17-AAG在SD大鼠短暂全脑缺血中的神经保护作用及机制研究

发布时间：2018-03-01 11:18

本文关键词： 17-AAG GCI OGD/R 大鼠海马神经元　出处：《重庆医科大学》2016年博士论文　论文类型：学位论文

【摘要】：背景与目的:在临床上,外科手术和心脏骤停导致的全脑低灌注是造成全脑缺血(Global cerebral ischemia,GCI)性损伤的重要原因。脑缺血后会引起神经细胞的不可逆死亡,从而导致神经病学和神经行为学上的障碍。在临床上,全脑缺血会导致记忆学习能力下降、痴呆等后遗症。因此,抑制神经细胞的不可逆死亡有望成为改善脑功能的的重要突破口。17-AAG(17-allylamino-demethoxygeldanamycin,17-AAG)即17-丙烯胺基,17-去甲氧基格尔德霉素作为Hsp90抑制剂在临床中是一种有效的抗肿瘤药物。据报道,17-AAG可以通过提高Hsp70的表达水平来保护创伤导致的脑损伤。因此,我们推测17-AAG在GCI中也可能具有重要的保护作用。本研究首先探索17-AAG在大鼠GCI模型中的神经保护作用;其次,分离培养大鼠的海马神经元,通过氧糖剥离/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)模拟大鼠在体GCI脑损伤,然后研究17-AAG对大鼠海马神经元凋亡的保护作用;最后,构建Hsp90表达和Hsp70干扰慢病毒,探讨17-AAG对大鼠海马神经元细胞保护作用的可能分子机制。方法:(1)将90只雌性SD大鼠随机的分成三组:假手术组(Sham)、全脑缺血再灌注组(GCI)、全脑缺血再灌注加17-AAG处理组(17-AAG),通过四血管闭塞法构建大鼠GCI模型。然后,通过TTC实验检测各组大鼠脑梗死体积,TUNEL实验检测神经元的凋亡情况,水迷宫实验检测各组大鼠的学习能力和记忆能力变化,Western blot检测各组大鼠脑组织中Hsp70、Hsp90、LC3B的蛋白表达。(2)分离培养大鼠海马神经元,通过OGD/R模拟大鼠在体GCI脑损伤,OGD时间为0.5h、1h、2h,再灌注时间为24h,TUNEL实验检测OGD/R对各组细胞凋亡的影响,然后Western blot检测OGD/R对各组细胞凋亡标志物Cleaved Caspase-3和自噬指标LC3B II、p62表达的影响。(3)通过TUNEL实验检测不同浓度的17-AAG(0.5μM、1.0μM、2.0μM)对OGD/R处理的神经元的凋亡的影响,然后Western blot检测凋亡标志物Cleaved Caspase-3和自噬指标LC3B II、p62表达的影响。然后,将细胞分为OGD/R组、OGD/R+17-AAG(1.0μM)组、OGD/R+17-AAG(1.0μM)+Rapamycin(1.0n M),通过TUNEL和Western blot研究自噬在17-AAG影响OGD/R处理的神经元凋亡中的角色。(4)通过Western blot研究17-AAG对OGD/R大鼠海马神经元中Hsp70和Hsp90表达的影响。构建Hsp90表达和Hsp70干扰慢病毒,将细胞分成四组:OGD/R+17-AAG+Con、OGD/R+17-AAG+Hsp70sh RNA、OGD/R+17-AAG+NC、OGD/R+17-AAG+Hsp90,通过TUNEL实验检测17-AAG是否通过Hsp90和Hsp70调节OGD/R大鼠海马神经元的凋亡,然后通过Western blot检测17-AAG是否通过Hsp90和Hsp70调节OGD/R大鼠海马神经元凋亡指标Cleaved Caspase-3和自噬指标LC3B II、p62的表达。结果:(1)与GCI组相比,17-AAG组GCI导致的梗死体积明显减少(P0.01),神经元存活明显增加(P0.01),大鼠的学习能力和记忆能力明显改善(P0.01,P0.01),同时Hsp90和LC3B蛋白表达明显降低(P0.01,P0.01),Hsp70蛋白表达明显增加(P0.01)。(2)与Con组相比,OGD时间为1h和2h时,OGD/R能够明显促进神经元的凋亡百分率的提高(P0.05,P0.01);OGD时间为0.5h,1h和2h时,OGD/R均能够明显增强凋亡标志物Cleaved Caspase-3的表达(P0.05,P0.01,P0.01)和自噬。(3)与OGD/R 1h相比,三种浓度的17-AAG(0.5μM、1.0μM、2.0μM)均可以降低OGD/R神经元的凋亡百分率(P0.05,P0.01,P0.01);17-AAG浓度为1.0μM、2.0μM时明显减少OGD/R大鼠海马神经元Cleaved Caspase-3蛋白的表达(P0.01,P0.01)和自噬。与OGD/R+17-AAG组相比,OGD/R+17-AAG+Rapamycin组神经元自噬、凋亡的百分率和Cleaved Caspase-3蛋白的表达、均明显升高(P0.05,P0.05)。(4)与Con组相比,OGD/R组Hsp70的表达明显降低(P0.01),而Hsp90的表达明显升高(P0.01);与OGD/R组相比,OGD/R+17-AAG组细胞中Hsp70的表达明显的升高(P0.05),而Hsp90的表达明显的降低(P0.05)。与OGD/R+17-AAG+Con相比,OGD/R+17-AAG+Hsp70 sh RNA组大鼠海马神经元凋亡率、Cleaved Caspase-3蛋白的表达和自噬均明显提高(P0.01,P0.05);与OGD/R+17-AAG+NC组相比,OGD/R+17-AAG+Hsp90大鼠海马神经元凋亡率、Cleaved Caspase-3蛋白的表达和自噬也均明显提高(P0.01,P0.05)。结论:在GCI动物模型中,GCI导致梗死、神经元死亡、大鼠的学习能力和记忆能力降低;在体外OGD/R实验中,OGD/R促进神经元凋亡,而17-AAG可以降低大鼠梗死体积和神经元死亡百分率并改善学习能力和记忆能力,同时通过Hsp70和Hsp90调节自噬来提高神经元的存活。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.31

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