Endoglin调控脑缺血后小胶质细胞TGF-β/ALK/Smad信号通路的机制研究

Endoglin is a transmembrane protein and an auxiliary receptor for TGF-β. Mutations in Endoglin cause type 1 hereditary hemorrhagic telangiectasia (HHT1), which causes stroke. Endoglin is involved in inflammatory response, but the mechanism is unknown. Our data indicated that compared to WT mice, Eng+/− mice had fewer CD68+ microphages at acute stage and more CD68+ macrophages at the post-acute stage of ischemic stroke, and impairs brain ischemic injury repair, which indicated that Eng-deficiency impairs macrophage homing to the injury site. Soon after an ischemic injury, a majority of microglia/macrophages migrating or infiltrating into the infarct areas initially assume the M2 phenotype which helps to reduce brain injury. M1 exacerbate ischemic injury. Endoglin modulate the activities of TGF-β/ALK signaling in endothelia cells. Both TGF-β and ALK are expressed by macrophages. We hypotheses that TGF-β/ ALK /Smad signal involved in the impaired microglia homing and polarization caused by Eng-deficiency. In this study, we will use Eng+/- mice, middle cerebral artery occlusion model as well as microglia transwell co- culture system, and with a variety of experimental techniques, to explore the association of macrophage homing and differentiation in Endoglin deficiency mice, and to invastigate whether Endoglin regulate microglia polarization through TGF-β/ALK /Smad signaling pathways. This will help to clarify the pathogenesis of HHT and related cerebral vascular disease.

Endoglin（ENG）是TGF-β共受体。ENG基因突变导致1型遗传性出血性毛细血管扩张症（HHT）并引起脑卒中。ENG参与炎性反应, 但机制不明。我们研究显示，Eng+/-鼠脑缺血急性期的CD68+细胞表达减少，恢复期仍有明显表达，且脑损伤更严重。提示ENG基因缺陷导致损伤加剧与炎性细胞浸润相关。研究表明脑缺血后早期小胶质细胞以M2型浸润为主，有保护作用。M1型则加剧脑损伤。在内皮细胞上，ENG通过TGF-β及其I型受体ALK1和ALK5传递信号。据此推测ENG基因缺陷可能促使激活的小胶质细胞向M1型转化，与TGF-β/ALK/Smad信号通路有关。本研究拟用Eng+/-小鼠，通过大脑中动脉缺血模型和细胞共培养系统等相关实验技术，研究脑缺血后小胶质细胞分型，揭示ENG基因通过TGF-β/ALK/Smad信号通路来调节小胶质细胞功能的机制。有助于阐明HHT及其所致脑卒中的发病机制。

Endoglin(CD105)是主要表达于血管内皮细胞的跨膜糖蛋白,也是转移生长因子β(TGF-β)超家族的Ⅲ型辅助受体。Endoglin是Ⅰ型膜蛋白，含有561氨基酸的细胞外结构域和疏水性的跨膜结构域。ENG基因突变导致1型遗传性出血性毛细血管扩张症（HHT）并引起脑卒中。ENG参与炎性反应, 但机制不明。本研究主要探讨ENG基因通过调节缺血后巨噬细胞的功能，参与缺血后炎性反应。方法：选制备阻断远端大脑中动脉的小鼠脑缺血模型(MCAO)。检测脑梗死/脑萎缩的体积、用墙角试验和移除黏附物能力测试检测脑缺血前后的动物行为学。免疫荧光染色检测脑缺血周边区的CD68+阳性细胞、M1和M2细胞表型及神经元凋亡。同时还用 Real-Time PCR的方法检测巨噬细胞M1和M2表型的表达。我们选用几个最具有特异性和广泛运用的M1和M2表型（CD206）的标记物等。CD31染色检测血管新生，BrdU/CD31共染色检测血管内皮增殖。我们研究显示Eng+/-鼠脑缺血急性期脑损伤严重而后期神经功能恢复差与急性期局部的炎性反应严重，恢复期炎症消退差有关。Eng+/-鼠脑缺血急性期的CD68+细胞表达减少，但恢复期仍有明显表达，而脑损伤更严重。Eng+/-鼠脑缺血急性期的IL-1ß, IL-6, 及TNF-α 水平较WT小鼠高。Eng+/-小鼠脑缺血60 天后，缺血边缘区的血管新生受损, BrdU/CD31阳性细胞较WT组少。炎性反应及M1和M2表型等改变会影响Endoglin信号通路，从而导致更严重的神经功能损伤有关。我们的研究初步提示Eng+/-鼠脑缺血后有更严重的炎性反应和神经功能恢复差。正常小鼠BM-来源的巨噬细胞能改善Endoglin基因缺陷鼠脑缺血的神经功能损伤，提示调节Endoglin相关信号可能成为治疗脑卒中的新靶点。