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目的探讨新型P2Y样G蛋白偶联受体GPR17对脑缺血及缺氧缺糖(OGD)诱导皮层混合培养细胞中神经元损伤及小胶质细胞激活的影响。方法①以线栓法制备大鼠局灶性脑缺血模型,采用免疫组织化学、Western blotting、RT-PCR以及免疫荧光等方法观察脑内GPR17的时空表达及细胞分布特点。大鼠侧脑室埋管给予GPR17 siRNA靶向沉默脑内GPR17表达,观察其对脑缺血急、慢性期神经元损伤和小胶质细胞激活的影响。②以OGD诱导大鼠皮层混合培养细胞的缺血性损伤,以GPR17 siRNA靶向沉默GPR17的表达,观察其对OGD诱导的原代皮层混合培养细胞中神经元损伤和小胶质细胞激活的影响。结果①缺血中心区,GPR17 mRNA及蛋白水平在再灌注24 h和7,14 d表达上调;缺血周边区,再灌注7,14 d表达上调。在正常大鼠脑组织,GPR17主要表达于神经元、少突胶质细胞。在缺血中心区,再灌注24 h GPR17主要表达于损伤的神经元、小胶质细胞和少突胶质细胞,再灌注14 d主要表达于增生激活的小胶质细胞,部分表达于少突胶质细胞;而在缺血周边区,再灌注24 h以及14 d,GPR17主要表达于神经元、小胶质细胞和少突胶质细胞。星形胶质细胞不表达GPR17。大鼠侧脑室给予GPR17 siRNA成功抑制脑内GPR17表达,显著改善再灌注24 h神经症状、减少脑梗死体积以及神经元损伤;同样,也改善再灌注14 d的脑萎缩和周边区的神经元损伤,并显著抑制小胶质细胞的增生激活。②大鼠原代皮层混合培养细胞中,OGD 1 h恢复24 h(OGD/R)诱导细胞活性降低,LDH释放增加,PI染色结果显示细胞坏死增加,以神经元死亡为主。GPR17 siRNA处理后减轻OGD/R诱导的细胞活性下降以及LDH释放,并减轻细胞坏死,以减轻神经元死亡为主;GPR17siRNA处理后能够改善小胶质细胞激活的形态变化。结论大鼠局灶性脑缺血后,脑内GPR17表达上调,介导缺血后急性神经元损伤以及亚急性/慢性期的小胶质细胞激活。GPR17还介导OGD诱导的大鼠皮层混合培养细胞中的神经元损伤和小胶质细胞激活。
Objective To investigate the effects of a novel P2Y-like G-protein coupled receptor GPR17 on neuronal damage and microglial activation in cultured cortical neurons induced by cerebral ischemia and oxygen-glucose deprivation (OGD). Methods ① The rat model of focal cerebral ischemia was established by thread occlusion. The temporal and spatial expression of GPR17 and the distribution of cells were observed by immunohistochemistry, Western blotting, RT-PCR and immunofluorescence. GPR17 siRNA was administered to the lateral ventricle of rats to silence the expression of GPR17 in the brain, and the effect of GPR17 siRNA on neuronal damage and microglial activation in acute and chronic phase of cerebral ischemia was observed. ② OGD-induced ischemic injury in rat cortical mixed culture cells, GPR17 siRNA targeted silencing of GPR17 expression was observed OGD-induced primary cortical mixed cultured cells in neuronal damage and microglial activation . Results ① In the ischemic center, the expression of GPR17 mRNA and protein was up-regulated at 24 h and 7 and 14 d after reperfusion. The expression of GPR17 mRNA and protein was up-regulated in the ischemic peripheral area at 7 and 14 d after reperfusion. In normal rat brain tissue, GPR17 is mainly expressed in neurons and oligodendrocytes. In ischemic center, GPR17 was mainly expressed in injured neurons, microglia and oligodendrocytes 24 h after reperfusion, and mainly expressed in proliferative activated microglia at 14 d after reperfusion, and partly expressed in oligodendrocytes Glial cells; while in the ischemic peripheral area, 24 h and 14 d after reperfusion, GPR17 mainly expressed in neurons, microglia and oligodendrocytes. Astrocytes do not express GPR17. Administration of GPR17 siRNA to the lateral ventricle in rats inhibited the expression of GPR17 in the brain and markedly improved the neurological symptoms at 24 h after reperfusion and decreased the volume of cerebral infarction and neuronal damage. Similarly, it also ameliorated the brain atrophy and peripheral neuronal damage 14 d after reperfusion , And significantly inhibited the proliferation of microglia activation. ② OGD induced a decrease of cell viability and LDH release after OGD recovery for 1 h at 1 h in rat primary cortical mixed culture cells, PI staining showed that cell death was increased and neuronal death was predominant. GPR17 siRNA treatment reduced the OGD / R-induced decreased cell viability and LDH release, reduced cell necrosis and decreased neuronal death. GPR17siRNA treatment could improve the morphological changes of microglial activation. Conclusion After focal cerebral ischemia in rats, the expression of GPR17 in the brain is up-regulated, which mediates the injury of acute neurons after ischemia and microglia activation in the subacute / chronic phase. GPR17 also mediates neuronal damage and microglial activation in OGD-induced rat cortical mixed culture cells.