Integrin β3调节血管平滑肌细胞源性外泌体分泌促进细胞迁移的研究

Vascular smooth muscle cell derived exosomes (VSMC-Exo) transfer cell signaling from vascular smooth muscle cells (VSMCs) to endothelial cells, fibroblasts and other target cells. VSMC-Exo participates in various cardiovascular pathological processes, including vascular calcification, endothelial inflammation and atherosclerosis. However, the specific mechanism of VSMC-Exo secretion and its related functions are still unclear. Our preliminary experiments showed that inhibition of endogenous integrin β3 suppressed VSMC-Exo secretion, suggesting that integrinβ3 could regulate VSMC-Exo secretion, and interfering/overexpressing of integrinβ3 could suppress/promote VSMCs migration, the mechanism may be related to VSMC-Exo secretion. Therefore, we propose our scientific hypothesis, integrin β3 regulates VSMC-Exo secretion and promotes cell migration. In this project, immunoprecipitation, enzyme-linked immunosorbent assay and nanoparticle tracking analysis will be used to clarify the regulatory effect of integrinβ3 on VSMC-Exo secretion, clarify its mechanism, and explore the effect of integrin β3 and VSMC-Exo on VSMCs migration, this project is expected to provide scientific basis for preventing and treating cerebrovascular diseases.

血管平滑肌细胞源性外泌体（VSMC-Exo）是平滑肌细胞与内皮细胞、成纤维细胞及其它靶细胞间信号传递的重要介质，其参与血管钙化、内皮炎症、动脉粥样硬化等心血管病理过程，但目前尚不清楚VSMC-Exo分泌的具体机制及相关功能。我们预实验发现干扰内源性integrin β3抑制VSMC-Exo分泌，提示integrin β3调控VSMC-Exo分泌的可能性，干扰/过表达integrin β3分别抑制/促进血管平滑肌细胞迁移，其机制可能与VSMC-Exo分泌有关，因此我们提出integrin β3调节VSMC-Exo分泌促进细胞迁移的科学假说。本项目拟采用免疫共沉淀、酶联免疫吸附、纳米粒子追踪分析等方法，明确integrin β3对VSMC-Exo分泌的调节作用，阐明相关机制，并探讨integrin β3、VSMC-Exo对血管平滑肌细胞迁移的影响，为寻找脑血管疾病防治新靶点提供科学依据。

血管平滑肌细胞源性外泌体（VSMC-Exo）是血管平滑肌细胞（VSMCs）与内皮细胞、成纤维细胞及其它靶细胞间信号传递的重要介质，其参与血管钙化、内皮炎症、动脉粥样硬化等心血管病理过程，但目前尚不清楚VSMC-Exo分泌的具体机制及相关功能，我们提出integrin β3调节VSMC-Exo分泌促进细胞迁移的科学假说。本项目采用免疫共沉淀、酶联免疫吸附、纳米粒子追踪分析等方法，研究发现integrinβ3参与调节VSMC-Exo分泌，其机制与TMEM16F介导的Ca2+依赖的磷脂翻转（Ca2+-dependent phospholipid scrambling，Ca2+-PLS）有关。随后我们对具体的机制进行研究，我们通过免疫共沉淀技术发现integrinβ3和TMEM16F存在相互作用，并且干扰和过表达VSMCs内源性TMEM16F不仅可分别抑制或促进VSMCs的外泌体分泌，还可分别逆转过表达integrinβ3导致的VSMC-Exo分泌的促进作用或干扰integrinβ3导致的VSMC-Exo分泌的抑制作用，此外我们通过流式细胞术在VSMCs中检测integrinβ3和TMEM16F对Ca2+-PLS的影响，结果提示在VSMCs中干扰或过表达integrinβ3可分别抑制或促进Ca2+-PLS，而过表达TMEM16F可逆转干扰integrinβ3导致的Ca2+-PLS抑制作用，干扰达TMEM16F可逆转过表达integrinβ3导致的Ca2+-PLS促进作用，这些结果提示integrinβ3对VSMC-Exo分泌的影响可能与TMEM16F介导的Ca2+-PLS有关。最后我们还探究了integrinβ3、VSMC-Exo与VSMCs迁移三者之间的内在联系，研究结果提示VSMC-Exo可促进VSMCs迁移，其作用机制与integrinβ3有关。本项目从理论上部分阐明了integrinβ3通过TMEM16F介导的Ca2+-PLS调控VSMC-Exo分泌，参与调节VSMCs迁移的新机制，为脑血管疾病的诊断、预防和治疗的新靶点、新机制提供了实验数据和理论依据。