SPK1对阿尔茨海默病细胞模型的保护作用及机制研究

发布时间：2018-04-05 23:26

本文选题：鞘氨醇激酶-1　切入点：阿尔茨海默病　出处：《华中科技大学》2013年硕士论文

【摘要】：目的：探讨鞘氨醇激酶-1（sphingosine kinase-1，SPK1）对β淀粉样蛋白诱导的阿尔茨海默病细胞模型是否具有保护作用及其可能的机制。方法：（1）以β-淀粉样肽片段25-35（amyloid β-peptide fragment25-35，Aβ25-35）处理已分化的小鼠神经瘤细胞（mouse Neuro-2a，N2a）作为阿尔茨海默病的细胞模型，将不同浓度的Aβ25-35加入培养的N2a细胞24h后通过四甲基偶氮唑（MTT）法检测细胞活性；Hoechst33258染色检测细胞凋亡率；透射电镜观察细胞核的超微结构。比较各组的差异。（2）将20μM的Aβ25-35加入培养的N2a细胞24h，应用免疫印迹技术检测SPK1蛋白水平的表达变化。然后构建腺病毒载体感染细胞干预SPK1的表达，通过MTT法检测细胞活性；Hoechst33258染色检测细胞凋亡率；透射电镜观察细胞核的超微结构。比较各组的差异，用以观察调节SPK1水平对Aβ25-35诱导的神经毒性的影响。（3）应用免疫印迹技术检测上述各组凋亡相关蛋白表达；应用透射电镜观察上述各组线粒体超微结构。比较各组的差异。结果：（1）随Aβ25-35浓度增加，，细胞活性下降，细胞凋亡率增加，20μM的Aβ25-35处理分化的N2a细胞24h符合阿尔茨海默病细胞模型。（2）Aβ组与对照组相比，SPK1蛋白水平下降；Ad-SPK1+Aβ组与Aβ组相比，细胞活性增高，凋亡率下降；SPK1-siRNA+Aβ组与Aβ组相比，细胞活性下降，凋亡率增高。（3）Aβ组与对照组相比，Bax/Bcl-2比例明显增加，出现结构损坏的线粒体；Ad-SPK1+Aβ组与Aβ组相比，Bax/Bcl-2的比例下降，线粒体结构明显改善； SPK1-siRNA组与Vector组相比，Bax/Bcl-2的比例增高，出现结构损坏的线粒体。结论：Aβ25-35诱导N2a细胞SPK1表达下降，上调SPK1的表达可以减轻Aβ25-35对N2a细胞的毒性损伤，其保护作用可能与调节凋亡相关蛋白Bax与Bcl-2的表达及保护线粒体结构有关。SPK1可作为对抗Aβ25-35神经毒性作用这一个靶点，达到预防和治疗阿尔茨海默病的目的。
[Abstract]:Aim: to investigate the protective effect of sphingosine kinase-1 SPK1 (sphingosine kinase-1) on Alzheimer's disease cell model induced by 尾 -amyloid protein and its possible mechanism.Methods 25-35(amyloid 尾 -peptide fragment of 尾 -amyloid peptide fragment 25-35 A 尾 25-35) was used to treat differentiated mouse neuro-2a N2a cells as a cell model of Alzheimer's disease.Different concentrations of A 尾 25-35 were added to cultured N2a cells for 24 hours. The apoptosis rate of N2a cells was detected by Hoechst33258 staining and the ultrastructure of the cell nucleus was observed by transmission electron microscope (TEM).The difference of each group was compared. (2) A 尾 25-35 (20 渭 M) was added to N2a cells for 24 h, and the expression of SPK1 protein was detected by Western blotting.Then the adenovirus vector infected cells were constructed to interfere with the expression of SPK1, the apoptosis rate was detected by MTT assay and the ultrastructure of the nucleus was observed by transmission electron microscope.To observe the effect of regulating the level of SPK1 on the neurotoxicity induced by A 尾 25-35. The expression of apoptosis-related protein was detected by Western blot and the ultrastructure of mitochondria was observed by transmission electron microscope.The differences of each group were compared.Results with the increase of A 尾 25-35 concentration, the cell activity decreased, and the apoptosis rate increased. The differentiated N2a cells treated with A 尾 25-35 at 20 渭 M for 24 h were in accordance with the Alzheimer's disease cell model. The protein level of SPK1 in Ad-SPK1 A 尾 group was lower than that in the control group, and that in Ad-SPK1 A 尾 group was higher than that in A 尾 group, and that in Ad-SPK1 A 尾 group was higher than that in A 尾 group.Compared with A 尾 group, the cell activity of SPK1-siRNA A 尾 group decreased, and the ratio of Bax-Bax / Bcl 2 in A 尾 group was significantly higher than that in A 尾 group, and the ratio of Bax-Bax / Bcl-2 in Ad-SPK1A 尾 group with structural damage was lower than that in A 尾 group.Compared with Vector group, the ratio of Bax-Bcl-2 in SPK1-siRNA group was higher than that in Vector group, resulting in structural damage of mitochondria.Conclusion the expression of SPK1 in N2a cells was induced by 1: a 尾 25-35, and up-regulation of SPK1 could attenuate the toxicity of A 尾 25-35 to N2a cells.Its protective effect may be related to the regulation of the expression of apoptosis-related protein Bax and Bcl-2 and the protection of mitochondrial structure. SPK1 may serve as a target to antagonize the neurotoxicity of A 尾 25-35 and to prevent and treat Alzheimer's disease.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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