胆盐依赖脂肪酶通过Wnt信号通路促进肠黏膜上皮细胞损伤修复的机制研究

Early enternal nutrition (EN) is beneficial for patients with intestinal failure to improve nutrition balance and to promote intestinal function recovery. However, patients with intestinal failure who receiving EN often have fat malabsorption, and their intestinal recovery and adaption take long time. A bile salt-dependent lipase (BSDL) exists in breast-milk. Our laboratory found that BSDL was not only able to help digest the fat, but also promote intestinal proliferation and barrier function, which hinted the prospect to be a supplement in EN formula.The underlying mechanism was proposed that BSDL could activate Wnt signaling pathway in intestinal epithelial cells by interacting with LRP6, but needs to be further studied. This project aims to validate and dynamically observe the binding between BSDL and LRP6 in CaCo-2 cells using biotin transfer and FRET methods. The interactive domains between LRP6 and BSDL are studied using gene mutagenesis method. This project aims to clarify the mechanism how BSDL activates Wnt signaling pathway and the downstream signal transduction cascade, as well as how BSDL signal transduction crosstalks with MAPK signaling pathway by Western snd immunoprecipitation. Animal model with short bowel is used to show whether oral administration of BSDL could promote mucosa regeneration and barrier repairing, and whether it could activate Wnt signaling pathway in vivo. It is of great significance to understand the mechanism how BSDL promotes intestinal repairing after failure, and to optimize the formulation of EN formula specific for intestinal failure.

早期肠内营养（EN）对促进肠衰竭患者能量代谢平衡和肠功能康复有积极作用，然而肠衰竭患者EN易发生脂肪吸收不良和脂肪泻，肠功能康复和肠代偿发生所需时间较长。预实验发现母乳中胆盐依赖脂肪酶（BSDL）不仅能够帮助脂肪消化，还可促进肠黏膜细胞增殖和增强肠屏障功能，提示可用于肠衰竭EN营养液添加物；同时发现BSDL可能与LRP6结合激活Wnt信号通路，但具体机制有待进一步阐明。本课题将通过生物素转移实验和FRET技术验证并动态反映LRP6与BSDL结合，以及基因突变研究两者相互作用结构域；采用Western和免疫共沉淀技术阐明BSDL激活Wnt信号通路逐级信号传导，以及与MAPK信号通路相互串话的分子机制；动物实验用以证明口服BSDL能够促进短肠大鼠肠黏膜再生和屏障修复，验证BSDL能够在体内激活Wnt信号通路。了解BSDL促肠黏膜损伤修复的分子机制为该蛋白在肠衰竭EN配方中的开发利用奠定基础。

早期肠内营养（EN）对于促进肠衰竭患者能量代谢平衡和肠功能康复有积极作用。然而肠衰竭患者 EN 易发生脂肪吸收不良和脂肪泻，肠功能康复和肠代偿发生所需时间较长。母乳中存在一种胆盐依赖脂肪酶（BSDL），经口服能帮助婴儿脂肪消化吸收。本课题通过BL21工程菌表达重组BSDL蛋白,蛋白分子量为67kD,蛋白电泳分析显示重组BSDL蛋白主要存在于包涵体中，通过镍离子亲和层析纯化重组蛋白，然后通过透析复性法对包涵体中的BSDL进行复性，复性后的BSDL蛋白活性达到5.358 U/ml。本课题发现 BSDL 可能与 LRP6 受体结合激活 CaCo-2 细胞 Wnt 信号通路，促进细胞增殖和增强屏障功能。采用 CCK-8 法和xCELLigence实时监测法测定BSDL处理后的细胞活力，结果显示细胞活力随着 BSDL 浓度的增加而增加；BSDL处理CaCo-2细胞后或促进Claudin-1，occludin 和 ZO-1 等紧密连接蛋白的表达；BSDL处理Caco-2细胞4小时后能够诱导β-catenin蛋白的入核，同时激活TCF/LEF激活的荧光素酶的表达。本课题通过生物素转移实验和免疫共沉淀技术验证了LRP6 与 BSDL 相互结合，BSDL通过LRP6介导被CaCo-2细胞胞吞，并激活 Wnt 信号通路分子逐级磷酸化进而促进β-catenin蛋白入核。通过动物实验进一步验证了细胞实验的结果，研究证明口服含有200ug/ml BSDL 的肠内营养液能够促进短肠综合症大鼠体重增长、肠黏膜再生和屏障修复，促进肠粘膜细胞分裂增殖抑制细胞凋亡，验证 BSDL 能够在体内激活 Wnt信号通路各级分子磷酸化，促进小肠隐窝内Lgr5+干细胞扩增。本课题对于了解 BSDL 促进肠黏膜损伤修复的分子机制，优化肠衰竭EN 配方有着重要意义。