Angptl4表达相关肠道细菌的确立及其介导的辐射抗性机制研究

Radiation enteritis is caused by intestinal damage due to the radiation therapy of pelvic, abdominal malignancies which is the main reason for the dose limitation of pelvic, abdominal radiotherapy. Recently, intestinal flora has become a research hotspot in radiation protection. Becides, Angiopoietin-like Protein 4 (Angptl4) has been reported to be positively correlated with intestinal radiation resistance. And its expression is regulated by intestinal bacteria. However, which intestinal bacteria can regulate Angptl4 expression and the mechanisms of Angptl4 mediates intestinal radiation resistance are still unknown. In previous work, we have predicted the intestinal bacterial components which may regulate the expression of Angptl4 through 16s rDNA amplicon high-throughput sequencing. We hypothesize that these intestinal bacteria can regulate the expression of Angptl4 and further can cause the different radiation response. On the basis of the previous work, the intestinal bacterial components that regulate the expression of Angptl4 would been established by isolating, identifying and function evaluating. Then, the established components would been used to regulate the expression of Angptl4, and the differential genes or signaling pathways related to the radiation response of different expression groups of Angptl4 would been analyzed and identified to demonstrate the mechanism of Angptl4-mediated radiation resistance. Our research will provide theory evidence for establishment of methods for improving radiation resistance by targeting intestinal microbiota to regulate the expression of Angptl4. In addition, our research can also provide theoretical basis for the development of radiaprotectants targeting Angptl4.

放射性肠病是由于腹、盆腔恶性肿瘤放疗导致的肠道并发症，是腹盆腔放疗剂量限制的主要原因。近年来肠道菌群成为辐射防护的研究热点。研究表明血管生成素样蛋白4Angptl4的表达与肠道辐射抗性正相关且受肠道细菌调控。而该蛋白受哪些菌群调控以及如何介导肠道辐射抗性机制仍不明确。在前期工作中，我们采用16s rDNA扩增子高通量测序技术筛选了可能调控Angptl4表达的肠道细菌，这些菌群可能通过调控Angptl4的表达而影响肠道的辐射抗性。在此基础上，我们将采用培养基预测、好厌氧培养等技术对这些细菌进行分离鉴定，并在体内体外进行功能评价，确立调控Angptl4表达的肠道菌群；此外，我们将通过比较转录组分析结合分子生物学技术鉴定Angptl4不同表达组辐射响应相关的差异基因和信号通路，明确Angptl4介导的辐射抗性机制。本研究将为肠道辐射防护新机制的探索提供理论基础，并为辐射防护药物的研发提供新靶点。

放射性肠损伤是由于盆腹腔恶性肿瘤经放射线治疗而引起的肠道损伤，是腹部肿瘤放疗剂量限制的主要原因而影响疗效。近年来，肠道菌群应用于辐射防护成为研究热点。研究表明血管生成素样蛋白4Angptl4的表达可能与肠道辐射抗性正相关，且其表达受肠道细菌的调控。本课题中我们的主要研究内容是：明确Angptl4表达与肠道辐射损伤程度之间的关系；采用16s rDNA扩增子高通量测序技术预测可能通过促进Angptl4表达进而起到影响肠道辐射抗性的肠道细菌；采用预测培养基或常用肠道细菌分离培养基对预测细菌进行厌氧分离纯化及鉴定；在体内动物实验和体外细胞实验水平对分离肠道细菌促进Angptl4表达和肠道辐射防护功能进行评价；在体外细胞水平，通过过表达、比较转录组分析等技术对Angptl4起到辐射防护的机制进行探索。经过实验研究，我们得到以下结果：Angptl4表达水平与肠道的辐射损伤程度负相关，Angptl4表达水平高的肠道辐射损伤较轻；通过高通量测序预测、肠道细菌的厌氧单菌分离纯化坚定、肠道单菌功能的体内体外验证，我们共分离到3株在体内体外水平均能促进Angptl4表达，且在体内动物实验水平具有辐射防护功能的肠道细菌，分别是Lactococcus sp.2-3; Lactobacillus reuteri 2-16和Ruminococcaceae sp. Rum；在采用体外细胞实验进行机制探索时，我们发现过表达Angptl4可提高肠上皮细胞存活率，且可降低照射引起的细胞凋亡，机制探索发现，Angptl4可能是通过调节PPAR信号通路和Rho信号通路，调控微管蛋白表达稳定细胞结构，降低辐射引起的细胞凋亡，进而起到辐射防护作用。本项目研究为肠道细菌作为辐射防护研究出发点提供实验依据，并为辐射防护药物的研发提供靶点，对事故造成肠道辐射损伤治疗或盆腹腔肿瘤放射治疗方案的优化及预后具有现实意义。