【摘要】：目的:為了明確記憶相關(guān)基因KIBRA在阿爾茨海默病(Alzheimer’s disease,AD)轉(zhuǎn)基因動物模型中是否存在表達改變及及其對神經(jīng)元凋亡的影響,進而探討KIBRA如何參與Aβ誘導的神經(jīng)元凋亡及其相關(guān)機制。方法:本研究首先通過免疫組織染色及免疫蛋白印跡的方法,觀察在不同月齡APP/PS1小鼠中是否存在KIBRA的表達改變。其次,通過TUNEL免疫熒光染色,觀察在KIBRA基因敲除小鼠中是否存在神經(jīng)元凋亡。最后,我們使用CRISPR/CAS9慢病毒及過表達慢病毒分別轉(zhuǎn)染HT22細胞,建立KIBRA基因敲除和KIBRA過表達細胞模型,進而探討KIBRA對Aβ誘導的神經(jīng)元凋亡及其相關(guān)機制的研究。結(jié)果:(1)KIBRA在APP/PS1轉(zhuǎn)基因小鼠腦內(nèi)特異性表達:選用9月齡及12月齡APP/PS1小鼠作為AD研究模型,通過KIBRA免疫組織染色分析,無論整個海馬,還是海馬亞區(qū)CA1區(qū)和CA3區(qū),9月齡和12月齡APP/PS1小鼠KIBRA陽性細胞數(shù)量均明顯低于對照組,其中12月齡尤為顯著(P0.01,P0.001)。免疫蛋白印跡結(jié)果同樣證實12月齡APP/PS1小鼠海馬區(qū)KIBRA的表達明顯低于野生型小鼠(P0.001)。隨著月齡的增加,KIBRA在APP/PS1轉(zhuǎn)基因小鼠海馬區(qū)的表達逐漸減少,提示KIBRA在AD小鼠海馬區(qū)的特異性降低,與學習記憶障礙密切相關(guān)。(2)KIBRA在神經(jīng)元凋亡中的重要作用:通過TUNEL熒光染色發(fā)現(xiàn),與野生型小鼠相比,12月齡APP/PS1小鼠海馬神經(jīng)元凋亡比例明顯增多(P0.01,P0.01)。4月齡KIBRA基因敲除小鼠海馬神經(jīng)元凋亡比例比野生型小鼠明顯增加(P0.05),提示KIBRA在神經(jīng)元凋亡的發(fā)生中扮演不可或缺的角色。(3)KIBRA對Aβ誘導的神經(jīng)元凋亡的保護作用:CCK8及免疫蛋白印跡結(jié)果顯示,與空病毒組相比,經(jīng)1μmol·L~(-1)的Aβ1-42寡聚體處理后的KIBRA敲除組細胞活力明顯降低(P0.01);凋亡相關(guān)蛋白(剪切的PARP、活化的caspase3)的表達量明顯增高(P0.05,P0.01)。KIBRA過表達組細胞存活率明顯優(yōu)于空病毒組(P0.05),凋亡相關(guān)蛋白較空病毒組顯著減少(P0.05),進一步提示KIBRA參與抑制Aβ誘導的神經(jīng)元凋亡,促進細胞增殖和存活。4.KIBRA通過激活Akt信號通路抑制Aβ誘導的神經(jīng)元凋亡:本實驗篩選凋亡相關(guān)信號通路,結(jié)果顯示,在1μmol·L~(-1) Aβ1-42寡聚體處理1 min后,KIBRA敲除組Akt Ser473位點磷酸化水平比空病毒組顯著降低(P0.05)。在Aβ1-42寡聚體處理1分鐘后,KIBRA過表達組Akt Ser473位點磷酸化明顯被激活,Akt Ser473位點磷酸化水平比空病毒組顯著增高(P0.01)。此外,與Aβ1-42寡聚體干預組相比,Akt特異性抑制劑(MK2206)預處理組KIBRA過表達細胞存活率明顯降低(P0.05);剪切的PARP表達量明顯增高(P0.05)。以上結(jié)果表明KIBRA通過激活Akt Ser473位點的磷酸化水平,抑制Aβ誘導的神經(jīng)元凋亡。結(jié)論:KIBRA作為一種神經(jīng)保護因子,通過激活Akt信號通路抑制Aβ誘導的神經(jīng)元凋亡,促進細胞增殖及存活,為AD發(fā)病機制及治療藥物的研究提供了新的靶點。
[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.
【作者單位】： 山東省立醫(yī)院
【分類號】：R749.16

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