可卡因-苯丙胺调节转录肽对氧葡萄糖剥夺的培养皮质神经元突触形成的影响  

Effect of cocaine-and amphetamine-regulated transcription peptide on synaptic formation in cultured cortical neurons subjected to oxygen-glucose deprivation

作　　者：王路娜[1] 曹翔[2] 张智[2] 钱健[1] 沙杜鹃[1] Wang Luna;Cao Xiang;Zhang Zhi;Qian Jian;Sha Dujuan(Department of General Medicine,Nanjing Drum Tower Hospital,the Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China;Department of Neurology,Nanjing Drum Tower Hospital,the Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China)

机构地区：[1]南京大学医学院附属鼓楼医院全科医学科,210008 [2]南京大学医学院附属鼓楼医院神经内科,210008

出　　处：《国际脑血管病杂志》2020年第6期433-439,共7页International Journal of Cerebrovascular Diseases

基　　金：国家自然科学基金(81571122);江苏省"六大高峰人才"高层次人才项目(2016-WSN-154);南京市医学科技发展项目(YKK17067)。

摘　　要：目的探讨可卡因-苯丙胺调节转录肽(cocaine-and amphetamine-regulated transcript,CART)对氧葡萄糖剥夺(oxygen-glucose deprivation,OGD)小鼠皮质神经元突触结构的影响。方法选用健康清洁级昆明小鼠16~17 d孕龄胚胎大脑皮质进行原代神经元培养,分为对照组、CART组、OGD组以及OGD+CART组。OGD+CART组在OGD处理后加入0.4 nmol/L CART55-102培养12 h;CART组予以等剂量CART55-102。采用流式细胞术检测神经元死亡率。通过免疫荧光分析观察神经元突触结构变化,并对轴突长度和突触蛋白Ⅰ阳性面积进行定量分析。采用实时荧光定量聚合酶链反应和蛋白质印迹分析检测脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)mRNA和蛋白表达。结果与对照组比较,OGD组神经元死亡率显著增高,神经元突触生长明显受到抑制,突触蛋白Ⅰ阳性面积显著缩小,BDNF mRNA和蛋白表达水平显著下调(P均<0.05)。与OGD组比较,加入CART55-102后能显著降低OGD神经元死亡率(P<0.05),逆转OGD对神经元突触生长的抑制作用,使神经元轴突长度及突触蛋白Ⅰ阳性面积显著增大(P均<0.05),并显著上调BDNF mRNA和蛋白表达水平(P均<0.05)。结论CART能保护OGD小鼠脑皮质神经元的突触结构,其机制可能与上调BDNF表达有关。Objective To investigate the effect of cocaine-and amphetamine-regulated transcript peptide(CART)on the synapse structure of mice cortical neuron subjected to oxygen-glucose deprivation(OGD).Methods Primary neurons of the embryonic cerebral cortex obtained from healthy and clean Kunming mice at gestational age of 16-17 d were cultured.They were divided into control group,CART group,OGD group,and OGD+CART group.0.4 nmol/L CART55-102 was added and cultured for 12 h after OGD treatment in the OGD+CART group;the CART group was given the same dose of CART55-102.The neuronal mortality was measured by the flow cytometry.The changes of synaptic structure were observed by immunofluorescence analysis,and the axon length and synapsinⅠpositive area were quantitatively analyzed.Real-time fluorescent quantitative polymerase chain reaction and Western blot analysis were used to identify the brain-derived neurotrophic factor(BDNF)mRNA and protein expression.Results Compared with the control group,the mortality of neurons in the OGD group was significantly increased,the neuronal synapse growth was significantly inhibited,the positive area of synapsinⅠwas significantly reduced,and the expression levels of BDNF mRNA and protein were significantly down-regulated(all P<0.05).Compared with the OGD group,adding CART55-102 significantly reduced the mortality of OGD neurons(P<0.05),reversed the inhibitory effect of OGD on neuronal synapse growth,significantly increased the length of neuron axons and the positive area of synapsinⅠ(all P<0.05),and significantly up-regulated BDNF mRNA and protein expression levels(all P<0.05).Conclusion CART can protect the synaptic structure of mice cortical neuron subjected to OGD,and its mechanism may be related to the up-regulation of BDNF expression.

关 键 词：神经元 原代细胞培养 氧 葡萄糖 细胞死亡 突触 可卡因-苯丙胺调节转录蛋白 突触蛋白类 脑源性神经营养因子 小鼠 

分 类 号：R743.3[医药卫生—神经病学与精神病学]