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目的探讨RIP1/3对创伤性脑损伤(TBI)后神经元的影响及其作用机制。方法将体外培养的皮层神经元分为4组:siRIP组、Nec-1预处理组、阴性质粒转染组和对照组。通过慢病毒转染法分别干涉RIP1、RIP3表达,建立离体TBI损伤模型,通过流式细胞学检测划伤后神经元存活情况。对体外培养的皮层神经元敲除RIP1/3,建立谷氨酸诱导神经元损伤模型,通过流式细胞学检测谷氨酸刺激后神经元存活情况。结果 siRIP组及Nec-1预处理组机械性损伤后神经元存活率高于阴性质粒转染组和对照组。Nec-1预处理组谷氨酸损伤后神经元存活率高于对照组,而siRIP组存活率与对照组和阴性转染组比较未见显著差异。结论 RIP1和RIP3可能对TBI诱导后神经元死亡有作用,而RIP1抑制剂Nec-1可能对TBI具有脑保护作用。RIP1/3与谷氨酸兴奋毒性诱导细胞死亡无关,而Nec-1对于谷氨酸损伤具有潜在保护作用,并可能存在特异性靶点。
Objective To investigate the effect of RIP1 / 3 on neurons after traumatic brain injury (TBI) and its mechanism. Methods Cortical neurons cultured in vitro were divided into four groups: siRIP group, Nec-1 preconditioning group, negative plasmid transfection group and control group. The expressions of RIP1 and RIP3 were respectively intervened by lentiviral transfection, and the injury model of TBI was established. The survival of the neurons was detected by flow cytometry. RIP1 / 3 was knocked out in cultured cortical neurons in vitro, and glutamate-induced neuronal injury was established. Neuronal survival after glutamate stimulation was detected by flow cytometry. Results The survival rates of neurons after mechanical injury in siRIP and Nec-1 pretreatment groups were higher than those in negative plasmid transfected and control groups. Nec-1 pretreatment group glutamate injury neuronal survival rate was higher than the control group, and siRIP group survival rate compared with the control group and negative transfected group no significant difference. Conclusion RIP1 and RIP3 may play an important role in the neuronal death after TBI induction, while RIP1 inhibitor Nec-1 may have a protective effect on TBI. RIP1 / 3 is not associated with glutamate-induced excitotoxicity, whereas Nec-1 has a potential protective effect on glutamate damage and may have specific targets.