本文關(guān)鍵詞： 17-AAG GCI OGD/R 大鼠海馬神經(jīng)元　出處：《重慶醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景與目的:在臨床上,外科手術(shù)和心臟驟停導(dǎo)致的全腦低灌注是造成全腦缺血(Global cerebral ischemia,GCI)性損傷的重要原因。腦缺血后會(huì)引起神經(jīng)細(xì)胞的不可逆死亡,從而導(dǎo)致神經(jīng)病學(xué)和神經(jīng)行為學(xué)上的障礙。在臨床上,全腦缺血會(huì)導(dǎo)致記憶學(xué)習(xí)能力下降、癡呆等后遺癥。因此,抑制神經(jīng)細(xì)胞的不可逆死亡有望成為改善腦功能的的重要突破口。17-AAG(17-allylamino-demethoxygeldanamycin,17-AAG)即17-丙烯胺基,17-去甲氧基格爾德霉素作為Hsp90抑制劑在臨床中是一種有效的抗腫瘤藥物。據(jù)報(bào)道,17-AAG可以通過(guò)提高Hsp70的表達(dá)水平來(lái)保護(hù)創(chuàng)傷導(dǎo)致的腦損傷。因此,我們推測(cè)17-AAG在GCI中也可能具有重要的保護(hù)作用。本研究首先探索17-AAG在大鼠GCI模型中的神經(jīng)保護(hù)作用;其次,分離培養(yǎng)大鼠的海馬神經(jīng)元,通過(guò)氧糖剝離/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)模擬大鼠在體GCI腦損傷,然后研究17-AAG對(duì)大鼠海馬神經(jīng)元凋亡的保護(hù)作用;最后,構(gòu)建Hsp90表達(dá)和Hsp70干擾慢病毒,探討17-AAG對(duì)大鼠海馬神經(jīng)元細(xì)胞保護(hù)作用的可能分子機(jī)制。方法:(1)將90只雌性SD大鼠隨機(jī)的分成三組:假手術(shù)組(Sham)、全腦缺血再灌注組(GCI)、全腦缺血再灌注加17-AAG處理組(17-AAG),通過(guò)四血管閉塞法構(gòu)建大鼠GCI模型。然后,通過(guò)TTC實(shí)驗(yàn)檢測(cè)各組大鼠腦梗死體積,TUNEL實(shí)驗(yàn)檢測(cè)神經(jīng)元的凋亡情況,水迷宮實(shí)驗(yàn)檢測(cè)各組大鼠的學(xué)習(xí)能力和記憶能力變化,Western blot檢測(cè)各組大鼠腦組織中Hsp70、Hsp90、LC3B的蛋白表達(dá)。(2)分離培養(yǎng)大鼠海馬神經(jīng)元,通過(guò)OGD/R模擬大鼠在體GCI腦損傷,OGD時(shí)間為0.5h、1h、2h,再灌注時(shí)間為24h,TUNEL實(shí)驗(yàn)檢測(cè)OGD/R對(duì)各組細(xì)胞凋亡的影響,然后Western blot檢測(cè)OGD/R對(duì)各組細(xì)胞凋亡標(biāo)志物Cleaved Caspase-3和自噬指標(biāo)LC3B II、p62表達(dá)的影響。(3)通過(guò)TUNEL實(shí)驗(yàn)檢測(cè)不同濃度的17-AAG(0.5μM、1.0μM、2.0μM)對(duì)OGD/R處理的神經(jīng)元的凋亡的影響,然后Western blot檢測(cè)凋亡標(biāo)志物Cleaved Caspase-3和自噬指標(biāo)LC3B II、p62表達(dá)的影響。然后,將細(xì)胞分為OGD/R組、OGD/R+17-AAG(1.0μM)組、OGD/R+17-AAG(1.0μM)+Rapamycin(1.0n M),通過(guò)TUNEL和Western blot研究自噬在17-AAG影響OGD/R處理的神經(jīng)元凋亡中的角色。(4)通過(guò)Western blot研究17-AAG對(duì)OGD/R大鼠海馬神經(jīng)元中Hsp70和Hsp90表達(dá)的影響。構(gòu)建Hsp90表達(dá)和Hsp70干擾慢病毒,將細(xì)胞分成四組:OGD/R+17-AAG+Con、OGD/R+17-AAG+Hsp70sh RNA、OGD/R+17-AAG+NC、OGD/R+17-AAG+Hsp90,通過(guò)TUNEL實(shí)驗(yàn)檢測(cè)17-AAG是否通過(guò)Hsp90和Hsp70調(diào)節(jié)OGD/R大鼠海馬神經(jīng)元的凋亡,然后通過(guò)Western blot檢測(cè)17-AAG是否通過(guò)Hsp90和Hsp70調(diào)節(jié)OGD/R大鼠海馬神經(jīng)元凋亡指標(biāo)Cleaved Caspase-3和自噬指標(biāo)LC3B II、p62的表達(dá)。結(jié)果:(1)與GCI組相比,17-AAG組GCI導(dǎo)致的梗死體積明顯減少(P0.01),神經(jīng)元存活明顯增加(P0.01),大鼠的學(xué)習(xí)能力和記憶能力明顯改善(P0.01,P0.01),同時(shí)Hsp90和LC3B蛋白表達(dá)明顯降低(P0.01,P0.01),Hsp70蛋白表達(dá)明顯增加(P0.01)。(2)與Con組相比,OGD時(shí)間為1h和2h時(shí),OGD/R能夠明顯促進(jìn)神經(jīng)元的凋亡百分率的提高(P0.05,P0.01);OGD時(shí)間為0.5h,1h和2h時(shí),OGD/R均能夠明顯增強(qiáng)凋亡標(biāo)志物Cleaved Caspase-3的表達(dá)(P0.05,P0.01,P0.01)和自噬。(3)與OGD/R 1h相比,三種濃度的17-AAG(0.5μM、1.0μM、2.0μM)均可以降低OGD/R神經(jīng)元的凋亡百分率(P0.05,P0.01,P0.01);17-AAG濃度為1.0μM、2.0μM時(shí)明顯減少OGD/R大鼠海馬神經(jīng)元Cleaved Caspase-3蛋白的表達(dá)(P0.01,P0.01)和自噬。與OGD/R+17-AAG組相比,OGD/R+17-AAG+Rapamycin組神經(jīng)元自噬、凋亡的百分率和Cleaved Caspase-3蛋白的表達(dá)、均明顯升高(P0.05,P0.05)。(4)與Con組相比,OGD/R組Hsp70的表達(dá)明顯降低(P0.01),而Hsp90的表達(dá)明顯升高(P0.01);與OGD/R組相比,OGD/R+17-AAG組細(xì)胞中Hsp70的表達(dá)明顯的升高(P0.05),而Hsp90的表達(dá)明顯的降低(P0.05)。與OGD/R+17-AAG+Con相比,OGD/R+17-AAG+Hsp70 sh RNA組大鼠海馬神經(jīng)元凋亡率、Cleaved Caspase-3蛋白的表達(dá)和自噬均明顯提高(P0.01,P0.05);與OGD/R+17-AAG+NC組相比,OGD/R+17-AAG+Hsp90大鼠海馬神經(jīng)元凋亡率、Cleaved Caspase-3蛋白的表達(dá)和自噬也均明顯提高(P0.01,P0.05)。結(jié)論:在GCI動(dòng)物模型中,GCI導(dǎo)致梗死、神經(jīng)元死亡、大鼠的學(xué)習(xí)能力和記憶能力降低;在體外OGD/R實(shí)驗(yàn)中,OGD/R促進(jìn)神經(jīng)元凋亡,而17-AAG可以降低大鼠梗死體積和神經(jīng)元死亡百分率并改善學(xué)習(xí)能力和記憶能力,同時(shí)通過(guò)Hsp70和Hsp90調(diào)節(jié)自噬來(lái)提高神經(jīng)元的存活。
[Abstract]:Background and objective: in clinical, surgery and cardiac arrest caused by cerebral hypoperfusion caused by cerebral ischemia (Global cerebral, ischemia, GCI) an important cause of injury. After cerebral ischemia can cause nerve cell death leading to irreversible disorder, neurology and neurological behavior. In clinic. Cerebral ischemia will lead to the decline of learning and memory ability, dementia and other sequelae. Therefore, inhibition of nerve cell death is expected to become an important breakthrough in irreversible.17-AAG improve the brain function of (17-allylamino-demethoxygeldanamycin, 17-AAG) 17- propylene amine, 17- demethoxygeldanamycin as Hsp90 inhibitors in clinical practice is an effective anti tumor drugs. According to reports, 17-AAG can improve the expression level of brain injury caused by trauma to protect Hsp70. Therefore, we hypothesized that 17-AAG in GCI may also have important. Support effect. This study first explores the neuroprotective effect of 17-AAG in rat GCI model; secondly, isolated and cultured rat hippocampal neurons by oxygen glucose release / reperfusion (oxygen-glucose deprivation/reperfusion OGD/R) simulation in rat GCI brain injury, and the protective effect of 17-AAG on apoptosis of rat hippocampal neurons; finally, we constructed the Hsp90 expression and Hsp70 interference lentivirus, and explore the possible molecular mechanism of 17-AAG cells in cultured hippocampal neurons of rats. Methods: (1) 90 female SD rats were randomly divided into three groups: sham operation group (Sham), cerebral ischemia reperfusion group (GCI), cerebral ischemia reperfusion plus 17-AAG treatment group (17-AAG), rat GCI model was established by four vessel occlusion method. Then, the cerebral infarction rats volume TTC assay and TUNEL assay, the apoptosis of neurons, the water maze test in rats Changes of learning ability and memory, Hsp70, brain tissue of rats Western blot detection in Hsp90, the expression of LC3B protein. (2) isolated and cultured rat hippocampal neurons of rat in vivo, simulated GCI brain injury through OGD/R, OGD for 0.5h, 1H, 2h, reperfusion for 24h, TUNEL assay. Detection of OGD/R on the apoptosis of cells in each group, and Western blot OGD/R to detect apoptosis were Cleaved markers Caspase-3 and LC3B indicators of autophagy expression. II, p62 (3) by different concentration TUNEL assay of 17-AAG (0.5 M, 1 M, 2 M) on the apoptosis of OGD/R treated neurons then, Western blot detection of apoptosis markers Cleaved Caspase-3 and LC3B II index of autophagy expression. Then, effects of p62, the cells were divided into OGD/R group, OGD/R+17-AAG (1 M) group, OGD/R+17-AAG (1 M) +Rapamycin (1.0N M), and Western blot of autophagy through TUNEL The influence of 17-AAG on neuronal apoptosis in OGD/R treatment role. (4) by Western blot the influence of 17-AAG on the expression of Hsp70 and Hsp90 neurons in the hippocampus of OGD/R rats. The expression of Hsp90 and Hsp70 to construct lentivirus, the cells were divided into four groups: OGD/R+17-AAG+Con, OGD/R+17-AAG+ Hsp70sh RNA, OGD/R+17-AAG+NC, OGD/R+17-AAG+Hsp90, apoptosis by TUNEL assay 17-AAG through Hsp90 and Hsp70 regulating OGD/R rat hippocampal neurons, and then through the Western blot 17-AAG and Hsp70 Hsp90 detection by regulating the apoptosis of hippocampal neurons in rats with OGD/R index Cleaved Caspase-3 and LC3B II p62's index of autophagy, expression. Results: (1) compared with GCI group, 17-AAG group, GCI in infarct volume was significantly reduce (P0.01), neuron survival significantly increased (P0.01), the ability of learning and memory ability of rats improved significantly (P0.01, P0.01), and Hsp90 and LC3B protein expression Was decreased significantly (P0.01, P0.01), the expression of Hsp70 protein increased significantly (P0.01). (2) compared with Con group, OGD for 1H and 2H, OGD/R can significantly promote the apoptosis percentage of neurons increased (P0.05, P0.01); OGD for 0.5h, 1H and 2H, OGD/R were able to significantly enhanced expression of apoptosis markers of Cleaved Caspase-3 (P0.05, P0.01, P0.01) and autophagy. (3) compared with OGD/R 1H, three concentrations of 17-AAG (0.5 M, 1 M, 2 M) can reduce the apoptosis percentage of OGD/R neurons (P0.05, P0.01, P0.01); 17-AAG concentration 1 M, 2 M significantly reduced the expression of Cleaved Caspase-3 protein in hippocampal neurons of OGD/R rats (P0.01, P0.01) and autophagy. Compared with OGD/R+17-AAG group, OGD/R+17-AAG+Rapamycin group, neuron autophagy, apoptosis rate and Cleaved expression of Caspase-3 protein, were Xian Shenggao (P0.05, P0.05). (4) compared with the Con group table Hsp70, OGD/R group Was decreased significantly (P0.01), and the expression of Hsp90 was significantly increased (P0.01); compared with OGD/R group, the expression of Hsp70 in OGD/R+17-AAG cells increased significantly (P0.05), and the expression of Hsp90 was significantly decreased (P0.05). Compared with OGD/R+17-AAG+Con, OGD/R+17-AAG+Hsp70 sh RNA group rat hippocampal neuronal apoptosis rate and expression. Autophagy in Cleaved Caspase-3 protein were increased significantly (P0.01, P0.05); compared with group OGD/R+17-AAG+NC, the apoptosis of hippocampal neurons in rats OGD/R+17-AAG+Hsp90 expression rate of Cleaved and autophagy Caspase-3 protein were significantly higher (P0.01, P0.05). Conclusion: in animal models of GCI, GCI induced neuronal death, reduce infarction, learning ability and the memory ability of rats; in vitro OGD/R, OGD/R promotes neuronal apoptosis, while 17-AAG can reduce the infarct volume and the percentage of neuronal death in rats and improve the learning ability and memory, at the same time Autophagy is regulated by Hsp70 and Hsp90 to improve the survival of the neurons.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R743.31

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳同度,張昌穎;素食大鼠的貧血現(xiàn)象[J];營(yíng)養(yǎng)學(xué)報(bào);1957年04期

2 陳偉強(qiáng);趙善廣;;自制注射用大鼠固定裝置[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);1992年04期

3 肖柳英,林培英,馮昭明,張丹;不同周齡的SD大鼠生理、生化及體重的正常值測(cè)定[J];中藥新藥與臨床藥理;1996年03期

4 李淑云;簡(jiǎn)易大鼠灌胃器的制作[J];錦州醫(yī)學(xué)院學(xué)報(bào);2001年04期

5 楊明智,陳積圣;一種大鼠抓取與固定的新工具介紹[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);2001年03期

6 戴英,陸群;復(fù)方H_(505)對(duì)Wistar大鼠外周血的血液流變學(xué)指標(biāo)的影響[J];中國(guó)血液流變學(xué)雜志;2001年01期

7 韋應(yīng)波,孫喜慶,曹新生,姚永杰,馮岱雅,楊長(zhǎng)斌;+Gz暴露時(shí)間對(duì)大鼠記憶功能和行為的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年01期

8 呂學(xué)軍,郭俊生,李敏,周利梅,張永娟;暈船大鼠體內(nèi)鐵含量的變化[J];中國(guó)職業(yè)醫(yī)學(xué);2003年04期

9 湯仁仙,王迎偉,王慧,周峰;201A中藥合劑對(duì)大鼠抗腎小球基底膜腎炎病變的影響[J];徐州醫(yī)學(xué)院學(xué)報(bào);2003年06期

10 孫同柱,付小兵,翁立新,梁雪梅,陳偉;介紹一種簡(jiǎn)易的大鼠保定方法[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);2004年01期

相關(guān)會(huì)議論文 前10條

1 尹音;孫振宇;胡敏;李冬霞;;持續(xù)性高正加速度對(duì)大鼠顳頜關(guān)節(jié)損傷的作用[A];第八屆全國(guó)顳下頜關(guān)節(jié)病學(xué)及（牙合）學(xué)大會(huì)論文匯編[C];2011年

2 祝~=驤;iJ梊霞;洃克琴;崔素英;文允摪;，

本文編號(hào)：1551695