调控Wnt信号通路在间充质干细胞用于禽流感治疗中的探讨

Avian influenza (AI) is a fulminating infectious disease which can threat animal husbandry and human heath seriously. Avian influenza mainly induce uncontrolled immune responses，course acute lung injury (ALI) and pulmonary fibrosis. Clinical treatments of AI including antivirus , expectant and supportive therapies , but the result of normal therapy is not good, mortality still highly. Our preliminary study showed that mesenchymal stem cells ( MSCs ) could inhibit the excessive immune response of AI in animal model, showen effect on AI therapy, but could not inhibit pulmonary fibrosis tendency in animals.Our study found, inhibit Wnt signal could prevent MSCs differentiate into fibroblast. In this project, MSCs and Wnt signal are the breakthrough points，we would study : ①Choose essential Wnt signal proteins during MSCs differentiate into fibroblast, construct their siRNA to study the therapeutic targets of pulmonary fibrosis. ② Prove that selectively block the Wnt signal during MSCs treatment in AI, could not only serves MSCs immune regulation function, but also inhibit MSCs differentiate into fibroblast, promote MSCs differentiate into into epithelial cells. Means avoid pulmonary fibrosis as well as alleviate the lung injury. This study would provide new ideas for clinical treatment of avian influenza.

禽流感（AI)是一种严重危害畜牧业和人类健康的烈性传染性疾病，以机体过度免疫应答导致急性肺损伤，肺纤维化为特征。临床治疗以抗病毒及对症支持治疗为主，疗效不佳，病死率极高。我们的前期研究发现骨髓间充质干细胞（MSCs）能抑制禽流感模型动物的过度免疫应答，对其有良好的治疗效果，但不能抑制肺纤维化倾向。研究发现抑制Wnt信号通路能够抑制MSCs向成纤维细胞分化。本项目将以MSCs及Wnt 信号通路为切入点,开展以下研究：①筛选在MSCs向成纤维细胞分化过程中起关键作用的Wnt蛋白,构建siRNA,寻找抑制肺纤维化的关键靶点。②在动物模型中证明,应用MSCs治疗AI的过程中,选择性阻断特异Wnt 蛋白能够在发挥MSCs免疫调节功能同时,抑制其向成纤维细胞分化，促进其向上皮细胞分化，从而在减轻肺损伤的同时，促进肺泡上皮结构重建，有效避免肺纤维化发生发展，为临床治疗禽流感等相关疾病提供新思路。

禽流感病毒(avian influenza virus，AIV)感染机体后，迅速在呼吸道复制病毒，同时诱导机体细胞炎症因子过度表达，导致过度免疫应答，进而损伤肺泡表面上皮细胞及毛细血管内皮细胞，出现急性肺损伤和肺纤维化。本研究以Wnt/β-catenin信号通路为切入点，在应用 MSCs治疗禽流感的过程中，通过下调Wnt/β-catenin信号通路蛋白的表达，实现抑制机体过度免疫反应的同时，减少肺纤维化发生，更有效的促进肺泡上皮结构重建。.结果发现：(1)证实在MSCs向成纤维细胞分化过程中抑制Wnt/β-catenin信号通路相关蛋白表达，可抑制MSCs过度增殖活力，减少纤维细胞转化。(2)证实骨髓间充质干细胞(MSCs)对H9N2 禽流感病毒(AIV)诱导的小鼠急性肺损伤有治疗作用。可显著降低促炎细胞因子水平以及肺部炎症细胞募集，有效减轻肺部炎症，改善肺功能，提高AIV感染小鼠的存活率。(3)筛选Wnt/β-catenin信号通路关键蛋白axin1为靶分子，设计特异性RNAi，转染MSCs，构建Axin1-RNAi-MSCs。Axin1-RNAi-MSCs的细胞过度增殖显著抑制，细胞形态维持正常，成纤维细胞表面分子标记Vimentin、α-SMA以及TE-7蛋白的表达均有显著降低。证实应用Axin1-RNAi-MSCs早期治疗AI动物，可显著降低感染动物的纤维化相关因子VEGF、bFGF、α-SMA、Vimentin和Collagen的表达。