDL1 and related components of the BMP signaling pathway by qRT-PCR, western blot and immunohistochemistry, respectively. Furthermore, we will study the target relationship between miR-217 and CHRDL1 gene, and clarify the miR-217-mediated BMP signaling pathway regulate antioxidant power of duck intestine epithelial cells. Parsing this scientific problem will be useful to providing theoretical basis and new ideas for dealing with transport stress.

长途运输导致鸭小肠产生持续氧化应激并造成黏膜损伤，影响鸭消化吸收和生产性能发挥，是规模化蛋鸭养殖面临的问题之一。miR-217和BMP信号通路在组织应激损伤中发挥重要调控作用。申请人前期研究结果表明，BMP信号通路抑制因子CHRDL1在运输应激鸭小肠中低表达，而CHRDL1是miR-217候选靶基因。据此我们推测：“长途运输应激鸭小肠中，miR-217下调CHRDL1基因表达，影响BMP信号通路激活，通过调控小肠上皮细胞抗氧化能力参与应激应答”。故本研究拟在miR-217、CHRDL1和BMP信号通路成员表达定位分析的基础上，确定miR-217对CHRDL1的靶向调控作用，并通过表达干预、BMP通路抑制等试验，解析miR-217介导BMP信号通路在小肠上皮细胞氧化应激中的作用，阐释miR-217在长途运输导致小肠损伤中的作用机制。研究结果将为运输应激的有效应对提供新靶点与新思路。

长途运输导致鸭小肠产生持续氧化应激并造成黏膜损伤，影响鸭消化吸收和生产性能发挥，是规模化蛋鸭养殖面临的问题之一。肠道是应激应答的靶器官，其在急性应激过程中的应答机制尚不甚明确。申请人前期研究结果和文献报道显示，miR-217和BMP信号通路在组织应激损伤中发挥重要调控作用。本研究旨在构建急性应激鸭小肠中miR-217通过下调靶基因调控BMP信号通路影响小肠上皮细胞抗氧化能力，参与应激应答的表达调控网络。具体研究内容包括：（1）分离、培养、鉴定鸭原代小肠上皮细胞，建立细胞氧化应激模型；（2）明确miR-217对氧化应激肠上皮细胞的作用；（3）明确miR-217与CHRDL1的靶向关系；（4）分析miR-217介导BMP信号通路在鸭小肠上皮细胞氧化应激损伤中的作用。经过三年的执行，本项目通过对miR-217定位、分析时空表达特征，研究其调控功能，取得如下结果：（1）成功构建了鸭原代小肠上皮细胞培养方法，建立小肠上皮细胞氧化应激模型，为鸭肠屏障功能研究奠定基础；（2）过表达miR-217能使CHRDL1表达量显著降低，根据miR-217与CHRDL1的结合位点，利用双荧光素酶检测法、点突变等手段，明确miR-217与CHRDL1的靶向关系；（3）在鸭小肠上皮细胞氧化应激模型中，过表达miR-217能够提高小肠上皮单层细胞屏障功能，而CHRDL1过表达载体与重组骨形态发生蛋白共转染细胞，能够抑制BMP诱导的肠屏障功能提升，说明在鸭小肠上皮细胞中，miR-217能够通过调控CHRDL1介导BMP信号通路的作用。综上所述，miR-217对雏鸭小肠上皮细胞屏障功能具有一定的维持作用，其调控机制主要通过抑制靶基因CHRDL1基因转录和蛋白表达，激活BMP信号通路实现。研究结果将为运输应激的有效应对提供新靶点与新思路。