DPP-4抑制劑對(duì)AD樣神經(jīng)退行性變的保護(hù)作用及其機(jī)制
發(fā)布時(shí)間：2018-08-01 14:44
【摘要】：目的:二肽基肽酶-4抑制劑對(duì)阿爾茨海默病樣神經(jīng)退行性變的保護(hù)作用及其機(jī)制。方法:1)SPF級(jí)10月齡雄性野生型C57BL/6小鼠和APP/PS1/Tau三重轉(zhuǎn)基因(3×Tg)小鼠隨機(jī)分為野生(WT)組,轉(zhuǎn)基因(Tg)組,西格列汀治療(Tg+SIT)組和沙格列汀治療(Tg+SAX)組,每組15只。待各組小鼠適應(yīng)飼養(yǎng)環(huán)境后,每天分別給予西格列汀2.6 mg/kg和沙格列汀3.5 mg/kg(約260μl)灌胃,WT組和Tg組每天予以等量無(wú)菌生理鹽水,持續(xù)8周。監(jiān)測(cè)各組小鼠體重和血糖水平;Morris水迷宮檢測(cè)小鼠空間學(xué)習(xí)記憶能力;免疫組化檢測(cè)小鼠腦內(nèi)Aβ42沉積;改良Bielschowsky染色檢測(cè)小鼠腦組織神經(jīng)原纖維纏結(jié)(NFTs)的數(shù)量;Fluoro-Jade B染色和尼氏染色標(biāo)記小鼠腦組織退行性變的神經(jīng)元;運(yùn)用微管結(jié)合實(shí)驗(yàn)檢測(cè)Tau與微管的結(jié)合能力,western blot和免疫熒光檢測(cè)小鼠腦內(nèi)GLP-1及其受體GLP-1R的表達(dá)水平、Tau蛋白和神經(jīng)絲(NFs)的磷酸化和糖基化修飾水平、突觸蛋白水平、胰島素受體底物-1(IRS-1)和GLP-1下游信號(hào)通路關(guān)鍵酶和蛋白PI3K、Akt、GSK-3β、CREB、JNK和ERK的磷酸化水平。2)取對(duì)數(shù)生長(zhǎng)期的人神經(jīng)母細(xì)胞瘤細(xì)胞SH-SY5Y分為6組:wortmannin干預(yù)組(W組,0.03μmol/L wortmannin處理12 h)、DPP-4I干預(yù)組(DPP-4I組,10μmol/L DPP-4I處理12 h)、DPP-4I與wortmannin共同干預(yù)組(DPP-4I+W組,10μmol/L DPP-4I預(yù)處理2 h,0.03μmol/L wortmannin處理12 h)、DPP-4I、wortmannin及Ex9-39共同干預(yù)組(DPP-4I+W+Ex9-39組,10μmol/L Ex9-39預(yù)處理2 h,10μmol/L DPP-4I作用2 h,最后0.03μmol/Lwortmannin處理12 h)、Ex9-39干預(yù)組(Ex9-39組,10μmol/L Ex9-39處理12h)、空白對(duì)照組(CON組,含1‰DMSO的PBS處理12 h)。MTT法檢測(cè)各組細(xì)胞活性的變化,Western blot法檢測(cè)各組微管相關(guān)的Tau總蛋白及其不同磷酸化位點(diǎn)、神經(jīng)絲(NFs)以及GLP-1下游信號(hào)通路PI3K-Akt-GSK-3β中關(guān)鍵酶的磷酸化水平。結(jié)果:1)各組小鼠體重和血糖間差異無(wú)統(tǒng)計(jì)學(xué)意義。與WT小鼠相比,Tg小鼠逃避潛伏期和游泳路徑長(zhǎng)度顯著延長(zhǎng),而穿越隱匿平臺(tái)次數(shù)和目標(biāo)象限游泳時(shí)間明顯縮短,小鼠腦內(nèi)海馬和皮質(zhì)區(qū)GLP-1及其受體GLP-1R的表達(dá)水平下降、淀粉樣蛋白Aβ42沉積、退行性變神經(jīng)元和NFTs數(shù)量增加,Tau蛋白、NFs磷酸化修飾增多而糖基化修飾減少,Tau蛋白與微管結(jié)合能力減弱,腦內(nèi)突觸蛋白水平下降,IRS-1和PI3K、Akt、GSK-3β、CREB及ERK磷酸化修飾水平下降,JNK磷酸化修飾水平上升;Tg小鼠經(jīng)西格列汀和沙格列汀處理后上述指標(biāo)明顯改善。2)與CON細(xì)胞相比,經(jīng)wortmannin處理的細(xì)胞活力下降,Tau蛋白在絲氨酸和蘇氨酸199、202、231和396位點(diǎn)以及NF-H/M的磷酸化水平升高,經(jīng)DPP-4抑制劑處理的細(xì)胞活力上升,上述指標(biāo)的磷酸化水平下降;與經(jīng)wortmannin處理的細(xì)胞相比,經(jīng)DPP-4抑制劑預(yù)處理的細(xì)胞活力上升,上述指標(biāo)的磷酸化水平下降。與CON細(xì)胞相比,經(jīng)Ex9-39處理的細(xì)胞活力下降,Tau蛋白在絲氨酸和蘇氨酸199、202、231和396位點(diǎn)以及NFs的磷酸化水平升高;與經(jīng)DPP-4抑制劑和wortmannin共同處理的細(xì)胞相比,經(jīng)Ex9-39預(yù)處理的細(xì)胞活力下降,上述指標(biāo)的磷酸化水平升高。與CON細(xì)胞相比,經(jīng)wortmannin處理的細(xì)胞PI3K、Akt、GSK-3β磷酸化水平下降,而經(jīng)DPP-4抑制劑處理的細(xì)胞PI3K、Akt、GSK-3β磷酸化水平上升;與經(jīng)wortmannin處理的細(xì)胞相比,經(jīng)DPP-4抑制劑預(yù)處理的細(xì)胞PI3K、Akt、GSK-3β磷酸化水平上升。結(jié)論:二肽基肽酶-4抑制劑通過(guò)提高GLP-1水平,改善腦內(nèi)GLP-1信號(hào)通路和腦內(nèi)糖代謝,提高AD小鼠學(xué)習(xí)記憶能力,改善神經(jīng)退行性變。
[Abstract]:Objective: the protective effect of two peptidyl peptidase -4 inhibitor on Alzheimer like neurodegeneration and its mechanism. Methods: 1) SPF grade 10 month old male wild type C57BL/6 mice and APP/PS1/Tau three weight transgenic mice (3 x Tg) mice were randomly divided into wild (WT) group, Tg group, Sig Leo Dean therapy (Tg+SIT) group and Shah Glenn Dean treatment (Tg+SAX). Groups of 15 rats in each group were treated with Sig Leo Dean 2.6 mg/kg and Shah Glenn Dean 3.5 mg/kg (about 260 L) every day after adapting to the feeding environment. Group WT and Tg were given aseptic saline for 8 weeks. The weight and blood glucose level of mice in each group were monitored, and Morris water maze was used to detect the ability of learning and memory in mice; The A beta 42 deposition in the brain of mice was detected; the number of neurogenic fibrous tangles (NFTs) in the brain tissue of mice was detected by modified Bielschowsky staining; Fluoro-Jade B staining and Nissl staining were used to mark the neurons of the degenerative brain tissue in mice; the binding ability of Tau and microtubules was detected by microtubule binding assay, and Western blot and immunofluorescence were used to detect GLP- in the brain of mice. 1 and its receptor GLP-1R expression level, phosphorylation and glycosylation level of Tau and NFs, synaptic protein level, insulin receptor substrate -1 (IRS-1) and key enzymes of the downstream signal pathway of GLP-1 and protein PI3K, Akt, GSK-3 beta, CREB, JNK, and ERK, from the logarithmic growth period of human neuroblastoma cells They were divided into 6 groups: wortmannin intervention group (group W, 0.03 mu mol/L wortmannin processing 12 h), DPP-4I intervention group (DPP-4I group, 10 micron mol/L DPP-4I processing 12 h), DPP-4I and wortmannin intervention group (10 mu 2, 0.03 micron treatment 12). Mol/L Ex9-39 pretreatment 2 h, 10 mol/L DPP-4I action 2 h, final 0.03 mol/Lwortmannin treatment 12 h), Ex9-39 intervention group (Ex9-39 group, 10 mu mol/L Ex9-39), the blank control group (1 per thousand, 12) The phosphorylation level of the same phosphorylation site, the nerve filament (NFs) and the key enzyme in the downstream signal pathway of GLP-1. Results: 1) there was no significant difference between the weight and blood sugar of the mice in each group. Compared with the WT mice, the escape latency and the swimming path length of the Tg mice were significantly prolonged, while the number of hidden platforms and the target quadrant were swimming in the Tg mice. The expression level of GLP-1 and its receptor GLP-1R in the hippocampus and cortex of mice decreased significantly, the deposition of amyloid A beta 42, the number of degenerative neurons and NFTs, the increase of Tau protein, NFs phosphorylation and glycosylation modification, the weakening of Tau protein and microtubule binding capacity, the decrease of synapse protein levels in the brain, IRS-1 and PI3K, Akt, GSK-3 beta, CREB and ERK phosphorylation modification level decreased, JNK phosphorylation modification level increased; Tg mice improved.2 after treatment with Western glipetine and Shah Glenn Dean. Compared with CON cells, wortmannin treated cell vitality decreased, Tau protein at serine and threonine 199202231 and 396 loci and NF-H/M phosphorylated water. The activity of the cells treated by DPP-4 increased and the phosphorylation level of the above index decreased. Compared with the cells treated with wortmannin, the activity of cells pretreated by the DPP-4 inhibitor increased and the phosphorylation level of the above index decreased. Compared with the CON cells, the cell vitality of the Ex9-39 treated cells decreased, and the Tau protein was in serine and SSU. The phosphorylation level of the 199202231 and 396 sites of ammonia and NFs increased. Compared with the cells treated with DPP-4 inhibitors and wortmannin, the activity of the cells pretreated by Ex9-39 decreased and the level of phosphorylation of the above indexes increased. The level of PI3K, Akt, GSK-3 beta phosphorylation of wortmannin treated cells decreased compared with CON cells, and the DPP-4 was reduced by DPP-4. The level of phosphorylation of PI3K, Akt, GSK-3 beta in the cells treated by inhibitors increased; compared with wortmannin treated cells, PI3K, Akt, and GSK-3 beta phosphorylation levels of cells treated by DPP-4 inhibitor increased. Conclusion: two peptidyl peptidase -4 inhibitors improve AD mice learning by improving GLP-1 level, improving the GLP-1 signaling pathway and brain glucose metabolism in the brain. Memory ability improves neurodegenerative changes.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R749.16
本文編號(hào)：2157910
[Abstract]:Objective: the protective effect of two peptidyl peptidase -4 inhibitor on Alzheimer like neurodegeneration and its mechanism. Methods: 1) SPF grade 10 month old male wild type C57BL/6 mice and APP/PS1/Tau three weight transgenic mice (3 x Tg) mice were randomly divided into wild (WT) group, Tg group, Sig Leo Dean therapy (Tg+SIT) group and Shah Glenn Dean treatment (Tg+SAX). Groups of 15 rats in each group were treated with Sig Leo Dean 2.6 mg/kg and Shah Glenn Dean 3.5 mg/kg (about 260 L) every day after adapting to the feeding environment. Group WT and Tg were given aseptic saline for 8 weeks. The weight and blood glucose level of mice in each group were monitored, and Morris water maze was used to detect the ability of learning and memory in mice; The A beta 42 deposition in the brain of mice was detected; the number of neurogenic fibrous tangles (NFTs) in the brain tissue of mice was detected by modified Bielschowsky staining; Fluoro-Jade B staining and Nissl staining were used to mark the neurons of the degenerative brain tissue in mice; the binding ability of Tau and microtubules was detected by microtubule binding assay, and Western blot and immunofluorescence were used to detect GLP- in the brain of mice. 1 and its receptor GLP-1R expression level, phosphorylation and glycosylation level of Tau and NFs, synaptic protein level, insulin receptor substrate -1 (IRS-1) and key enzymes of the downstream signal pathway of GLP-1 and protein PI3K, Akt, GSK-3 beta, CREB, JNK, and ERK, from the logarithmic growth period of human neuroblastoma cells They were divided into 6 groups: wortmannin intervention group (group W, 0.03 mu mol/L wortmannin processing 12 h), DPP-4I intervention group (DPP-4I group, 10 micron mol/L DPP-4I processing 12 h), DPP-4I and wortmannin intervention group (10 mu 2, 0.03 micron treatment 12). Mol/L Ex9-39 pretreatment 2 h, 10 mol/L DPP-4I action 2 h, final 0.03 mol/Lwortmannin treatment 12 h), Ex9-39 intervention group (Ex9-39 group, 10 mu mol/L Ex9-39), the blank control group (1 per thousand, 12) The phosphorylation level of the same phosphorylation site, the nerve filament (NFs) and the key enzyme in the downstream signal pathway of GLP-1. Results: 1) there was no significant difference between the weight and blood sugar of the mice in each group. Compared with the WT mice, the escape latency and the swimming path length of the Tg mice were significantly prolonged, while the number of hidden platforms and the target quadrant were swimming in the Tg mice. The expression level of GLP-1 and its receptor GLP-1R in the hippocampus and cortex of mice decreased significantly, the deposition of amyloid A beta 42, the number of degenerative neurons and NFTs, the increase of Tau protein, NFs phosphorylation and glycosylation modification, the weakening of Tau protein and microtubule binding capacity, the decrease of synapse protein levels in the brain, IRS-1 and PI3K, Akt, GSK-3 beta, CREB and ERK phosphorylation modification level decreased, JNK phosphorylation modification level increased; Tg mice improved.2 after treatment with Western glipetine and Shah Glenn Dean. Compared with CON cells, wortmannin treated cell vitality decreased, Tau protein at serine and threonine 199202231 and 396 loci and NF-H/M phosphorylated water. The activity of the cells treated by DPP-4 increased and the phosphorylation level of the above index decreased. Compared with the cells treated with wortmannin, the activity of cells pretreated by the DPP-4 inhibitor increased and the phosphorylation level of the above index decreased. Compared with the CON cells, the cell vitality of the Ex9-39 treated cells decreased, and the Tau protein was in serine and SSU. The phosphorylation level of the 199202231 and 396 sites of ammonia and NFs increased. Compared with the cells treated with DPP-4 inhibitors and wortmannin, the activity of the cells pretreated by Ex9-39 decreased and the level of phosphorylation of the above indexes increased. The level of PI3K, Akt, GSK-3 beta phosphorylation of wortmannin treated cells decreased compared with CON cells, and the DPP-4 was reduced by DPP-4. The level of phosphorylation of PI3K, Akt, GSK-3 beta in the cells treated by inhibitors increased; compared with wortmannin treated cells, PI3K, Akt, and GSK-3 beta phosphorylation levels of cells treated by DPP-4 inhibitor increased. Conclusion: two peptidyl peptidase -4 inhibitors improve AD mice learning by improving GLP-1 level, improving the GLP-1 signaling pathway and brain glucose metabolism in the brain. Memory ability improves neurodegenerative changes.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R749.16
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 郭愛(ài);宋奇駿;彭鵬;高新宇;陳書(shū)儀;趙綱;鄧艷秋;;抗T2DM藥治療阿爾茨海默病研究進(jìn)展[J];生命的化學(xué);2017年04期
2 Emanuel Monteiro Candeias;Inês Carolina Sebastio;Susana Maria Cardoso;Sónia Catarina Correia;Cristina Isabel Carvalho;Ana Isabel Plácido;Maria Sancha Santos;Catarina Resende Oliveira;Paula Isabel Moreira;Ana Isabel Duarte;;Gut-brain connection: The neuroprotective effects of the anti-diabetic drug liraglutide[J];World Journal of Diabetes;2015年06期
3 孫潔;陳書(shū)儀;路素素;鄭軍;鄧艷秋;;利拉魯肽對(duì)2型糖尿病阿爾茨海默病樣三重轉(zhuǎn)基因小鼠學(xué)習(xí)記憶的影響[J];天津醫(yī)藥;2015年07期
4 郭敏;李剛;;突觸可塑性相關(guān)蛋白的研究進(jìn)展[J];神經(jīng)藥理學(xué)報(bào);2013年06期
5 熊慧;鄧艷秋;;用抗糖尿病藥物GLP-1及其類(lèi)似物治療阿爾茨海默病[J];生命的化學(xué);2013年06期
6 趙綱;戎凱;鄧艷秋;;阿爾茨海默病與腦內(nèi)胰島素信號(hào)轉(zhuǎn)導(dǎo)通路的關(guān)系[J];山東醫(yī)藥;2012年29期
7 劉慧莉;趙剛;;運(yùn)動(dòng)、阿爾茨海默病與突觸可塑性[J];中國(guó)康復(fù)理論與實(shí)踐;2012年03期
8 崔冉亮;胡海燕;呂樸;戎凱;陳寧;鄧艷秋;;神經(jīng)絲蛋白質(zhì)糖基化與磷酸化的相互調(diào)節(jié)和神經(jīng)退行性疾病[J];生命的化學(xué);2009年06期
9 ;Overexpression of dishevelled-1 attenuates wortmannin-induced hyperphosphorylation of cytoskeletal proteins in N2a cell[J];Acta Pharmacologica Sinica;2005年06期
10 周建軍;樂(lè)秀芳;韓家嫻;楊蔚怡;;評(píng)價(jià)抗癌物質(zhì)活性的改良MTT方法[J];中國(guó)醫(yī)藥工業(yè)雜志;1993年10期
，本文編號(hào)：2157910
本文鏈接：http://sikaile.net/yixuelunwen/jsb/2157910.html
最近更新
教材專(zhuān)著
