發(fā)布時間：2019-06-03 06:43

【摘要】：目的:阿爾茨海默氏病(Alzheimer's disease,AD)的病因及發(fā)病機制仍未完全清楚,極大的限制了臨床治療藥物的開發(fā),由于β-淀粉樣肽(β-Amyloid peptide,Aβ)的神經(jīng)毒性作用是AD發(fā)病過程的關(guān)鍵環(huán)節(jié),因此,本課題采用傳統(tǒng)上降低膽固醇的史他汀類藥物一洛伐他汀,研究其對抗Aβ引起的神經(jīng)毒性作用,特別是對抗Aβ神經(jīng)毒性作用過程中毒蕈堿型乙酰膽堿能受體(Muscarinic acetylcholine receptors,mAChRs)表達和氧化應激水平等的改變,探討他汀類藥物在AD治療中的非膽固醇依賴性神經(jīng)保護作用。方法:選擇體外培養(yǎng)的原代大鼠海馬神經(jīng)細胞和人骨髓神經(jīng)母細胞瘤細胞(SH-SY5Y),采用免疫熒光雙染法檢測NeuN和GFAP進行原代大鼠海馬神經(jīng)細胞純度鑒定。用洛伐他汀、Aβ寡聚體(β-Amyloid peptide oligomers,AβOs)分別或合并處理細胞24 h～48 h,采用CCK-8法檢測細胞的存活率;蛋白印跡法(Western blotting)和實時熒光定量PCR(Real-time PCR)檢測細胞中 M1 mAChR 和 M3 mAChR 的蛋白及 mRNA表達水平;生物化學方法測定細胞內(nèi)膽固醇、丙二醛(Malondialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)和谷胱甘肽過氧化物酶(Glutathione peroxidase,GSH-Px)活性及氧自由基(OH-、H2O2、O2·-)等的變化。結(jié)果:(1)原代培養(yǎng)的大鼠海馬神經(jīng)細胞純度達到85%以上;0.O1μmol/L或0.1μmol/L洛伐他汀處理細胞,不會引起細胞活性和膽固醇水平明顯改變(P0.05)。(2)洛伐他汀對 mAChRs 的作用:0.01μmol/L 或 0.1μmol/L 洛伐他汀處理神經(jīng)細胞24 h,M1 mAChR和M3 mAChR的蛋白及mRNA表達水平明顯增加(P0.05)。(3)洛伐他汀抗AβOs對mAChRs的作用:0.5μmol/L AβOs處理神經(jīng)細胞48 h,M1 mAChR和M3 mAChR的蛋白及mRNA表達水平明顯下降(P0.05),但預先用0.11μmol/L洛伐他汀處理細胞24h,可減輕AβOs導致的M1 mAChR和M3 mAChR的蛋白及mRNA表達水平改變(P0.05)。(4)洛伐他汀對杭AβOs的細胞毒性作用:0.5μmol/L AβOs處理兩種神經(jīng)細胞48 h,MDA含量增加、SOD和GSH-Px活性降低、氧自由基(OH-、H2O2、O2·-)水平明顯增加(P0.05);給予AβOs前預先用0.1μmol/L洛伐他汀處理神經(jīng)細胞24 h,可減弱AβOs引起的MDA含量、SOD及GSH-Px活性、氧自由基水平等改變(P0.05)。結(jié)論:(1)0.01μmol/L及0.1μmol/L洛伐他汀處理細胞,不會引起細胞活性和膽固醇水平明顯改變。(2)洛伐他汀可能通過上調(diào)M1 mAChR和M3 mAChR的表達對抗Aβ毒-性作用。(3)洛伐他汀可緩解AβOs引起的MDA含量增加、SOD及GSH-Px活性降低、氧自由基(OH-、H2O2、O2·-)水平升高等毒性作用。綜上,洛伐他汀可能以非膽固醇依賴的方式,緩解Aβ引起的mAChRs表達降低及氧化應激水平升高,這為他汀類藥物治療AD提供了實驗依據(jù)。
[Abstract]:Objective: the etiology and pathogenesis of Alzheimer's disease (Alzheimer's disease,AD) are still not fully understood, which greatly limits the development of clinical therapeutic drugs, due to 尾-Amyloid peptide,. The neurotoxicity of A 尾) is the key link in the pathogenesis of AD. Therefore, this paper studied the neurotoxicity induced by A 尾 by using Lovastatin, a traditional cholesterol lowering drug, to study the neurotoxicity induced by A 尾. In particular, the expression of muscarinic acetylcholine receptor (Muscarinic acetylcholine receptors,mAChRs) and the change of oxidative stress water during antagonizing A 尾 neurotoxicity were studied to explore the cholesterol-independent neuroprotective effect of statins in the treatment of AD. Methods: primary rat hippocampal neurons and human bone marrow neuroblastoma cells (SH-SY5Y) were cultured in vitro. NeuN and GFAP were detected by immunofluorescence double staining to identify the purity of primary rat hippocampal neurons. Lovastatin and A 尾 oligomer (尾-Amyloid peptide oligomers, A 尾 Os) were used to treat the cells for 24 h and 48 h, respectively. The survival rate of the cells was detected by CCK-8 assay. The expression of M1 mAChR and M3 mAChR protein and mRNA in cells were detected by (Western blotting) and real-time fluorescence quantitative PCR (Real-time PCR). Determination of intracellular cholesterol, malondialdehyde (Malondialdehyde,MDA) content, activities of Superoxide Dismutase,SOD and Glutathione peroxidase,GSH-Px and oxygen free radical (OH-,H2O2,) by biochemical method The change of O2 -), etc. Results: (1) the purity of primary cultured rat hippocampal neurons was over 85%; 0.O1 渭 mol / L or 0.1 渭 mol / L lovarastatin, There was no significant change in cell activity and cholesterol level (P 0.05). (2). The effect of lovarastatin on mAChRs was 0.01 渭 mol / L or 0.1 渭 mol / L for 24 h. The expression levels of M1 mAChR and M3 mAChR protein and mRNA were significantly increased (P 0.05). (3). The effect of lovarastatin on mAChRs was treated with 0.5 渭 mol / L A 尾 Os for 48 h. The expression levels of M1 mAChR and M3 mAChR protein and mRNA were significantly decreased (P 0.05), but the cells were treated with 0.11 渭 mol / L lovarastatin for 24 h. It could reduce the expression of M1 mAChR and M3 mAChR protein and mRNA induced by A 尾 Os (P 0.05). (4). The cytotoxicity of lovarastatin on hang A 尾 Os was increased after treatment with 0.5 渭 mol / L A 尾 Os for 48 h. The activities of SOD and GSH-Px decreased and the level of oxygen free radical (OH-,H2O2, O2 -) increased significantly (P 0.05). Pretreatment with 0.1 渭 mol / L lovarastatin for 24 h before administration of A 尾 Os could weaken the changes of MDA content, SOD and GSH-Px activity and oxygen free radical level induced by A 尾 Os (P 0.05). Conclusion: (1) the cells were treated with 0.01 渭 mol / L and 0.1 渭 mol / L lovarastatin. It did not cause significant changes in cell activity and cholesterol level. (2) Lovastatin may antagonize A 尾 toxicity by up-regulating the expression of M1 mAChR and M3 mAChR. (3) Lovastatin can alleviate the increase of MDA content induced by A 尾 Os. The activities of SOD and GSH-Px decreased and the level of oxygen free radical (OH-,H2O2, O 2 -) increased. In summary, lovarastatin may alleviate the decrease of mAChRs expression and the increase of oxidative stress level induced by A 尾 in a cholesterol-independent manner, which provides experimental basis for statins in the treatment of AD.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

【參考文獻】

相關(guān)期刊論文 前10條

1 王惠國;趙小紅;張楠楠;秦海宏;盧軒;馮寶民;;阿爾茨海默病發(fā)病機制概況[J];遼寧中醫(yī)雜志;2016年10期

2 周嬋;蘭文杰;;老年人阿爾茨海默病的病因病機及認知治療研究進展[J];大家健康(學術(shù)版);2016年09期

3 鄭玲艷;韓瑞蘭;曹俊彥;;β-淀粉樣蛋白在阿爾茨海默病中的作用[J];內(nèi)蒙古醫(yī)科大學學報;2016年02期

4 蔡佳;賀娟;徐貴麗;;他汀類藥物的臨床應用研究進展[J];中國醫(yī)院用藥評價與分析;2015年02期

5 尹喜娟;;阿托伐他汀對急性冠脈綜合征患者C-反應蛋白的作用分析[J];中外醫(yī)學研究;2014年02期

6 趙虹;駱慶和;殷明;趙文娟;;氧化應激與阿爾茨海默病[J];中國老年學雜志;2013年16期

7 姜斌;馬瑞;杜繼臣;;阿托伐他汀對急性腦梗死患者氧化應激指標及頸動脈斑塊的影響[J];臨床薈萃;2013年07期

8 何玲;王聰;孫寶娟;;針對阿爾茨海默病治療的G蛋白偶聯(lián)受體及其藥物研究進展[J];國際藥學研究雜志;2013年03期

9 孔繁軍;陳強;周亮;崔德華;;單體、寡聚體及纖維狀Aβ毒性作用的比較研究[J];中國實驗診斷學;2012年09期

10 范紅波;;氧自由基在老年癡呆發(fā)病機制中的作用[J];中國現(xiàn)代醫(yī)藥雜志;2012年04期

，

本文編號：2491744