摘要
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16 d)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,q RT-PCR检测SYK和凋亡相关的原癌基因Fra-1 m RNA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 m RNA(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。
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 easpase-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 immunofluorescenee assay. Anoxia/hypoglycemia injury induced increased SYK expression in the neurons （ P 〈 0.01 ）. SYK silencing by RNA interference significantly reduced easpase-3 activity （ P 〈 0.05 ） and cell apoptosis （ P 〈 0. 01 ）, while the mRNA （ P 〈 0.01 ） andprotein （ 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 co- silencing of SYK and Fra-1. Conclusion Fra-I 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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2017年第13期1381-1386,共6页
Journal of Third Military Medical University
关键词
SYK
Fra-1
缺氧/缺糖损伤
大脑皮质神经元
凋亡
spleen tyrosine kinase
Fra-1
anoxia/hypoglycemia injury
cerebral cortical neurons
apoptosis
