脑缺血再灌注中TIGAR蛋白调控的分子机制

发布时间：2018-05-15 19:59

本文选题：TIGAR + 葡萄糖　；参考：《苏州大学》2015年硕士论文

【摘要】：目的:研究脑缺血再灌注损伤对TIGAR蛋白的调节及相应的分子机制。方法:采用小鼠短暂性大脑中动脉阻塞再灌注(transient middle cerebral artery occlusion/reperfusion,MCAO/R)模型和体外培养原代神经元缺糖缺氧再复糖复氧(oxygen glucose dripivation/reoxygenation,OGD/R)模型。使用Western blot法检测缺血后小鼠大脑皮层和原代皮层神经元中TIGAR和p53蛋白的表达;在缺血前0.5 h给予p53抑制剂pifithrin-alpha(PFT-α,30μM),Western blot法检测PFT-α对原代皮层神经元中TIGAR以及p53靶蛋白Bax表达的影响;构建p53慢病毒感染原代皮层神经元细胞敲除p53蛋白,检测细胞内TIGAR、p53、Bax蛋白的水平。使用强生血糖仪检测小鼠缺血前后的血糖水平,Western blo法检测胰岛素(insulin,1 U/kg)对小鼠缺血皮层区TIGAR表达的影响。小鼠尾静脉注射50%的葡萄糖溶液(500 g/kg),检测血糖水平和大脑皮层区TIGAR的表达。采用不同浓度的葡萄糖溶液(0-150μM)刺激HT22细胞2 h,Western blot法检测细胞内TIGAR的水平,CCK8检测细胞的活性以及在荧光显微镜下观察HT22细胞的形态。小鼠侧脑室注射肾上腺素(0.12mg/kg)、尾静脉注射氢化可的松(50,100,200,400 mg/kg)以及胰高血糖素(0.1,0.5,1,5,10 mg/kg),Western-blot法检测大脑皮层区TIGAR的水平;采用不同浓度的肾上腺素(0.1,1,10,50μM)、氢化可的松(0.01,0.1,1,10,100μM)、胰高血糖素(0.001,0.01,0.1,1,10μM)以及胰岛素(0.0625,0.125,0.25,0.5,1μM)刺激HT22细胞3 h,检测细胞内TIGAR的水平。体外培养原代皮层神经元细胞,采用过氧化氢(H2O2,10,30,90μM)共同培养(3,6,12 h)造氧化应激模型,CCK8检测细胞存活率,GSH/GSSG试剂盒检测细胞内的GSH水平,DHE探针标记检测细胞内ROS变化以及Western-blot法检测细胞内TIGAR的水平。提前4 h给予抗氧化剂NADPH(10μM),检测细胞内TIGAR和ROS的水平。使用Western blot法检测缺血后小鼠大脑皮层和原代皮层神经元中SP1蛋白的表达;在缺血前24 h给予SP1抑制剂MIT(mithramycin A,300 n M),Western blot法检测MIT对原代皮层神经元中TIGAR表达的影响;构建SP1慢病毒感染原代皮层神经元敲除SP1蛋白,检测细胞内TIGAR和SP1的蛋白水平。结果:小鼠脑缺血再灌注后,TIGAR水平明显升高,在3 h达到峰值(P0.001)。原代皮层神经元在OGD/R后,TIGAR和p53蛋白水平明显升高,分别在3 h和6 h达到峰值(P0.01,P0.001)。Western blot结果显示:使用p53抑制剂PFT-α后对缺血再灌注诱导的TIGAR的上升没有影响,但是能够明显抑制p53靶蛋白Bax的水平(P0.001)。构建的p53慢病毒能够显著抑制原代皮层神经元细胞中p53和Bax的蛋白水平,而对TIGAR的表达没有明显影响。小鼠脑缺血再灌注后,血糖水平明显上升,在0.5 h达到峰值(P0.001),使用胰岛素控制血糖水平后(P0.001),TIGAR的诱导表达被部分抑制了(P0.01)。给小鼠尾静脉注射50%的葡萄糖溶液,血糖水平明显上升,但是TIGAR的水平没有明显变化。体外培养海马神经元细胞系HT22细胞,加入葡萄糖继续培养2 h,对TIGAR的表达、细胞活力以及形态均未造成明显影响。HT22细胞在无糖预培养4 h后加入葡萄糖继续培养2 h,Western blot结果显示TIGAR的水平没有变化。在体内实验中,Western blot结果显示:肾上腺素能够明显诱导TIGAR蛋白(P0.001,P0.001,P0.001);3 h时氢化可的松能诱导TIGAR蛋白表达,且呈现剂量依赖性,50 mg/kg的氢化可的松在给药后1 h和2 h时明显激活TIGAR表达(P0.01,P0.01);0.5和1 mg/kg的胰高血糖素在给药后2 h时能够诱导TIGAR的表达(P0.05,P0.05)。体外实验中,HT22细胞加入激素后继续培养3 h,Western blot结果显示:1μM的肾上腺素、0.1μM的氢化可的松和1μM的胰高血糖素能够增加TIGAR的表达(P0.01,P0.001,P0.01),同时,低剂量的胰岛素在一定低浓度范围内抑制TIGAR蛋白的表达而高剂量的胰岛素能激活TIGAR(P0.01,P0.001)。培养原代皮层神经元细胞,加入H2O2继续培养造氧化应激模型,CCK-8、GSH、DHE和Western blot结果显示:30和90μM的H2O2有效抑制了神经元细胞的活力(P0.001,P0.001),降低了细胞内的GSH水平(P0.05,P0.001),增加了细胞内的ROS水平(P0.01,P0.01),同时在3 h时H2O2能够明显激活TIGAR的表达,且呈现剂量依赖性。提前4 h加入抗氧化剂NADPH,Western blot和DHE结果显示:NADPH能够部分抑制TIGAR的上调以及ROS的增加(P0.001,P0.05)。Western blot结果显示:脑缺血再灌注后,小鼠大脑皮层区和原代皮层神经元细胞中SP1蛋白明显被激活,分别在0.5 h和1 h达到峰值(P0.001,P0.001),使用SP1抑制剂MIT后能够明显抑制缺血再灌注诱导的TIGAR的上升(P0.001),构建的SP1慢病毒能够明显抑制原代皮层神经元细胞中SP1蛋白的表达(P0.001),同时能够明显抑制缺血再灌注诱导的TIGAR的上升(P0.001)。结论:脑缺血再灌注激活TIGAR和p53,并且TIGAR的变化不依赖于p53蛋白。脑缺血再灌注诱导血糖上升,控制血糖水平能够部分抑制TIGAR的表达;葡萄糖不能直接调控TIGAR;升血糖激素(肾上腺素、氢化可的松、胰高血糖素)能够诱导TIGAR,而降血糖激素胰岛素在一定剂量范围内抑制TIGAR的表达。ROS能够诱导TIGAR的表达,抑制ROS则抑制TIGAR的水平。脑缺血再灌注诱导SP1的表达,体外实验证实,TIGAR受SP1的调节。
[Abstract]:Objective: To study the regulation and molecular mechanism of cerebral ischemia reperfusion injury to TIGAR protein. Methods: transient middle cerebral artery occlusion/reperfusion (MCAO/R) model of transient middle cerebral artery (artery occlusion/reperfusion, MCAO/R) model in mice and in vitro cultured primary neurons were cultured in vitro and oxygen deficiency and anoxia reoxygenation (oxygen glucose dripivation/re). Oxygenation, OGD/R) model. Western blot was used to detect the expression of TIGAR and p53 protein in the cerebral cortex and primary cortical neurons of the mice after ischemia. The p53 inhibitor pifithrin-alpha (PFT- alpha, 30 mu M) was given at 0.5 h before ischemia. The effect of alpha on the expression of the primary cortical neurons and the expression of the target protein was detected by Western restriction. P53 protein was knocked out of the primary cortical neurons of p53 lentivirus infection, and the level of TIGAR, p53 and Bax protein in the cells was detected. The blood glucose level of mice before and after ischemia was detected by Johnson glucose meter. The effect of insulin (insulin, 1 U/kg) on the expression of TIGAR in the ischemic cortex area of mice was detected by Western blo method. The injection of glucose in the tail vein of mice was 50%. The liquid (500 g/kg) was used to detect the level of blood sugar and the expression of TIGAR in the cerebral cortex area. The glucose solution of different concentrations (0-150 mu M) was used to stimulate the HT22 cell 2 h, the Western blot method was used to detect the level of TIGAR in the cells, the activity of the cells and the morphology of the HT22 cells were observed by the fluorescence microscope. The mice were injected with adrenaline (0.12mg/kg) in the lateral ventricle. The caudal vein was injected with hydrocortisone (50100200400 mg/kg) and glucagon (0.1,0.5,1,5,10 mg/kg), and Western-blot was used to detect the level of TIGAR in the cerebral cortex, with different concentrations of adrenaline (0.1,1,10,50 mu M), hydrocortisone (0.01,0.1,1,10100 u M), glucagon (0.001,0.01,0.1,1,10 mu M) and insulin (0.0625,0.) (0.0625,0.). 125,0.25,0.5,1 mu M) stimulated the HT22 cell 3 h to detect the level of TIGAR in the cells. The primary cultured cortical neurons were cultured in vitro, and the oxidative stress model was created by co culture of hydrogen peroxide (H2O2,10,30,90 M). The survival rate of the cells was detected by CCK8. The GSH level of the cells was detected by GSH/GSSG reagent box. The level of intracellular TIGAR was detected by Western-blot method. Antioxidant NADPH (10 u M) was given 4 h ahead of time to detect the level of TIGAR and ROS in cells. Western blot method was used to detect the expression of SP1 protein in the cerebral cortex and primary cortical neurons of the mice after ischemia, and 24 h before ischemia. The effect of MIT on the expression of TIGAR in the primary cortical neurons was detected by T, and the protein level of TIGAR and SP1 in the primary cortical neurons of the SP1 lentivirus infection was detected and the protein level of TIGAR and SP1 in the cells was detected. Results: after cerebral ischemia reperfusion in mice, the level of TIGAR increased significantly, and the peak value reached the peak at 3 h (P0.001). The white level was significantly higher, at the peak of 3 h and 6 h respectively (P0.01, P0.001).Western blot results showed that the use of p53 inhibitor PFT- alpha had no effect on the increase of TIGAR induced by ischemia-reperfusion, but could obviously inhibit Bax level of p53 target protein (P0.001). The constructed slow virus could significantly inhibit the primary cortical neurons cells. The protein level of p53 and Bax had no significant influence on the expression of TIGAR. After cerebral ischemia and reperfusion in mice, the blood glucose level increased significantly, reached a peak at 0.5 h (P0.001). After using insulin to control the blood glucose level (P0.001), the induced expression of TIGAR was partially inhibited (P0.01). The glucose level of the tail vein of mice was injected with 50% glucose solution. The blood glucose level was clear. There was no obvious change in the level of TIGAR. The cultured hippocampal neuron cell line HT22 cells were cultured in vitro, and the glucose continued to be 2 h. The expression of TIGAR, cell vitality and morphology did not significantly affect the.HT22 cells after the glucose free pre culture 4 h to add glucose to 2 h, Western blot results showed the level of TIGAR. In the in vivo experiment, Western blot results showed that adrenaline could obviously induce TIGAR protein (P0.001, P0.001, P0.001), and hydrogenated cortisone could induce TIGAR protein expression at 3 h, and showed a dose dependence. The 50 mg/kg hydrocortisone activated TIGAR expression obviously at 1 h and 2 h after administration; 0.5 and 1. Glucagon was able to induce TIGAR expression (P0.05, P0.05) at 2 h after administration. In vitro, HT22 cells added 3 h after adding hormone, and Western blot showed that 1 micron of adrenaline, 0.1 micron hydrocortisone and 1 micron M glucagon could increase the expression of TIGAR. Meanwhile, low doses of insulin In a certain low concentration, the expression of TIGAR protein was inhibited and the high dose of insulin activated TIGAR (P0.01, P0.001). The primary cultured cortical neurons were cultured, and H2O2 was added to the oxidative stress model. The results of CCK-8, GSH, DHE and Western blot showed that 30 and 90 mu M H2O2 effectively inhibited the activity of neuron cells. The intracellular GSH level (P0.05, P0.001) was reduced, and the ROS level in the cell (P0.01, P0.01) was increased. At the same time, the H2O2 could obviously activate the expression of TIGAR at the time of 3 h, and showed a dose dependence. 4 h added to the antioxidant NADPH. .Western blot results showed that after cerebral ischemia-reperfusion, the SP1 protein in the cerebral cortex and primary cortical neurons of the mice was activated obviously, reaching the peak value at 0.5 h and 1 h respectively (P0.001, P0.001). The SP1 inhibitor MIT could obviously inhibit the increase of TIGAR induced by ischemia-reperfusion (P0.001), and the SP1 lentivirus could be obvious. Inhibition of the expression of SP1 protein (P0.001) in the primary cortical neuron cells (P0.001), and can obviously inhibit the increase of TIGAR induced by ischemia-reperfusion (P0.001). Conclusion: cerebral ischemia reperfusion activates TIGAR and p53, and the changes of TIGAR are not dependent on p53 protein. Cerebral ischemia reperfusion induces blood glucose increase, and the control of blood glucose level can partly inhibit TIGAR The expression of glucose can not directly regulate TIGAR, and the hyperglycemic hormone (adrenaline, hydrocortisone, glucagon) can induce TIGAR, while hypoglycemic hormone insulin inhibits the expression of TIGAR in a certain dose and induces the expression of TIGAR and inhibits the level of ROS to inhibit TIGAR. The expression of SP1 is induced by cerebral ischemia-reperfusion. In fact, TIGAR is regulated by SP1.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3

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