糖浓度增加致海马神经元MLCK升高对细胞微丝骨架的影响

发布时间：2018-03-22 09:08

本文选题：细胞原代培养　切入点：海马神经元　出处：《贵州医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:原代培养SD新生鼠海马神经元,倒置显微镜观察海马神经元的形态,透射电镜观察其超微结构;Western blotting检测MLCK、p-MLC表达水平;免疫荧光染色,激光共聚焦显微镜下观察F-actin的变化;探讨糖浓度增加海马神经元中MLCK升高对神经元微丝骨架的影响,推测其改变在糖尿病认知功能障碍中的机制。方法:1.原代培养SD新生鼠海马神经元及其纯度鉴定:无菌条件下分离SD新生鼠海马并进行原代培养,倒置相差显微镜观察细胞形态;每2天采用CCK-8检测海马神经元活性,细胞培养至第7天,NSE免疫组化染色鉴定其纯度;2.实验分组:对照组、高糖组、ML-7组。其中,对照组用葡萄糖浓度为25mmol/L的培养基培养;高糖组细胞培养至第5天换用糖浓度为45mmol/L培养基培养48小时;ML-7组在高糖作用的同时加入MLCK抑制剂ML-7(20μmol/L)作用8小时。3.Western blot检测各组细胞内MLCK、p-MLC表达水平。4.透射电镜观察各组海马神经元超微结构改变。5.进行免疫荧光染色,激光共聚焦显微镜下观察各组微丝成分F-actin的变化。结果:1.成功培养海马神经元,倒置相差显微镜下观察培养7d的神经元形态成熟,体积较大,聚集存活,光晕明显;突起发达并向外延伸,互相连接形成密集交错的网络。且培养7天的海马神经元细胞活性达到最高,之后产生短暂的平台期,11d后活性逐渐下降,经鉴定细胞纯度达90%以上;2.与对照组相比,高糖组细胞内p-MLC、MLCK均上调(P0.05),ML-7组p-MLC、MLCK均较高糖组表达下调(P0.05);3.与对照组相比,高糖组海马神经元突触缩短,超微结构显示海马神经元细胞核膜皱缩,细胞核形态不规则,核内染色质分布不均、颗粒化、凝集成块并边集,细胞内质网,线粒体有囊泡化扩张,伴随有自噬现象的产生,且微丝成分减少;与高糖组相比,ML-7组在细胞及线粒体方面无明显变化,细胞中出现极少自噬现象,内质网等细胞器改变不大,微丝更丰富。4.免疫荧光显示高糖组细胞形态改变,F-actin发生解聚、重排,且轴突、树突退缩;与高糖组比较ML-7组细胞形态完整,F-actin带清晰。结论:1.糖浓度增加使海马神经元内MLCK、p-MLC表达增加;2.糖浓度增加导致海马神经元形态及超微结构发生改变;3.MLCK和p-MLC表达升高,引起F-actin解聚,神经元细胞骨架重排;MLCK抑制剂ML-7可阻断F-actin的解聚。
[Abstract]:Objective: to observe the morphology of hippocampal neurons in primary cultured neonatal SD rats, observe the morphology of hippocampal neurons by inverted microscope, detect the expression of MLCK-p-MLC by transmission electron microscopy (TEM), detect the expression of MLCK-p-MLC by Western blotting, and observe the changes of F-actin by immunofluorescence staining and confocal laser microscopy. To investigate the effect of increased glucose concentration on the microfilament skeleton in hippocampal neurons. Methods 1. Primary culture of neonatal SD rat hippocampal neurons and their purity identification: isolation and primary culture of SD newborn rat hippocampus under aseptic condition. The morphology of the cells was observed by inverted phase contrast microscope, the activity of hippocampal neurons was detected by CCK-8 every 2 days, and the purity of neurons was identified by immunohistochemical staining on the 7th day of culture. The experiment was divided into two groups: control group, high glucose group and ML-7 group. The control group was cultured on 25mmol/L medium with glucose concentration. Cells in high glucose group were cultured on 45mmol/L medium for 48 hours. ML-7 group was treated with MLCK inhibitor ML-7(20 渭 mol / L for 8 hours. 3. Western blot was used to detect the expression level of MLCKp-MLC. 4. Transmission electron microscope was used to observe the expression of MLCKp-MLC in cells of each group. The ultrastructural changes of hippocampal neurons in group A. 5. immunofluorescence staining, Results 1. Hippocampal neurons were successfully cultured. The neurons cultured under inverted phase contrast microscope were observed to be mature in shape, large in size, aggregating and surviving, and obvious halo. The neurites developed and extended outward, connected with each other to form a dense interlaced network. The activity of hippocampal neurons reached its highest level after 7 days of culture, and then decreased gradually after 11 days of transient plateau phase. The purity of the cells was over 90%. Compared with the control group, the expression of p-MLCnMLCK in the high glucose group was significantly up-regulated than that in the high glucose group. Compared with the control group, the synapses of hippocampal neurons in the high glucose group were shorter than those in the high glucose group, and the expression of p-MLCU MLCK in the ML-7 group was significantly lower than that in the high glucose group. The ultrastructure showed that the nuclear membrane of hippocampal neurons shrank, the shape of nucleus was irregular, the distribution of chromatin in nucleus was uneven, granulation, agglutination, endoplasmic reticulum, cysts and mitochondria, accompanied by autophagy. Compared with high glucose group, ML-7 group had no obvious changes in cell and mitochondria, few autophagy in cells, and little change in endoplasmic reticulum and other organelles. Immunofluorescence showed that F-actin was depolymerized, rearranged, axon and dendritic retreated in high glucose group. Conclusion 1. The increase of glucose concentration can increase the expression of MLCKP-MLC in hippocampal neurons. 2. The increase of glucose concentration leads to changes in morphology and ultrastructure of hippocampal neurons. 3. The expression of MLCK and p-MLC increases and F-actin deaggregates. Neuronal cytoskeleton rearrangement (MLCK) inhibitor ML-7 can block the depolymerization of F-actin.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2;R749.1

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