脑源性神经营养因子对皮层神经元的保护作用及其机制

发布时间：2018-08-06 21:11

【摘要】： 第一部分体外培养皮层神经元及显微镜形态观察目的:研究皮层神经元体外培养方法,了解其超微结构特点方法:取新生的SD大鼠,应用机械分离法取下皮层组织,剪碎并应用胰酶消化,离心取细胞悬液用含10%胎牛血清培养,12小时之内换用含2%B27无血清培养,每三天换液一次,并在显微镜下观察细胞形态结构特征结果:皮层神经元刚接种时呈圆球形,4小时后就开始长出轴突,不过有点短,12个小时轴突进一步长长,胞体立体感强,透明发亮。到第4天轴突之间建立初步的联系,第7天轴突之间已经充分建立联系,密密交织成网。结论:在培养环境稳定,无污染的条件下,12小时内换液换用2%的B27来培养就可以成功的培养神经元。第二部分脑源性神经营养因子对谷氨酸损伤的皮层神经元有保护作用目的:探讨脑源性神经营养因子(BDNF)对由谷氨酸损伤的皮层神经元有保护作用方法:分组分别建立正常对照组,谷氨酸组和BDNF组,正常对照组就是按原先的方法培养皮层神经元至第七天,谷氨酸组即为在培养至第六天的皮层神经元中加入50μmol/L谷氨酸,30min后原培养基继续培养1天。BDNF组是在培养第5天的皮层神经元的培养液中加入50 ng/ml的BDNF,作用24小时后换用含50μmol/L的谷氨酸培养基,30min后原培养基培养24小时备检测。用AO/EB免疫荧光染色法在荧光显微镜下观察细胞形态上的变化,应用MTT来检测细胞凋亡率的改变。结果:谷氨酸组可见细胞突起皱缩、断裂,并可见凋亡细胞产生,由于细胞膜的通透性被破坏,被EB染成红色可见细胞核固缩状或呈片段状(图3C)。BDNF组:破坏的细胞相对较少。MTT显示:BDNF组的细胞凋亡较谷氨酸组明显减低. MTT空白对照组(1.26±0.06)相比,谷氨酸组细胞活力(0.72±0.10)显著降低(P0.05);而与谷氨酸组比较,BDNF组(1.14±0.06)可显著提高细胞活力(P0.01)。谷氨酸组细胞凋亡比BDNF组明显增多(P㩳0.05)。讨论:BDNF能够保护皮层神经元免受谷氨酸损伤第三部分BDNF保护皮层神经元的作用机制研究目的:探讨BDNF保护皮层神经元免受谷氨酸损伤的机制方法:将培养的细胞分为正常对照组,BDNF组,谷氨酸组,分别将各组细胞研磨,提取蛋白,用western-blot的方法检测BDNF的低亲和力受体p75NTR,下游信号分子JNK,ERK。结果:BDNF组与谷氨酸组相比,p75NTR表达无明显差异,BDNF组与谷氨酸组相比JNK表达明显降低,ERK表达明显增加。讨论:BDNF能够保护谷氨酸引起的大脑皮层神经元的损伤,其机制是通过调节神经元受体之间的比例,上调ERK,下调JNK,从而促进细胞的存活,降低细胞的凋亡。
[Abstract]:Part one: morphological observation of cortical neurons in vitro and microscopic observation objective: to study the methods of cortical neurons culture in vitro and to understand the ultrastructural characteristics of cortical neurons. The cortical tissue was removed by mechanical separation, the tissue was shredded and digested by trypsin, the cell suspensions were obtained by centrifugation and cultured with 10% fetal bovine serum for 12 hours with 27 serum-free culture. The morphological and structural characteristics of the cells were observed under microscope: the cortical neurons began to grow into axons 4 hours after inoculation, but the axons were a little short, and the axons were longer at 12 hours and had a strong sense of stereosome. Transparent and shiny. By the 4th day, the primary connection between axons was established, and on the 7th day, the connections between axons were fully established and closely intertwined into networks. Conclusion: under the condition of stable culture environment and no pollution, the neurons could be cultured successfully by using 2% B27 in 12 hours. Part two the protective effect of brain-derived neurotrophic factor (BDNF) on cortical neurons injured by glutamate objective: to investigate the protective effect of brain-derived neurotrophic factor (BDNF) on cortical neurons injured by glutamate. Methods: normal control group was established respectively. The glutamate group and the BDNF group, the normal control group, cultured cortical neurons in the same way until the seventh day. Glutamic acid group added 50 渭 mol/L glutamate to cortical neurons for 30 minutes after culture. BDNF group continued to culture for 1 day. BDNF group added 50 ng/ml BDNF into the culture medium of cortical neurons on the 5th day. After 24 hours of exposure, BDNF was added to the culture medium of cortical neurons. The glutamate medium containing 50 渭 mol/L was used for 30 minutes and the original medium was cultured for 24 hours. AO/EB immunofluorescence staining was used to observe the changes of cell morphology under fluorescence microscope and MTT was used to detect the change of apoptosis rate. Results: in Glutamic acid group, the processes were crumpled, broken, apoptotic cells were produced, and the permeability of cell membrane was destroyed. The nuclei were pyknotic or segmented by EB staining (fig. 3C) .BDNF group: the number of damaged cells was relatively small. MTT showed that the apoptosis of cells in the WBDNF group was significantly lower than that in the glutamic acid group. Compared with the control group (1.26 卤0.06), the cell viability of glutamate group (0.72 卤0.10) was significantly decreased (P0.05), while that of MTT group (1.14 卤0.06) was significantly higher than that of glutamate group (P0.01). Apoptosis in glutamate group was significantly higher than that in BDNF group (P0. 05). Study on the mechanism of BDNF protecting cortical neurons from glutamic acid damage part 3: objective: to explore the protective effects of BDNF on cortical neuron immunity Mechanism of Glutamic Acid injury: cultured cells were divided into normal control group and BDNF group. In the glutamate group, the cells of each group were ground, the protein was extracted, the low affinity receptor p75NTR of BDNF was detected by western-blot, and the downstream signal molecule JNKN ERK was detected. Results there was no significant difference in the expression of p75NTR between the two groups. The expression of JNK in the BDNF group was significantly lower than that in the glutamate group. It is discussed that: BDNF can protect cerebral cortical neurons from glutamic acid-induced damage by regulating the ratio of neuron receptors, upregulating ERK and down-regulating JNKs, thus promoting cell survival and reducing cell apoptosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R33

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