摘要
【目的】研究体外培养的大鼠皮层神经元氧糖剥夺(OGD)处理后,Sema3A、Nrp-1mRNA的表达变化及其与神经元轴突网络损伤的关系。【方法】体外培养新生SD大鼠皮层神经元,随机分为正常对照组和OGD处理组:噻唑蓝(MTT)比色法测定神经元活性;βⅢ-tubulin免疫荧光染色观察轴突网络形态学变化,Real-timePCR检测Sema3A及其受体Nrp-1mRNA的表达;βⅢ-tubulin免疫荧光染色观察外源性Sema3A蛋白对神经元轴突生长的影响。【结果】随着OGD时间的延长,神经元活性呈梯度下降(P<0.01);OGD处理4h可导致神经元轴突网络发生明显的崩解损伤;OGD处理4h、6h后,Sema3AmRNA表达水平显著上调(P<0.01);OGD处理6h后,Nrp-1mRNA表达水平显著上调(P<0.01);经外源性Sema3A(5mg/mL)蛋白处理,可观察到神经元的轴突生长及延伸受到明显抑制(P<0.01)。【结论】OGD处理可诱导Sema3A、Nrp-1mRNA表达上调,并且外源性Sema3A蛋白可在体外抑制轴突生长,提示Sema3A/Nrp-1可能参与OGD处理后轴突网络崩解、细胞凋亡等病理生理过程。
[Objective] To investigate the relationship between the alteration of Sema3A and Nrp-1 mRNA expression and the neurite network damage after oxygen glucose deprivation（OGD） in cultured rat cortical neurons.[Methods] Cultured cortical neurons of newborn SD rats were randomly divided into control group and OGD treatment group.Neuronal survival rate was assayed with MTT method.Morphological changes of neurite network were observed with beta Ⅲ tubulin（βⅢ-tubulin） immunofluorescence staining.The expression of Sema3A and Nrp-1 mRNA were detected by real-time PCR.The effect of exogenous Sema3A protein on the axonal outgrowth was measured with βⅢ-tubulin immunofluorescence staining.[Results] Treated with OGD,the neuron survival rate reduced in a time-dependent manner（P 0.01）.OGD 4 h led to serious damage of neurite network.After OGD 4 h and 6 h,the mRNA expression of Sema3A obviously increased（P 0.01）;and after OGD 6 h,the mRNA expression of Nrp-1 was also significantly up-regulation（P 0.01）.The outgrowth and extension of neuron axon were obviously inhibited by the treatment with exogenous Sema3A（5 mg/mL）（P 0.01）..[Conclusion] OGD can induce up-regulation of Sema3A and Nrp-1 mRNA,and exogenous Sema3A protein inhibits axon outgrowth in vitro.Sema3A/Nrp-1 may participate in the pathophysiology process of neurite damage and neuron apoptosis in cultured neuron of OGD treatment.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2011年第2期186-190,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金(9151066302000004)
广东省自然科学基金(06021357)
