细菌介导肠道C-反应蛋白参与免疫代谢调控的机制研究

According to our previous study, C-reactive protein (CRP) , located in the lesion, is involved in the pathological process directly, while the plasma CRP is often used to evalute the inflammation degree of the body with no specifity. The abundant immune response in intestinal tract is very suitable for the functional studies of CRP. Therefor we created a intestinal epithelial cells with a human crp inserted random transgene mouse by using a special intestinal promoter (IEC-huCRPtg), combined 2% DSS induced Inflammatory Bowel Disease (IBD) and high fat diet induced obesity animal model to simulate the intestinal CRP respones in the these diseases. The preliminary experiments evince IEC-huCRPtg mice have IBD suppression and obesity promotion, but the invovled pathological mechanism still need to be studied. From preliminary mechanism exploration, we found that CRP can bind the intestinal bacteria selectively. Then we will identify the bacteria species bonded CRP by FACS separation and 16S rRNA-V4 sequencing. Next, we will use mall-molecule inhibitors, RAN interference (RNAi), transplantation of identified bacteria, appropriate antibiotic treatment, etc. to improve the signal transfer process of intestinal CRP in IBD suppression and obesity promotion and define the main function of CRP in intestinal immune and metabolic homeostasis. Meanwhile, we hope that the design thinking of CRP and the binding bacteria as a drug target could provide fundamental basis for the treatment and prevention of intestinal diseases.

课题组研究认为，局部病灶C-反应蛋白（CRP）才会直接参与疾病的病理过程，而血浆CRP更多是非特异性系统评估机体炎症程度；肠道内丰富的免疫应答非常适合CRP的功能研究，因此我们构建了肠上皮细胞特异性CRP转基因（IEC-huCRPtg）小鼠，并结合2% DSS诱导急性肠炎和高脂饮食诱导肥胖模型，模拟病理条件下的肠道CRP应答。预实验显示，IEC-huCRPtg小鼠表现出明显的肠炎抑制与肥胖促进，但涉及的分子机理并不清楚。初步机制探索发现，CRP可以选择性结合肠道细菌来发挥功能。之后，我们将借助流式分选和16S rRNA-V4测序，鉴定CRP结合细菌种属；利用小分子抑制剂、RNA干扰、鉴定细菌移植和相应抗生素处理等，完善肠道CRP抑制肠炎与促进肥胖涉及的信号传递过程，明确CRP在肠道免疫与代谢稳态中的关键位置，同时以CRP及其结合细菌为药物靶点的设计思路，为肠道疾病的治疗与预防提供基础。

与以往血液CRP功能研究不同，本项目关注肠道CRP对炎症性肠病的影响，因为血液水平的CRP只能整体反应机体炎症状况，而局部病灶组织的CRP才是真正参与体内炎症免疫应答的。在本项目研究中，我们利用肠道CRP高表达小鼠（IEC-huCRPtg）和CRP敲除小鼠（Crp-/-）发现，CRP能够促进2% DSS诱导的急性炎症性肠病的发展，并且这种结果在上述两种小鼠中相互印证。与此同时，免疫组化结合粘液特染发现，肠道上皮内CRP与肠道粘液在分布上完全吻合，并且动物模型中CRP对于肠道粘液的改变异常显著，因此我们推测，CRP可以通过调控肠道粘液的分泌参与炎症性肠病的发生。随后，我们使用FACS检测证实了CRP与肠道内特殊细菌的选择性结合，借助16S RNA V3+V4区测序发现，Akkermansia muciniphila（Akk）菌丰度在三种小鼠中存在显著的差异，进一步的粪菌移植（FMT）实验证实CRP引起的肠道菌群重塑确实造成了三种小鼠在2% DSS诱导的急性肠炎的病情改变。然而，已有众多研究表明，Akk菌是一种嗜黏蛋白菌，能够降解肠道上皮中的黏蛋白，进而对肠道黏膜免疫产生影响，于是我们认为CRP通过结合特殊细菌Akk影响肠道粘液的合成与分解代谢，最终影响炎症性肠病的发生。在后期的机制研究中，我们检测了CRP对Akk细菌的生长的影响，CRP与AKK细菌的结合，CRP对黏蛋白MUC2表达的影响，CRP对DSS诱导肠炎的黏膜免疫应答等，最终解释CRP如何影响粘液改变调控炎症性肠病的发生。通过本项目的研究，我们证实了CRP在炎症性肠病中的关键作用和CRP参与炎症性肠病调控的病理机制，而CRP本身和CRP结合特异细菌则被证实是各种炎症性肠病的潜在治疗靶点，这将有利于建立各种炎症性肠病及其并发症的预防与治疗的新策略。