清化活血法基于microRNA 介导SDF-1/CXCR4通路干预BMSCs归巢修复 UC的机制研究

Ulcerative colitis (UC) is a chronic inflammation bowel disease in the rectum and colon without clear and definite pathogenesis, therefore it is currently classified as a hardly treated disease by WHO. Recently, a great number of researches have proved that bone marrow mesenchymal stem cells (BMSCs) transplantation is effective and safe for treating UC. Lots of researches have discovered that SDF-1/CXCR4 plays an important role in BMSCs migration, miR-27b down-regulating SDF-1 interferes BMSCs migrating toward liver. The representative Supplemented Pulsatilla Decoction has a benefit effect on treating UC with method of clearing heat and activating blood. Our previous researches have found that CXCR4 and SDF-1 is highly expressed in the inflammatory tissue of UC rat, therefore microRNA dominating SDF-1/CXCR4 pathway to intervene BMSCs migration might be a new possible pathway for the method of clearing heat and activating blood treating UC. This study aims to establish an UC rat model and discuss the mechanism of clearing heat and activating blood treating UC based on microRNA dominating SDF-1/CXCR4 pathway to intervene BMSCs migration, with application of RT-PCR, WB, ELISA, and IHC, in order to build a solid scientific theory for traditional Chinese medicine to prevent and treat UC and intervene BMSCs transplantation.

溃疡性结肠炎是一种原因尚不明确的慢性直肠和结肠炎症性疾病，被WHO列为现代难治病之一。近年来，大量研究证实了骨髓间充质干细胞（BMSCs）移植治疗UC的有效和安全性。研究表明SDF-1/CXCR4轴在BMSCs归巢中发挥重要作用，miR-27b负调控SDF-1干预 BMSCs向肝脏迁移。以加味白头翁汤为代表的清化活血法对UC有良好疗效，我们前期研究发现UC小鼠炎症部位CXCR4、SDF-1表达升高，因此我们推断microRNA 介导SDF-1/CXCR4轴促进BMSCs归巢亦可能是清化活血法治疗UC的另一可能分子生物学途径。本研究以UC模型小鼠为研究对象，采用RT-PCR、蛋白印迹、ELISA及免疫荧光等技术，从基因、分子、细胞及整体水平探索清化活血法通过microRNA 介导SDF-1/CXCR4轴干预BMSCs归巢修复UC机制，为中医药防治UC和干预BMSCs移植治疗UC提供科学依据。

背景：清化活血中药和BMSC对UC有治疗作用，机制未明，BMSC在UC中的归巢机制亦未明。研究表明microRNA参与UC的发病机制中，体外实验表明miR-27b调控SDF-1干预BMSC向肝脏迁移 ，miR-27b是否通过SDF-1/CXCR4干预BMSC在UC小鼠归巢，目前尚无研究。研究表明中医药可以通过SDF-1/CXCR4干预BMSC归巢，清化活血中药能否通过miR-27b/SDF-1/CXCR4通路干预BMSC在UC小鼠归巢亦无研究。主要研究内容：清化活血中药和BMSC治疗DSS诱导UC的实验研究；清化活血中药干预BMSC归巢修复UC机制的初步研究；采用HT29进行体外实验，研究中药含药血清对HT29 miR-27b/SDF-1/CXCR4表达，以及Tanswell实验研究中药含药血清调节HT29细胞对BMSC迁移能力的影响。重要结果：1、清化活血中药和BMSC移植治疗DSS诱导UC有良好疗效，清化活血中药和BMSC分别干预后，模型小鼠体重增加，DAI降低，病理组织学获得改善，还可降低TNF-α、IL-6，增加IL-4，IL-10。体内实验中药干预后，DSS+TCM组肠道miR-27b表达较DSS组降低，SDF-1、CXCR4表达较DSS组升高；与DSS+BMSC相比，DSS+TCM+BMSC组肠道荧光信号明显增加；与DSS+TCM+MSC组相比，DSS+TCM+MSC+AMD3100、DSS+TCM+BMSC+LV-miR-27b组荧光信号明显降低，肠道SDF-1、CXCR4表达明显降低；体外实验，miR27b mimic组的SDF-1、CXCR4表达较miR27b mimic对照组明显降低，含药血清组对BMSC的迁移能力大于对照血清组，以上均提示BMSC归巢至肠道可能受miR-27b/SDF-1/CXCR4轴调控，清化活血中药可能通过下调miR-27b，进一步调控SDF-1/CXCR4轴促进BMSC归巢至肠道。 科学意义：本研究为中医药防治UC和BMSC移植治疗UC提供基础理论支持。