UC-MSC靶向修复非炎性血栓形成的免疫调控机制与体内转归

Autoimmune diseases often cause non-inflammatory thrombosis due to immune dysregulation and aggravate the involving vital organs function even sudden death.Although current treatment make patients tend to be stable, it can not terminate the non-inflammatory thrombosis,so we need to find a new therapeutic approach.The preliminary studies treat SLE associated with DVT regional ulcer and dry gangrene using umbilical cord mesenchymal stem cells (UC-MSCs).The results showed that UC- MSCs inhibit the secretion of inflammatory cytokine and affect activated expression of B cell,but the regulatory mechanism of the therapy for non-inflammatory thrombosis is not clear.The experiment will take lupus-prone （NZW/BXSB）F1 hybrid male mice and rat CIA model as subjects through molecular biology methods to explore the modulation mechanism of UC-MSC to targeted repair B-cell active signaling pathway and the immune repaire and revascularizational mechanism of the UC-MSC for non-inflammatory thrombosis also with SPION method to trace UC-MSC. In vitro, UC-MSC intervened modling the micro-inflammatory situation then screening the important cell factors which influence the thrombosis,and constructed interfering RNA-expressing vector and over expression vector to explore the functional mechanism of these cell factors.Confirming the quantity of UC-MSC that effective targeted repair the injured area , we can produce a new therapy for non-inflammatory thrombosis.

自身免疫性疾病由于免疫调节异常而常频发非炎性血栓形成，加重已受累的重要脏器功能，甚至导致突然死亡。目前的治疗方案虽使病情能趋向稳定，但不能根治，更不能终止非炎性血栓形成。故需要寻找新的治疗途径。本项目前期采用脐带间充质干细胞（UC-MSC）治疗SLE伴发DVT性局部溃疡、干性坏疽等，结果发现UC-MSC抑制炎症因子分泌和影响B细胞活化表达。但其治疗非炎性血栓机制不清楚。本项目拟采用分子生物学方法和SPION标记 UC-MSC示踪法，以（NZW/BXSB）F1雄性狼疮小鼠及CIA大鼠为实验对象，探索UC-MSC靶向修复B细胞活化信号通路的调节机制及UC-MSC对非炎性血栓形成的免疫修复和血管重建机制。通过体外模拟UC-MSC干预炎症微环境，筛查关键影响细胞因子，并构建细胞因子过表达和干涉表达载体，探究其功能机制。确定UC-MSC靶向修复损伤部位的有效细胞量，提出治疗非炎性血栓形成全新方法.

免疫炎性微环境异常所致广泛性非炎性易栓症，频发于以SLE和RA为首的自免性疾病。大量致病性自身抗体和免疫复合物损伤皮肤、血管、关节、内脏等多个重要器官，导致全身非炎性免疫性血管炎，形成有利发生复发性致死性动静脉血栓形成的炎症微环境，奠定了诱发广泛性非炎性血栓形成的病理基础。. 本课题成功制备能表现体内免疫炎性微环境紊乱的动物模型CIA大鼠和选择表现自发性狼疮的MRL/lpr小鼠模型为研究对象，选择不受伦理限制的脐带来源的脐带间充质干细胞（UC-MSCs），制成浓度为2×10^6/ml的P3代UC-MSCs细胞悬液，采用超顺氧化铁磁性纳米粒子（SPION）作为体内新型示踪剂，建立了可重复应用的UC-MSCs移植新型示踪评价方法。从体内免疫炎症微环境、淋巴细胞表达、易栓症基础、UC-MSCs靶向修复等方面，探索UC-MSCs归巢和体内转归机制，完成了课题预期的研究目标，取得如下结论：1）体内免疫炎性微环境异常可导致RA和SLE等免疫紊乱性疾病，T和B细胞功能异常参与该过程，并成为免疫炎性易栓症的重要病理基础；2）UC-MSCs通过向炎症部位特异性归巢而调节免疫炎性微环境，其归巢作用可被AMD3100阻断，SDF-1/CXCR4轴是介导和促进UC-MSCs向炎性损伤部位归巢的重要机制；3）UC-MSC通过靶向修复免疫炎性微环境的调控作用，从而参与阻止免疫炎性易栓症的发生过程；4）SPION作为UC-MSCs的新型标记示踪剂，使动态随访UC-MSCs体内靶向修复成为现实。且SPION、UC-MSCs、AMD3100阻断剂等均未影响肝肾功能，为体内应用安全性提供了依据。. 研究结果从细胞和蛋白水平为调控免疫炎性微环境和干预免疫炎性易栓症的新型治疗提供了理论依据。已发表课题论文3篇，2篇论文被SCI期刊接受并审稿中，1篇待发表。并发表3篇课题相关论文。出席学术会议4次，培养6名硕士研究生。