放射损伤中肠微生态调控肠干细胞的机制研究

More than 50% patients develop intestinal complications with a high risk of mortality after abdominal/pelvic radiotherapy. Irradiation leads to two prominent alterations in the gut: 1) massive loss of intestinal stem cells which govern the homeostasis and replenishment of the intestinal epithelia; and 2) disequilibrium of the intestinal microbiota. The work of the applicant in Germany has shown that irradiation induced up-regulation of Wnt/beta-catenin signaling activity sensitizes intestinal stem cells to apoptosis (The EMBO Journal，2015). Moreover, our unpublished data indicates that irradiation significantly impairs self-renewal and proliferation capacity of intestinal stem cells. However, how gut microbiota regulates intestinal stem cells upon irradiation remains to be determined. Given that intestinal microbiota impacts on Wnt signaling, we hypothesize that irradiation induced dysregulation of intestinal microbiota regulate the fate and function of intestinal stem cells through Wnt signaling pathway. To verify the hypothesis, the project intends to study: 1) the dynamic changes of intestinal microbiota, Wnt signaling, and the loss and impairment of intestinal stem cells upon irradiation; 2) impact of modulating microbiota on intestinal stem cell function and Wnt signaling; 3) the regulatory link between microbiota and Wnt signaling on intestinal stem cells using APCmin mice (high-Wnt activity) and LRP6 neutralization antibody injected mice (low-Wnt activity). Overall, this project will provide new insights into understanding the mechanisms underlying irradiation induced gut injury, thus suggest potential new means of prevention.

腹/盆腔放疗引起的放射损伤并发症中，肠道发病率超过50%且致死率高。放射线照射造成肠微生态紊乱，同时大量杀伤肠上皮稳态维持和损伤后修复的核心--肠干细胞。申请人在德国的工作证实照射上调Wnt信号途径而加剧肠干细胞凋亡（The EMBO Journal，2015）；此外，我们预实验提示照射可显著损伤肠干细胞自我更新与增殖能力。但放射损伤中肠微生态是否调控肠干细胞及机制尚不清楚。已知肠微生态可调节Wnt信号途径。据此我们提出假说：照射后肠微生态紊乱通过调控Wnt信号途径决定肠干细胞的命运及功能。为验证该假说，本项目拟研究放射损伤中：1）肠微生态、肠干细胞、Wnt信号途径的动态变化及三者间的相关性；2）利用肠干细胞培养技术研究调控肠微生态对肠干细胞及Wnt信号途径的作用；3）利用高/低Wnt活性小鼠模型研究肠微生态调控肠干细胞及其调控Wnt信号途径的关系。本项目将为肠道放射损伤防治提供新视角。

肠干细胞（ISC）衰老是许多肠道衰老相关疾病的主要发病基础。然而ISC衰老的机制尚不清楚。本项目通过体外三维培养，发现衰老ISC存在显著的分化障碍，我们进一步通过实时定量PCR明确了衰老ISC上调Wnt信号途径表达而下调分化相关基因表达，培养体系中下调Wnt信号途径活性可拯救衰老ISC分化缺陷的表型。这项研究首次揭示了衰老ISC对Wnt/beta-catenin信号途径的过高敏感性介导了其分化缺陷，这对老年人中肠道肿瘤发病率显著升高、肠道放、化疗损伤后修复障碍有重要的启示意义，为探索可能的改善途径提供了重要靶点。.老年人体重过重、脂肪堆积等问题常见。本项目探索了肠道微生态在其中的作用，以及可能的抗衰老途径，我们发现，相比年轻小鼠，老年小鼠肠道微生态中与促进炎症和肥胖相关的菌群显著增加，与肥胖呈正相关的厚壁菌/拟杆菌比值显著升高。对老年小鼠进行短期限食可逆转老年小鼠肠道微生态异常，使其接近年轻小鼠，并明显改善老年小鼠肥胖、减少脂肪堆积。.放射线照射与化疗药物均可引起DNA损伤，使ISC凋亡及功能受损。本项目在接受超大剂量MTX的自由饮食小鼠中观察到致死率约90%，而化疗前进行2周限食的小鼠致死率降低至10%以下。限食显著减轻了肠道炎症，减少了ISC丢失，并改善了ISC功能。进一步研究发现，限食显著上调了肠道微生态中乳杆菌。通过广谱抗生素清除肠道微生物后，限食对MTX化疗损伤的保护作用完全消失，而胃饲鼠李糖杆菌（一种乳杆菌）则可部分模拟限食对MTX化疗损伤的保护作用。这说明化疗前短期限食可显著增加肠道微生态中保护性菌群，从而大大减少化疗肠毒性，改善ISC功能，促进化疗损伤后肠道修复重建。化疗相关肠毒性是最常见的化疗不良反应之一，这项研究成果对临床上改善化疗相关肠道不良反应，保证化疗按期规范实施，改善预后具有重要意义。.