慢性低灌注白质损伤CD73高表达的分子机制及其保护效应研究

Proliferation and differentiation of oligodendrocyte precursor cells (OPC) plays an important role in white matter lesions induced by chronic hypoperfusion. Adenosine promotes OPC differentiation and myelination. CD73 is the rate limiting enzyme for the production of extracellular adenosine. Our previous study suggested that CD73 expression and activity significantly increased in astrocytes in white matter lesions induced by chronic cerebral hypoperfusion and exerts protective effect; however, the specific molecular mechanisms are unknown. Our preliminary study through the bioinformatics analysis and cell experiments indicated that STAT3 may regulate the expression of CD73 in astrocytes. Further study suggested that leukemia inhibition factor (LIF), a STAT3 upstream signaling molecule significantly increased in white matter lesions induced by chronic cerebral hypoperfusion. Accordingly, we formulate a hypothesis: elevated LIF in cerebral white matter lesions could induce transcriptional factor pSTAT3 expression in astrocytes and then promote the expression of CD73. Extracellular adenosine mediated by elevated CD73 promotes OPC differentiation into mature oligodendrocytes and increase myelination. In order to prove our hypothesis, we use different molecular biological technique, such as EMSA and Chip assays to clarify the molecular mechanism of elevated CD73 in white matter lesions induced by chronic hypoperfusion and provide new strategies for the treatment of ischemic leukoencephalopathy.

少突胶质前体细胞OPC的增殖和分化在慢性低灌注白质损伤后髓鞘修复中发挥重要作用。腺苷促进OPC分化为成熟的少突胶质细胞OL，同时促进OL包绕轴突形成髓鞘。CD73是胞外腺苷产生的限速酶。我们前期研究首次发现，慢性低灌注白质损伤小鼠脑白质星形胶质细胞CD73表达和活性增高并且发挥保护作用，但具体分子机制不明。我们通过生物信息学和细胞实验初步研究表明STAT3可能调控CD73表达。进一步研究发现STAT3上游信号分子白血病抑制因子LIF在慢性低灌注白质损伤中表达增高。据此，我们假设：慢性低灌注白质损伤小鼠脑白质LIF表达增高，上调星形胶质细胞pSTAT3，启动CD73的转录，通过介导胞外腺苷的产生，促进OPC的分化和髓鞘的形成，完成受损白质再髓鞘化。本课题拟运用EMSA、Chip等技术阐明慢性低灌注白质损伤中CD73表达上调并发挥保护作用的分子机制，为缺血性脑白质损伤的治疗提供新策略和靶点

随着我国社会人口构成的老龄化，认知功能障碍对人群健康的影响日益突出。缺血性白质脑病是血管性认知障碍的主要病因之一。目前对慢性缺血性白质脑病病理机制的研究相对较少迫切需要从细胞和分子机制进一步探索缺血性白质脑病的治疗新策略和途径。既往我们发现慢性低灌注脑白质损伤小鼠胼胝体脑白质 CD73表达和活性明显增高，但是慢性低灌注脑白质损伤小鼠胼胝体脑白质星形胶质细胞CD73表达上调的机制不清楚。在本研究中，我们通过双侧颈总动脉套扎内径为0.18毫米的弹簧圈稳定构建小鼠慢性低灌注性脑白质损伤模型，八臂迷宫实验以及Y型迷宫表明慢性低灌注小鼠存在工作记忆损伤，Kluver-Barrera 染色可见模型小鼠胼胝体神经纤维稀疏、空泡形成、空隙增加，GFAP、CD11b 免疫组织化学染色显示模型小鼠胼胝体星型胶质细胞和小胶质细胞增生明显。慢性低灌注白质损伤小鼠胼胝体LIF及pSTAT3表达增加。LIF可通过促进星形胶质细胞STAT3的磷酸化，促进STAT3结合到CD73的启动子上，继而促进CD73的表达。此外，我们还发现沉默CD73加剧慢性低氧介导的星形胶质细胞炎症反应，低氧可以抑制A1AR的表达，而激活A1AR可显著抑制低氧介导的炎症反应。低氧下可促进星形胶质细胞膜上腺苷受体A1AR和A2AR形成异二聚体，导致A1AR的功能被抑制，进而导致EAAT2介导的谷氨酸摄取受到抑制。进一步研究发现异二聚体是通过上调YY1，进而使其募集HDAC1到PPARγ启动子上，抑制PPARγ的转录，进而阻止 PPARγ对EAAT2的正向调控，这可能是导致低氧/损伤下胞外谷氨酸堆积，损害神经细胞的重要机制之一。通过对腺苷产生的限速酶CD73以及腺苷受体在慢性低灌注白质损伤中的机制研究为血管性认知障碍的治疗提供可能的药物靶点