HD-5,6调控肠道微生态-TLRs-免疫细胞轴促进电离辐射后肠粘膜内稳态重建的作用及机制

Many patients suffered from radiation enteritis after radiotherapy or exposure in nuclear accidents. The prevention and treatment of radiation enteritis has become a difficult problem, of which the pathogenisis is yet to be elucidated. Lately studies suggested that enteric α-defensins have anti-inflamational function through selectively inhibiting bacteria in intestinal microbiota, regulating TLRs-MyD88 pathway bactericide and immune cell activation. Based on the phenomenon of the tightly association between dysfunction of intestinal bacterial ecology, aberrant inflammation and chaos expression of intestinal α-defensins in patients received radiotherapy, we hypothesized that enteric defensins can facilitate the reestablishment of intestinal mucosal homeostasis post ionization radiation through regulating the axis of intestinal microbiota- TLRs- immune cells. So, this project will select human enteric α-defensin HD5,6 transgenic MMP7-/- mice as abdomen-pelvic radiotherapy model and cell lines of IEC-6 and THP-1 to explore the function and mechanism of HD5,6 in facilitating the reestablishment of intestinal mucosal homeostasis after ionization exposure. Further, expression level of HD5,6, the structure and function of intestinal bacteria, and activation & distribution of immune cells, and inflammation reaction in intestinal mucosa will be investigated by applying the analyzing methods of bacterial metagenome and Macro-metabolism group, qRT-PCR, Western blot and ChIP method and so on. Also, the effect of exogenous intake HD5,6 to prevent and treatment radiation enteritis will be confirmed. Hopefully, the occurrence mechanism of radiation enteritis would be disclosed, which may beneficial to explore better clinical strategies to prevent and cure radiation- enteric syndromes.

放疗或核事故受照者患放射性肠炎较为常见，防治已成难题，发病机制尚未阐明。新近研究提示，肠道α-防御素具有选择性抗菌，调节TLRs-MyD88通路和T细胞活化的抗炎作用。基于我们前期发现放疗后肠菌群失调、粘膜炎症失控与防御素表达紊乱密切相关的现象，提出防御素调控肠道微生态-TLRs-免疫细胞轴促进放射肠内稳态重建的作用机制假设。本项目拟以人肠道α-防御素（HD-5,6）双转基因MMP7-/-小鼠腹盆腔模拟放疗照射模型和IEC-6、THP-1细胞为研究对象，采用菌群宏基因组与代谢组分析、qRT-PCR、Western blot、ChIP和流式细胞术等方法，从HD-5,6表达水平、肠菌群结构功能改变、粘膜免疫细胞活化和炎症反应等方面，探讨HD-5,6促进放射后肠道内稳态的重建作用和摄入HD-5,6对放射性肠炎的防治效果。本研究有望深入揭示放射性肠炎的发生机制，为放射性肠道综合症的防治提供新策略。

放疗或核事故受照者患放射性肠炎较为常见，防治已成难题，发病机制尚未阐明。本项目完成了以下研究内容：(1)口服后可在肠腔内缓释HDs (HD5和HD6)纳米颗粒的设计、制备和表征分析；(2）含HDs纳米颗粒防治小鼠放射性肠炎的效果及作用机制; (3) HD5+/+MMP7-/-、HD6+/+MMP7-/-、HD5+/+HD6+/+MMP7-/- 和MMP7-/- 4种基因修饰小鼠种群的建立与繁育；HDs在小鼠肠道上皮转录和翻译表达的检测与分析；HDs对小鼠肠道微生态、肠粘膜免疫细胞亚群的影响和在体研究HDs防治放射性肠炎的作用及机制。..成功设计并制备胰蛋白酶敏感的肠道缓释HDs纳米递送系统。纳米颗粒直径约为50-80 nm，孔径约为5.5nm，颗粒大小均匀，分散性好。HDs装载率约为49.7%。在肠道环境中释放HDs具有时间依赖性，在6 h左右可释放约50%靶分子的优点。外源口服HDs具有调节小鼠肠道微生态，恢复Lactobacillus等益生菌丰度，减轻受照小鼠的全身性的炎症反应，调节肠上皮Glut1、TFF3和Muc2基因的表达从而促进再生修复，可显著提高受照小鼠的存活率。提示HDs具有通过口服防治肠炎的潜在应用前景。..成功繁育和建立HD5,6转基因小鼠种群。采用RT-PCR和免疫组化验证了HDs在小鼠肠上皮的转录和翻译。发现HDs可改变肠粘膜DCs、ILC3s和Treg/Th 17免疫细胞亚群活化状态及数量。在体研究证实HDs具有提高Dubosiella等益生菌属含量，稳定和平衡肠道菌群的作用。结果表明HDs具有调控肠上皮免疫细胞和微生态的双重功能，提示HDs可能是防治肠炎相关疾病的潜在靶标。