芬戈莫德调节免疫功能治疗创伤性脑损伤的机制研究与临床概念验证

The excessive inflammatory response post traumatic brain injury(TBI) is an important factor of secondary brain injury, and the neurological dysfunction caused by it still lacks effective treatment.The colleagues in our insitute have confirmed that the injured neural function of the cerebral hemorrhage patients is greatly improved by early and short-term application of Fingolimod. Fingolimod also shows good prospects to improve the impaired neural function in the secondary brain injury in TBI mice.However, different timing and different terms of administration of Fingolimod result in significant ouctome in TBI mice. An in-depth study of its active mechanism in TBI is urgent. This study plans to be performed based on the in vitro cell culture system and TBI mice models. 1) Exploration of the modulating ability of Fingolimod on the mobility, invasion and proliferation of Treg and NK cells. The modulation of Fingolimod on the subtypes conversion between M1 and M2 of microglia is also inspected. The role of the chemokine expression in these processes is preliminarily confirmed too; 2) The flow cytometry, cytokine array and other facilities are applied to monitor the function changes of the immunological cells and the released cytokines in the injured brain tissue, lung, spleen and peripheral blood in the TBI mice. The in vivo modulation potential of Fingolimod will then be evaluated. 3 ) The therapeutic effects of the Fingolimod on the impaired neural function of the TBI mice will be compared, which is administrated with different initial time and different administrating term. 4) In accordance with the POC standards, the preliminary clinical validation studies of Fingolimod treatment for TBI patients will be carried out. The POC study will provide the theoretical foundation for developing new therapeutic strategies for TBI.

脑外伤（TBI）过度炎症反应是继发性脑损伤的重要因素，其所致的神经功能障碍尚缺少有效治疗方法。本单位前期证实脑出血后早期、短时间应用芬戈莫德可显著改善患者神经功能，而应用此药干预TBI小鼠的继发性脑损伤也显示了良好前景，但不同给药时机，不同给药时程对TBI小鼠的作用差异较大，急需对其在TBI中的作用机制进行深入研究。拟通过体外细胞培养和TBI小鼠模型：1）探索芬戈莫德对Treg、NK迁移浸润，小胶质细胞M1和M2亚型转换等功能调控，以及趋化因子表达在此过程中的作用；2）应用流式细胞仪、Cytokine Array等监测局灶脑组织、肺、脾和周围血中免疫细胞和炎性细胞因子变化，探明芬戈莫德对TBI在体免疫调控作用; 3）比较TBI小鼠伤后不同时机、不同疗程给予芬戈莫德干预对神经功能修复的效果；4）按照概念验证研究（POC）规范，开展芬戈莫德治疗TBI的临床验证研究，为开发TBI新疗法奠定基础。

芬戈莫德（Fingolimod）是溶血磷脂—鞘氨醇-1-磷酸（S1P）类似物，阻止免疫细胞释放，有很强的免疫调节功能。脑外伤（TBI）后的过度免疫炎症反应是继发性脑损伤的重要因素。本研究基于不同的脑外伤模型（eCCI和DBI）证实应用芬戈莫德抑制免疫对于不同类型脑外伤产生截然不同的作用结果。对于局灶性TBI（eCCI）,芬戈莫德的可有效降低TBI后损伤灶的免疫炎症反应，减少CD4+、CD8+ T、NK细胞浸润，增加脑组织内Treg细胞含量，减轻TBI后的白质损害、血脑屏障破坏，促进神经功能修复；在大鼠弥漫性脑损伤模型（DBI）中应用芬戈莫德虽降低脑组织炎性因子含量，及外周血中CD4+、CD8+ T细胞数量，增加Treg细胞含量，但却导致了更严重的神经细胞损伤，神经功能的损害，加重了损伤后肺部感染，增加了死亡率。在基于eCCI的TBI小鼠模型对TBI后出现的外周脏器损伤的研究中，发现了Fingolimod对于局灶性脑外伤所继发的心肺损伤具有缓解作用，改善了TBI后的肺部蛋白渗出，中性粒细胞浸润，肺泡间隔通透性，降低肺组织炎性因子表达，明显缓解TBI后出现的心功能不全，减少心肌细胞凋亡坏死及间质纤维化，降低心肌组织炎性因子含量。在对脑创伤研究过程中，对于硬脑膜淋巴管（mLVs）研究热点进行了积极探索，发现mLVs是硬膜下血肿吸收引流的重要途经，阻断该通路会明显减慢血肿的吸收。且硬膜下的血肿会影响mLVs的功能结构，阻碍其正常引流功能。为进一步研究他汀类药物治疗CSDH的机制研究打开思路，为团队继续探索mLVs功能调控与脑外伤关系打下一定理论及技术基础。项目原定目标发表SCI论著5篇，中文论著或综述5篇，获得国家级项目1项和省部级项目1项，培养博士生2名，硕士生5名，项目研究实际发表SCI论著15篇，中文论著5篇，研究部分成果参获天津市科技进步特等奖1项，培养博士生5名，硕士生6名，已超额完成任务。