记忆相关基因KIBRA抑制Aβ诱导的神经元凋亡研究

发布时间：2018-08-15 19:14

【摘要】：目的:为了明确记忆相关基因KIBRA在阿尔茨海默病(Alzheimer’s disease,AD)转基因动物模型中是否存在表达改变及及其对神经元凋亡的影响,进而探讨KIBRA如何参与Aβ诱导的神经元凋亡及其相关机制。方法:本研究首先通过免疫组织染色及免疫蛋白印迹的方法,观察在不同月龄APP/PS1小鼠中是否存在KIBRA的表达改变。其次,通过TUNEL免疫荧光染色,观察在KIBRA基因敲除小鼠中是否存在神经元凋亡。最后,我们使用CRISPR/CAS9慢病毒及过表达慢病毒分别转染HT22细胞,建立KIBRA基因敲除和KIBRA过表达细胞模型,进而探讨KIBRA对Aβ诱导的神经元凋亡及其相关机制的研究。结果:(1)KIBRA在APP/PS1转基因小鼠脑内特异性表达:选用9月龄及12月龄APP/PS1小鼠作为AD研究模型,通过KIBRA免疫组织染色分析,无论整个海马,还是海马亚区CA1区和CA3区,9月龄和12月龄APP/PS1小鼠KIBRA阳性细胞数量均明显低于对照组,其中12月龄尤为显著(P0.01,P0.001)。免疫蛋白印迹结果同样证实12月龄APP/PS1小鼠海马区KIBRA的表达明显低于野生型小鼠(P0.001)。随着月龄的增加,KIBRA在APP/PS1转基因小鼠海马区的表达逐渐减少,提示KIBRA在AD小鼠海马区的特异性降低,与学习记忆障碍密切相关。(2)KIBRA在神经元凋亡中的重要作用:通过TUNEL荧光染色发现,与野生型小鼠相比,12月龄APP/PS1小鼠海马神经元凋亡比例明显增多(P0.01,P0.01)。4月龄KIBRA基因敲除小鼠海马神经元凋亡比例比野生型小鼠明显增加(P0.05),提示KIBRA在神经元凋亡的发生中扮演不可或缺的角色。(3)KIBRA对Aβ诱导的神经元凋亡的保护作用:CCK8及免疫蛋白印迹结果显示,与空病毒组相比,经1μmol·L~(-1)的Aβ1-42寡聚体处理后的KIBRA敲除组细胞活力明显降低(P0.01);凋亡相关蛋白(剪切的PARP、活化的caspase3)的表达量明显增高(P0.05,P0.01)。KIBRA过表达组细胞存活率明显优于空病毒组(P0.05),凋亡相关蛋白较空病毒组显著减少(P0.05),进一步提示KIBRA参与抑制Aβ诱导的神经元凋亡,促进细胞增殖和存活。4.KIBRA通过激活Akt信号通路抑制Aβ诱导的神经元凋亡:本实验筛选凋亡相关信号通路,结果显示,在1μmol·L~(-1) Aβ1-42寡聚体处理1 min后,KIBRA敲除组Akt Ser473位点磷酸化水平比空病毒组显著降低(P0.05)。在Aβ1-42寡聚体处理1分钟后,KIBRA过表达组Akt Ser473位点磷酸化明显被激活,Akt Ser473位点磷酸化水平比空病毒组显著增高(P0.01)。此外,与Aβ1-42寡聚体干预组相比,Akt特异性抑制剂(MK2206)预处理组KIBRA过表达细胞存活率明显降低(P0.05);剪切的PARP表达量明显增高(P0.05)。以上结果表明KIBRA通过激活Akt Ser473位点的磷酸化水平,抑制Aβ诱导的神经元凋亡。结论:KIBRA作为一种神经保护因子,通过激活Akt信号通路抑制Aβ诱导的神经元凋亡,促进细胞增殖及存活,为AD发病机制及治疗药物的研究提供了新的靶点。
[Abstract]:Aim: to investigate the expression of memory associated gene KIBRA and its effect on neuronal apoptosis in Alzheimer's disease AD transgenic animal models. Then it is discussed how KIBRA participates in A 尾 -induced neuronal apoptosis and its related mechanism. Methods: the expression of KIBRA in APP/PS1 mice of different months was observed by immunohistochemistry and Western blot. Secondly, TUNEL immunofluorescence staining was used to observe whether there were neuronal apoptosis in KIBRA knockout mice. Finally, CRISPR/CAS9 lentivirus and overexpression lentivirus were used to transfect HT22 cells respectively to establish KIBRA gene knockout and KIBRA overexpression cell models, and then to investigate the mechanism of apoptosis induced by KIBRA in A 尾 -induced neurons. Results: (1) the specific expression of KIBRA in the brain of APP/PS1 transgenic mice: 9 months old and 12 months old APP/PS1 mice were selected as AD research model, and analyzed by KIBRA immunohistochemical staining, regardless of the whole hippocampus, The number of KIBRA positive cells in 9 and 12 month old APP/PS1 mice was significantly lower than that in the control group, especially in 12 month old APP/PS1 mice (P0.01, P0.001). Western blot also confirmed that the expression of KIBRA in hippocampus of 12 month old APP/PS1 mice was significantly lower than that of wild type mice (P0. 001). The expression of KIBRA in hippocampus of APP/PS1 transgenic mice decreased with the increase of age, suggesting that the specificity of KIBRA in hippocampus of AD mice was decreased. (2) the important role of KIBRA in neuronal apoptosis: it was found by TUNEL fluorescence staining, The percentage of apoptosis of hippocampal neurons in 12-month-old APP/PS1 mice was significantly higher than that in wild-type mice (P0.01, P0.01). The percentage of apoptosis of hippocampal neurons in KIBRA knockout mice was significantly higher than that in wild-type mice (P0.05), suggesting that KIBRA was involved in the development of neuronal apoptosis. (3) the protective effect of KIBRA on A 尾 -induced apoptosis of neurons: CCK8 and Western blot showed that, Compared with the empty virus group, The cell viability of KIBRA knockout group treated with 1 渭 mol L ~ (-1) A 尾 1-42 oligomer was significantly decreased (P0.01), and the expression of apoptosis-related protein (P0.05, P0.01) was significantly increased in KIBRA knockout group (P0.05, P0.01). The cell survival rate of KIBRA overexpression group was significantly better than that of empty virus group (P0.05). The protein level was significantly lower than that in the empty virus group (P0.05), which suggested that KIBRA was involved in the inhibition of A 尾 -induced neuronal apoptosis. Promote cell proliferation and survival. 4. KIBRA inhibits neuronal apoptosis induced by A 尾 by activating Akt signaling pathway. After 1 min treatment with 1 渭 mol L ~ (-1) A 尾 1-42 oligomer, the phosphorylation level of Akt Ser473 site in KIBRA knockout group was significantly lower than that in empty virus group (P0.05). After treatment with A 尾 1-42 oligomer for 1 minute, the phosphorylation level of Akt Ser473 site in KIBRA overexpression group was significantly higher than that in empty virus group (P0.01). In addition, compared with the A 尾 1-42 oligodepressant intervention group, the survival rate of KIBRA overexpression cells was significantly decreased in MK2206 preconditioning group (P0.05), and the expression of shearing PARP was significantly increased (P0.05). These results suggest that KIBRA inhibits the apoptosis of neurons induced by A 尾 by activating the phosphorylation level of Akt Ser473 sites. Conclusion as a neuroprotective factor, the Akt signaling pathway inhibits the apoptosis of neurons induced by A 尾, promotes cell proliferation and survival, and provides a new target for the study of the pathogenesis and therapeutic drugs of AD.
【作者单位】：山东省立医院
【分类号】：R749.16

【相似文献】