SYK调控缺氧/缺糖损伤诱导的大脑皮质神经元凋亡及其机制

目的 探讨抑制脾酪氨酸激酶(spleen tyrosine kinase,SYK)对缺氧/缺糖损伤诱导的大脑皮质神经元的凋亡的影响及相关机制.方法 分离培养10只SD怀孕大鼠(190 ～230 g,胎龄16d)胎鼠的大脑皮质神经元细胞并用MAP2免疫荧光法进行鉴定.建立缺氧/缺糖损伤的体外模型,caspase-3荧光检测试剂盒检测caspase-3活性,Annexin-V FITC/PI法检测细胞凋亡,qRT-PCR检测SYK和凋亡相关的原癌基因Fra-1 mRNA的表达,Western Blot检测SYK、Bcl-2和Fra-1蛋白的表达.Control组:无转染处理的缺氧/缺糖损伤的神经元;SYK siRNA组:转染SYK siRNA的缺氧/缺糖损伤的神经元;SYK-Fra-1 siRNA组:同时转染SYK siRNA和Fra-1 siRNA的缺氧/缺糖损伤的神经元;non-specific siRNA组:转染non-specific siRNA的氧/缺糖损伤的神经元.结果 MAP2免疫荧光鉴定为神经元细胞.缺氧/缺糖损伤诱导神经元SYK表达(P＜0.01),RNA干扰抑制SYK表达后caspase-3活性(P＜0.05)和细胞凋亡(P＜0.01)均显著降低,但是Fra-1 mRNA (P ＜0.01)和蛋白(P＜0.05)表达量明显上升.用RNA干扰技术同时抑制SYK和Fra-1后,caspase-3活性(P＜0.01)显著上升但Bcl-2蛋白表达(P＜0.01)显著降低.结论 Fra-1介导了SYK对缺氧/缺糖损伤诱导的神经元凋亡的调节,为脑卒中所致的神经元缺血性损伤的治疗提供了新的靶点.

Spleen tyrosine kinase regulates anoxia/hypoglycemia-induced apoptosis in cultured cerebral cortical neurons

Objective To investigate the effect of inhibiting spleen tyrosine kinase (SYK) on apoptosis in cerebral cortical neuron induced by anoxia/hypoglycemia (A/H) injury and explore the related mechanism.Methods The fetal rat cerebral cortical neurons were isolated from 10 pregnant SD rats (weighting 190 ～ 230 g,gestational age of 16 d),cultured and then identified by MAP2 immunofluorescence assay.The cerebral cortical neuron model with A/H injury was established in vitro.Then the cells were divided into A/H injured neurons (control group),A/H injured neurons with SYK siRNA transfection (SYK siRNA group),A/H injured neurons with co-transfection of SYK siRNA and Fra-1 siRNA (SYK-Fra-1 siRNA group),and A/H injured neurons with non-specific siRNA transfection (non-specific siRNA group).Caspase-3 activity was detected by the caspase-3 kit.Cell apoptosis was determined by Annexin-V FITC/PI assay.The mRNA expressions levels of SYK and apoptosis-related proto-oncogene Fra-1 were measured by qRT-PCR.The protein levels of SYK,Bcl-2 and Fra-1 were detected by using Western blotting.Results The neurons were identified by MAP2 immunofluorescence assay.Anoxia/hypoglycemia injury induced increased SYK expression in the neurons (P ＜ 0.01).SYK silencing by RNA interference significantly reduced caspase-3 activity (P ＜ 0.05) and cell apoptosis (P ＜ 0.01),while the mRNA (P ＜ 0.01) and protein (P ＜ 0.05) levels of Fra-1 were obviously up-regulated.Caspase-3 activity was significantly increased (P ＜ 0.01) and Bcl-2 protein expression was markedly decreased (P ＜ 0.01) in the cells after the cosilencing of SYK and Fra-1.Conclusion Fra-1 mediates SYK regulation in the apoptosis of cerebral cortical neurons induced by A/H injury,which provides the new target for A/H injury in neurons caused by cerebral stroke.