腺苷A2A受体缺失致周细胞凋亡在慢性低灌注脑白质损伤中的作用及机制

Damage of blood brain barrier is an early event of white matter injury induced by chronic cerebral hypoperfusion. Recently, it is found that the decrease of pericytes is the key link in the damage of blood brain barrier in white matter injury induced by chronic cerebral hypoperfusion. Our results indicated that apoptosis was an important reason for the reduction of pericytes in white matter injury induced by chronic cerebral hypoperfusion. A2A receptor gene knockout promote apoptosis of pericyte , suggesting that the A2A receptor play a key role in peripheral cell apoptosis. Since the modulation of A2A receptor on pericytes paly an important role in maintaining the integrity of the brain blood brain barrier. Therefore, we speculate that the deletion of A2A receptor promotes apoptosis of pericytes in white matter injury induced by chronic cerebral hypoperfusion, but the mechanism is not clear. The pre-experiment found that upregulation of A2A receptor deletion after the mitochondrial apoptosis protein Bax expression, and down-regulation of VEGF-A expression. As known, VEGF-A can inhibit apoptosis of pericytes. In this project, we comprehensively applied microimaging, morphology and molecular biology technology to elucidate the mechanism of promotion of A2A receptor to pericyte apoptosis from two aspects of mitochondrial apoptotic pathway and VEGF-A/VEGFR2 anti apoptotic pathway. It lays a theoretical foundation for understanding the pathological mechanism of white matter injury induced by chronic cerebral hypoperfusion and exploring the prevention and treatment measures.

血脑屏障损害是慢性低灌注脑白质损伤的早期事件。新近发现在慢性低灌注脑白质损伤中周细胞减少是血脑屏障损害的关键环节。本项目组前期研究发现在慢性低灌注脑白质损伤中细胞凋亡是周细胞减少的重要原因，A2A受体基因敲除后周细胞凋亡更加显著，提示A2A受体是周细胞凋亡的关键环节。鉴于A2A受体调控周细胞在维持脑血脑屏障完整中发挥核心作用，因此我们推测在慢性低灌注脑白质损伤中A2A受体缺失促进周细胞凋亡，但其机制不清。预实验发现A2A受体缺失后周细胞线粒体凋亡蛋白Bax表达上调，且VEGF-A表达下调。已知VEGF-A可抑制周细胞凋亡。为此，本项目综合应用显微成像、形态学、分子生物学等技术从线粒体凋亡途径和VEGF-A/VEGFR2抗凋亡通路两方面阐明在慢性低灌注脑白质损伤中A2A受体缺失促进周细胞凋亡的机制。为深入理解慢性低灌注脑白质损伤的病理机制、探索防治措施奠定理论基础。

利用腺苷A2A受体基因敲除小鼠构建慢性低灌注脑白质损伤小鼠模型，发现在白质损伤区域小胶质细胞/巨噬细胞显著增生活化，并且随着建模时间延长存在M1向M2的转化。腺苷A2A受体基因缺失抑制了活化的小胶质细胞/巨噬细胞向M2型转化，诱导其向M1型转化从而加重局部炎症反应，增强低灌注脑白质损伤。进一步利用腺苷A2A受体基因敲除小鼠做供体构建骨髓细胞腺苷A2A受体-/-小鼠，构建慢性低灌注脑白质损伤模型。结果发现骨髓来源细胞腺苷A2A受体基因敲除诱导小胶质细胞/巨噬细胞向M1型转化。培养巨噬细胞系RAW264.7，在低糖低氧培养基中培养1h，4h，12h，24h后采用免疫荧光双标染色、免疫定量PCR检测M1和M2标记物的蛋白及mRNA表达，结果发现M1标记物iNOS的表达明显增高，CD16和CD32的表达明显增高，Argine-1的表达下降。CD206的表达无明显差异。构建PPARγ的iRNA慢病毒载体，成功转染巨噬细胞，采用ELISA检测巨噬细胞在低糖低氧培养下炎性因子的表达，结果发现抑制PPARγ的表达显著降低了腺苷A2A受体介导的M2型巨噬细胞的诱导，降低了炎性因子的表达。电镜下观察到慢性脑血流低灌注状态下腺苷A2A受体基因敲除小鼠的周细胞肿胀变大，随着低灌注时间延长周细胞肿胀程度逐渐加重。周细胞在低糖低氧培养后凋亡细胞显著增多，凋亡蛋白BAX，Caspase3蛋白表达显著增加，抗凋亡蛋白BCL2蛋白表达显著减少，但是VEGF-A的表达显著增加。激活腺苷A2A受体显著抑制低糖低氧培养的周细胞凋亡，抑制周细胞凋亡蛋白BAX，Caspase3蛋白表达，抑制VEGF-A的蛋白表达，该结果提示激活腺苷A2A受体发挥着抗凋亡的作用，其中作用机制可能与VEGF-A有关。进一步构建VEGF-A的iRNA慢病毒载体，成功转染周细胞后检测发现VEGF-A表达缺失后激动腺苷A2A受体的抗凋亡现象减弱，低糖低氧培养的周细胞凋亡细胞显著增多，上述结果提示VEGF-A可能参与了腺苷A2A受体对低糖低氧培养周细胞的抗凋亡作用。最后，收集入组了不同狭窄程度的单侧颈动脉狭窄无症状患者各20人，分析了认知功能检测量表和常见血管危险因素的相关关系，并且采用VBM软件查找异常白质/灰质脑区，结果发现中重度的单侧颈动脉狭窄无症状患者即可出现认知功能障碍，血管狭窄程度是单侧颈动脉狭窄无症状患者认知功能损害的独立