重组表达IL-22的双歧杆菌促进IBD肠干细胞自我更新机制的研究

The imbalance of intestinal flora leads to the disorder of differentiation and self-renewal of intestinal stem cells (ISC) in inflammatory bowel disease (IBD). Probiotics and anti-inflammatory factor IL-22 can promote intestinal mucosal healing and protect intestinal tract in IBD. For the first time, the applicant has constructed the bifidobacteria expressing anti-inflammatory factor at home and abroad, and the effect of the treatment of IBD is significant effective. Meanwhile, this project had been obtained the excellent doctoral thesis of the province. The early cell experiments showed that the recombinant bifidobacterium expressing IL-22 (BL-IL-22) could activated STAT3, and which could promote the proliferation of intestinal epithelial cells (IEC) and ISC, the effect was better than that of IL-22 alone. We also found that BL-IL-22 could change the direction of differentiation of ISC by inhibiting autophagy. Therefore, we speculate that Bifidobacterium as carrier and IL-22 might active STAT3 together to promote the self-renewal and differentiation of ISC by acrosstalk of regulating autophagy of IEC, which have an obvious synergistic effect. This project aiming to isolate and culture ISC and mouse models in vitro by gene intervention verify the mechanism that BL-IL-22 can promote the proliferation and differentiation of ISC and change ISC differentiation direction by regulating autophagy. In order to provide theoretical basis for clinical application, the core signal pathway of this biological system will be analyzed, and its network regulation of the new molecular mechanism be clarified.

在炎症性肠病（IBD）中肠道菌群失调伴随肠道干细胞（ISC）的分化及自我更新的紊乱，益生菌和抗炎因子IL-22等可促进肠道黏膜愈合，保护IBD肠道。申请人率先构建表达抗炎因子的双歧杆菌，其治疗IBD效果显著（获省优秀博士论文）。前期细胞实验发现重组表达IL-22双歧杆菌（BL-IL-22）生物系统，激活STAT3，促进肠上皮细胞（IEC）及ISC的增殖，且效果优于单独使用IL-22。还发现BL-IL-22可抑制IEC自噬，并改变其分化方向。据此推测双歧杆菌作为载体与IL-22共同激活STAT3，协同调节IEC自噬，促进ISC的自我更新。本项目拟通过基因干预等方法，在体外肠道系统分离培养ISC及IBD小鼠模型中，验证BL-IL-22促进ISC增殖和分化及调节自噬对ISC分化方向改变的具体机制。深入分析本生物系统协同作用的核心信号通路，阐明其网络调控的新分子机制，为临床应用提供理论依据。

在炎症性肠病（IBD）中肠道菌群失调伴随肠道干细胞（ISC）的分化及自我更新的紊乱，与肠道内稳态失调密切相关，益生菌和抗炎因子IL-22等可促进肠道黏膜愈合，保护IBD肠道。长非编码RNA（lncRNAs）的异常表达已在多种炎症性疾病以及UC中发现。小鼠腹腔注射IL-22或PBS，然后用DSS诱导结肠炎。采用组织学和分子检测方法测定小鼠的组织病理学参数。然后，进行RNA测序以筛选对照组、IL-22治疗组前后小鼠中lncRNAs的差异表达谱。采用实时定量PCR（qRT PCR）和慢病毒技术，确定LncRNA溃疡性结肠炎LncRNA（LncRNA UCL）为结肠炎小鼠的调节分子。使用RNA下拉分析和验证LncRNA UCL和mmu-miR-568之间的相互作用。同时，通过双荧光素酶分析预测并确认claudin-1为mmu-miR-568的靶分子。IL-22通过增强肠上皮细胞紧密连接和粘附连接蛋白的表达，促进肠上皮细胞增殖，抑制其凋亡，从而显著改善DSS诱导的结肠炎小鼠的组织病理学特征，减少促炎细胞因子的产生。在DSS诱导的结肠炎小鼠中，LncRNA UCL表达显著下调，而在IL-22的作用下，LncRNA UCL表达显著恢复。在机制方面，lncRNA UCL通过隔离mmu-miR-568和维持紧密连接（TJ）蛋白claudin1的表达来调节肠上皮内环境平衡。我们的研究结果表明，IL-22通过维持紧密连接和粘附连接蛋白的表达，上调lncRNA UCL的表达，从而减轻DSS诱导的结肠炎症状。在IL-22的作用下，lncRNA/miR-568/claudin1轴在调节肠上皮细胞内稳态中起着关键作用。重组表达IL-22双歧杆菌（BL-IL-22）生物系统，激活STAT3，促进肠上皮细胞（IEC）及ISC的增殖，且效果优于单独使用IL-22。还发现BL-IL-22可抑制IEC自噬，并改变其分化方向。双歧杆菌作为载体与IL-22共同激活STAT3，协同调节IEC自噬，促进ISC的自我更新。

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