磁刺激对小鼠海马原代神经元即刻早期基因和细胞骨架蛋白表达的影响  

Influence of magnetic stimulation on the expression of immediate early genes and cytoskeletal protein in primary cultured mouse hippocampal neurons

作　　者：张展翅[1,2] 马隽[1,2,3] 栾峰[4] 康林[1] 苏玉红[1] 王彦永[2] 王铭维[1,2] 崔慧先[1,2] 

机构地区：[1]河北医科大学解剖学教研室,石家庄050017 [2]河北省脑老化与认知神经科学实验室,石家庄050031 [3]河北体育学院,石家庄050041 [4]河北医科大学第三医院,石家庄050051

出　　处：《解剖学杂志》2012年第3期327-331,369,F0004,共7页Chinese Journal of Anatomy

基　　金：国家973计划前期研究专项（2010CB535005）

摘　　要：目的：观察磁刺激对小鼠海马原代神经元c-fos、活性调节的细胞骨架蛋白（Arc）和微管相关蛋白2（MAP2）的表达及神经元突起生长的影响，探讨磁刺激对细胞突起生长的影响机制。方法：体外培养的海马原代神经元，分为对照组，假刺激组，20％（20％强度组）、30％（30％强度组）、40％最大刺激强度组（40％强度组），24h后开始刺激，频率1Hz，最大输出强度3．7T。连续刺激5d后应用荧光显色检测c-fos、Arc和MAP2阳性神经元免疫荧光强度，计数MAP2阳性神经元多突起神经元个数及细胞突起长度，并应用免疫印迹和RT-PCR技术对免疫荧光显色结果进行验证。结果：磁刺激能促进小鼠海马原代神经元突起数目和长度的生长，3个强度组多突起神经元（n≥2）占总神经元的比例以及海马原代神经元突起长度均高于对照组，且30％强度组高于20％、40％强度组，结果有统计学意义。免疫荧光显色30％强度组c-fos阳性神经元比例、Arc和MAP2免疫荧光强度高于相关对照组，差异有统计学意义。免疫印迹和RT-PCR结果同免疫荧光显色结果一致。结论：磁刺激能促进体外培养的海马原代神经元突起生长，其机制可能和c-fos、Arc和MAP2的表达上调有关。Objective： To observe the effects of magnetic stimulation on the expression of c-fos, activity regulated cytoskeletal protein, microtubule-associated protein 2 and neurite growth in primary cultured mouse hippocampal neurons, and to explore the possible mechanism of magnetic stimulation on cell neurite growth. Methods： The primary cultured hippocampal neurons were divided into five groups： control group, sham group, 200% maximum stimulus intensity group （200/00 intensity）, 300/00 maximum stimulus intensity group （30% intensity）, and 40% maximum stimulus intensity group （40% intensity）. The neurons were stimulated at a rate of 1 Hz after 24h, and the maximum output intensity of magnetic field was 3.7 Tesla. Continuous stimulation for 5d, the stimulation coil was held paralleled 1 cm above the dish. Cellular immunofluorescence staining was executed immediately in the fifth day after stimulation; the immunofluorescence intensity of cffos, Arc and MAP2-positive neurons was detected, and the multiple neurites neurons and the cell neurite length of MAP2 positive neurons were counted. Meanwhile, semiquantitative RT-PCR and Western blotting were applied to verify the results. Results： The per- centage of multiple neurites neurons（n^2）and the cell neurite length in each stimulation group was significantly higher than that in the control group. Meanwhile, it was higher in the 30% intensity group than that in 20% and 40% intensity groups, and there was significant difference in the results. C-fos positive neurons proportion and the immunofluorescence intensity of Arc and MAP2 in the 30% intensity group was significantly higher than that in the related control group. The results of Western blotting and RT-PCRwere consistent with the immunofluorescence. Conclusion： Magnetic stimulation can promote the neurite outgrowth of primary cultured hippocampus neuron, and the mechanism may be related to the upregulation of c-los, Are and MAP2.

关 键 词：磁刺激 海马神经元 免疫荧光 c-fos活性调节的细胞骨架蛋白 微管相关蛋白2 

分 类 号：R454.1[医药卫生—治疗学]