体外培养大脑皮质神经元加入淀粉样β蛋白25～35诱导细胞周期蛋白的异常表达

Abnormal expression of cyclin in cerebral cortical neurons cultured in vitro induced by beta-amyloid 25-35

目的:观察加入神经毒性较强的淀粉样β蛋白25～35多肽片断诱导神经元表达细胞周期相关蛋白的变化,以及细胞周期相关蛋白表达与神经损伤之间的关系。方法:实验于2004-10/12在武汉大学医学院神经生物学实验室完成。取妊娠18d昆明小鼠,在无菌条件下分离胚脑皮质,制成单细胞悬液,在有血清培养基中培养。神经细胞进行神经元特异性烯醇化酶免疫荧光检测呈阳性后,第4天实验组加入4μL淀粉样β蛋白25～35(2g/L)继续培养,对照组加入4μL生理盐水,在第7天行免疫荧光检测,并分析细胞周期相关蛋白A和B1的表达。结果:实验组加入淀粉样β蛋白25～35继续培养3d后,神经元行细胞周期相关蛋白A和B1免疫荧光染色,神经元内分别出现蓝色颗粒(FITC荧光素标记二抗)和红色颗粒(Cy3荧光素标记二抗),即细胞周期相关蛋白A、细胞周期相关蛋白B1在神经元内呈阳性表达。对照组细胞周期相关蛋白A和B1在神经元内呈阴性表达。结论:在体外神经细胞培养过程中,加入淀粉样β蛋白25～35后,细胞周期相关蛋白A和B1在神经元内异常表达,提示淀粉样β蛋白的毒性机制中有细胞周期机制或部分机制的异常激活。

AIM:To observe the changes of cyclin expressing neurons induced by betaamyloid 25-35 and study the association of cyclin expression with neural injury.METHODS:The experiment was finished in the Laboratory of Neurobiology, Wuhan University from October to December 2004.We isolated the cells from the cerebral cortex of 18day cyetic mice and then planted them into culture medium with growth factors and serum in experimental group, and added 4 μL betaamyloid 25-35(2 g/L) in it at the 4th day after primary culture, whereas the control group was added with 4 μL saline,and detected by using immunofluorescence method at the 7th day.The cortical neurons and the expression of cyclin were evaluated with neuronspecific enolase immunocytochemistry to analyze the expression of cyclin A and cyclin B1,respectively.RESULTS:①The cyclin A and cyclin B1 of neurons were detected by using immunofluorescence;blue granules(the secondary antibody was labeled by FITC fluorescence) and red granules(the secondary antibody was labeled by Cy3 fluorescence) were found in the neuronal cells at the 3rd day after induced with betaamyloid 25-35 in the experimental group,which indicated the positive expression of cyclin A and cyclin B1.②The cyclin A and cyclin B1 expressed negatively in control group.CONCLUSION:Betaamyloid 25-35 can induce the abnormal expression of cyclin A and cyclin B1 in cortical neurons cultured in vitro,which indicates that the toxic mechanism of betaamyloid 25-35 is related to abnormal activation of cells.