调节性T细胞外泌体抑制TBI后小胶质细胞过度活化作用的研究

Traumatic brain injury (TBI) leaded to the excessive activation of microglia, which releases a variety of pro-inflammatory cytokines. How to maintain moderate inflammatory response is a key step after TBI. Our team has demonstrated that increased Tregs post-injury could reduce the activation of microglia, and switch M1 phenotype to M2 phenotype. But the mechanism is not very clear. Exosomes, which contained miRNA, proteins and mRNA etc., are flat extravescular vesicles releasing from cells. Our group has sorted Tregs by magnetic beads and proved that Tregs could release the exosomes through the electron microscope, western blot and Nanoparticle tracking technology. And we also demonstrated the PKH67-labeled exosome were taken up by microglia with high efficiency after 6h of incubation. Besides, microarray analysis showed miR-16 is the most abundant miRNA in Treg derived exosomes. In this project, we will further investigate: 1). the inhibitory effects of Treg-exosomes in vitro by the culture of microglia; 2).transfect Treg with miR-16 agomir/antagomir to determine the important effects of miR-16 in exosome; 3). the inhibitory effect of Treg-exosomes on the activation of microglia in vivo. The outcome will offer new understanding of inflammation after TBI.

颅脑创伤（TBI）后过度活化的小胶质细胞可加重病情，如何调控过度炎症反应是TBI救治关键。课题组前期通过药物诱导和细胞移植增加TBI后小鼠体内Treg数量，可减轻小胶质细胞的过度活化，促进M1向M2型小胶质细胞转化，但其机制并不十分清楚。外泌体是细胞主动分泌的大小均一、可携带多种生物学信息的囊泡样小体，课题组前期成功获取Treg来源外泌体并进行鉴定，利用PKH67标记外泌体证明其可被共培养的小胶质细胞大量吞噬，结合文献全基因组分析筛出外泌体中表达丰富的miRNA-16，本项目将在此基础上借助细胞培养及小鼠TBI模型进一步研究：1.体外实验证实Treg外泌体可抑制小胶质细胞过度活化；2.调控Treg及外泌体中miR-16（agomir/antagomir）表达水平，验证其为外泌体介导抑炎作用的关键miRNA；3.动物实验证实Treg外泌体可抑制小胶质细胞过度活化；为TBI的临床治疗提供依据。

颅脑创伤（TBI）是中青年人群中致残、致死的重要原因，除原发性损伤外，TBI后继发性炎性损害也起着至关重要的作用，而调控TBI后急性期小胶质细胞的过度激活被认为是促进TBI预后的重要措施，课题组前期通过体内药物诱导（IL-2/anti-IL-2 complex、Atorvastatin、Erythropoietin）和体外Treg细胞移植的方法增加TBI后小鼠体内Treg细胞数量，发现其可减轻小胶质细胞的过度活化，促进M1型向M2型小胶质细胞转化，促进TBI小鼠的预后。为进一步探明其作用机制，我们聚焦于外泌体这一前沿领域，外泌体是细胞主动分泌的大小均一、可携带多种生物学信息的囊泡样小体。本项目通过免疫磁珠分选小鼠脾脏内Treg细胞进行培养，收集其培养基，选用超速离心法获取Treg外泌体，并利用透射电镜、Western Blot、纳米颗粒示踪技术进行鉴定。后续实验通过：1）PKH67标记外泌体并加入到体外培养的BV2细胞培养基内孵育，证明其可被小胶质细胞大量内在化，从而发挥其生物学信息转运功能；2）对低氧诱导损伤的BV2细胞进行预处理，发现其可以明显减少M1型小胶质细胞相关标志物的表达；3）收集步骤2）中不同处理组BV2细胞培养基加入到体外培养的小鼠脑微血管内皮细胞中，发现来源于外泌体预处理组的BV2培养基可以明显减轻内皮细胞间紧密连接蛋白的降解；4）利用miRNA-195抑制剂转染Treg细胞，获取低表达miR-195的Treg外泌体，重复上述步骤2）及步骤3）中实验，在细胞水平证实miR-195是外泌体发挥抑炎及血脑屏障保护作用的关键；5）通过尾静脉移植Treg外泌体干预TBI模型小鼠证实，Treg来源外泌体可以促进小胶质细胞向抗炎表型转化，并减轻血脑屏障的破坏，从而促进TBI后神经功能恢复。通过本项目的研究为Treg抑制小胶质细胞过度活化机制提供了新的理论依据。