發(fā)布時間：2018-03-01 11:18

  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)報道,17-AAG可以通過提高Hsp70的表達(dá)水平來保護創(chuàng)傷導(dǎo)致的腦損傷。因此,我們推測17-AAG在GCI中也可能具有重要的保護作用。本研究首先探索17-AAG在大鼠GCI模型中的神經(jīng)保護作用;其次,分離培養(yǎng)大鼠的海馬神經(jīng)元,通過氧糖剝離/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對大鼠海馬神經(jīng)元凋亡的保護作用;最后,構(gòu)建Hsp90表達(dá)和Hsp70干擾慢病毒,探討17-AAG對大鼠海馬神經(jīng)元細(xì)胞保護作用的可能分子機制。方法:(1)將90只雌性SD大鼠隨機的分成三組: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),通過四血管閉塞法構(gòu)建大鼠GCI模型。然后,通過TTC實驗檢測各組大鼠腦梗死體積,TUNEL實驗檢測神經(jīng)元的凋亡情況,水迷宮實驗檢測各組大鼠的學(xué)習(xí)能力和記憶能力變化,Western blot檢測各組大鼠腦組織中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OGD/R模擬大鼠在體GCI腦損傷,OGD時間為0.5h、1h、2h,再灌注時間為24h,TUNEL實驗檢測OGD/R對各組細(xì)胞凋亡的影響,然后Western blot檢測OGD/R對各組細(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TUNEL實驗檢測不同濃度的17-AAG(0.5μM、1.0μM、2.0μM)對OGD/R處理的神經(jīng)元的凋亡的影響,然后Western blot檢測凋亡標(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Western blot研究17-AAG對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TUNEL實驗檢測17-AAG是否通過Hsp90和Hsp70調(diào)節(jié)OGD/R大鼠海馬神經(jīng)元的凋亡,然后通過Western blot檢測17-AAG是否通過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時間為1h和2h時,OGD/R能夠明顯促進神經(jīng)元的凋亡百分率的提高(P0.05,P0.01);OGD時間為0.5h,1h和2h時,OGD/R均能夠明顯增強凋亡標(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實驗中,OGD/R促進神經(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,同時通過Hsp70和Hsp90調(diào)節(jié)自噬來提高神經(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)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R743.31

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