外泌体介导的CCL2/CCR2信号对颅脑损伤后炎性单核细胞趋化作用机制研究

Traumatic brain injury, or TBI, is one of the most threats to patients death and disability. There is currently no effective treatment. Recent studies have shown that neuroinflammation after TBI is a key reason to aggravate secondary brain injury. And it is the key factor to induced neuroinflammation, which a large number of inflammatory monocytes are recruited to the injury. Although chemokine CCL2/CCR2 signaling is known to be involved in its recruitment, the exact transport mechanism is not well understood. It has been reported in the literature that microglial can secrete exosomes that carry both CCL2 and CD47. Preliminary work suggests that CD47 is involved in inhibiting recruitment and activation of inflammatory monocytes and inhibiting macrophage phagocytosis. Therefore, a scientific hypothesis is proposed that CCL2 and CD47 exosomes are involved in the chemotaxis of CCL2/CCR2 signals to inflammatory monocytes after TBI. This project intends to establish a model of TBI in vivo and in vitro, and to study the mechanism of inflammatory monocyte chemotaxis involved in exosomes after TBI using transgenic mice, two-photon laser in vivo imaging and mRNA-array techniques to elucidate the mechanism of recruitment of inflammatory monocytes after TBI, the mechanism of occurrence and development of secondary cerebral edema, an provide a theoretical basis for finding effective therapeutic targets.

创伤性颅脑损伤（TBI）是威胁患者生命和致残的主要原因之一，目前尚无有效的救治方法。研究表明TBI后神经炎性反应是加重继发性脑损伤的关键因素，而大量炎性单核细胞被招募至损伤部位是神经炎性反应的重要诱因。尽管已知趋化因子CCL2/CCR2信号参与其招募，但具体递送机制尚不清楚。文献报道小胶质细胞等可分泌同时携带CCL2和CD47的外泌体。前期工作提示CD47参与抑制炎性单核细胞的招募和活化，并抑制巨噬细胞对靶细胞的吞噬清除。因此提出科学假说：TBI后小胶质细胞等释放携带CCL2和CD47的外泌体介导了CCL2/CCR2信号对炎性单核细胞的趋化。本项目拟建立TBI体内、外模型，应用转基因小鼠、双光子激光活体成像等技术研究TBI后外泌体参与的炎性单核细胞趋化作用机制，从而为阐明TBI后炎性单核细胞的招募机制，为探索继发性脑水肿的发生发展机制，以及寻找相应有效治疗靶点提供理论依据。

外泌体是大小介于30-100nm之间的细胞外囊泡，可以作为体内远距离物质交换、信号传递的媒介。但是，外泌体在创伤性颅脑损伤后的炎症信号传递以及炎症微环境调控中的作用以及调节机制尚不清楚。在本项目中，我们发现（1）炎性小胶质细胞外泌体形态饱满，并且出现一群粒径较大的波峰外泌体群。炎症刺激会减少外泌体的分泌量，但是会增加外泌体被巨噬细胞的吞噬效率。并且吞噬炎性外泌体后，巨噬细胞的M1促炎表面标志物CD86表达显著升高，TNF-α、iNOS等促炎基因表达显著升高。外泌体在小胶质细胞与单核巨噬细胞间具有炎性信号传递的作用。（2）炎性小胶质细胞分泌外泌体中的Esyt1, Uba2, lqgap2, Slc9ar3r1, Polr2a, Slc15a3, Rcc1, Flna, Tgm1等蛋白的表达显著升高。接收炎性外泌体后，巨噬细胞的Cxcl2, Ccl2, Acod1, Ccl3, Csf3等基因的表达改变显著，同时细胞在凋亡、免疫反应、细胞形态改变以及增殖等生物过程基因表达呈现显著差异。另外TNF , IL-17, NF-KB, Toll-like receptor, NOD-like receptor等信号通路显著参与此过程。这些关键蛋白和基因通路可能是炎症信号传递的分子机制。（3）我们通过局部移植神经组织工程微单元来改善创伤性颅脑后过度的炎性环境。在接受炎性微环境改善治疗后，小鼠运动和学习记忆功能恢复更快，损伤处小胶质细胞数量显著减少，并且其中M2抗炎型细胞比例显著升高，胶质瘢痕厚度减少，内源性再生神经细胞数量增多。因而，炎性微环境改善对于促进脑损伤恢复具有积极作用。我们的研究为创伤性脑损伤后外泌体介导的炎性微环境形成机制奠定了理论基础，同时也为以炎性外泌体信号阻断或炎性微环境改善为靶点的创伤性颅脑损伤新疗法的开发提供重要支撑点。