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(jī)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(dá)和氧化應(yīng)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(hù)作用。方法:選擇體外培養(yǎng)的原代大鼠海馬神經(jīng)細(xì)胞和人骨髓神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y),采用免疫熒光雙染法檢測NeuN和GFAP進(jìn)行原代大鼠海馬神經(jīng)細(xì)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(xì)胞24 h～48 h,采用CCK-8法檢測細(xì)胞的存活率;蛋白印跡法(Western blotting)和實(shí)時熒光定量PCR(Real-time PCR)檢測細(xì)胞中 M1 mAChR 和 M3 mAChR 的蛋白及 mRNA表達(dá)水平;生物化學(xué)方法測定細(xì)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(xì)胞純度達(dá)到85%以上;0.O1μmol/L或0.1μmol/L洛伐他汀處理細(xì)胞,不會引起細(xì)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(xì)胞24 h,M1 mAChR和M3 mAChR的蛋白及mRNA表達(dá)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(xì)胞48 h,M1 mAChR和M3 mAChR的蛋白及mRNA表達(dá)水平明顯下降(P0.05),但預(yù)先用0.11μmol/L洛伐他汀處理細(xì)胞24h,可減輕AβOs導(dǎo)致的M1 mAChR和M3 mAChR的蛋白及mRNA表達(dá)水平改變(P0.05)。(4)洛伐他汀對杭AβOs的細(xì)胞毒性作用:0.5μmol/L AβOs處理兩種神經(jīng)細(xì)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(yù)先用0.1μmol/L洛伐他汀處理神經(jīng)細(xì)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(xì)胞,不會引起細(xì)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(dá)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(dá)降低及氧化應(yīng)激水平升高,這為他汀類藥物治療AD提供了實(shí)驗(yàn)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(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

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