本文選題：TIGAR + 葡萄糖　； 參考：《蘇州大學》2015年碩士論文

【摘要】：目的:研究腦缺血再灌注損傷對TIGAR蛋白的調(diào)節(jié)及相應的分子機制。方法:采用小鼠短暫性大腦中動脈阻塞再灌注(transient middle cerebral artery occlusion/reperfusion,MCAO/R)模型和體外培養(yǎng)原代神經(jīng)元缺糖缺氧再復糖復氧(oxygen glucose dripivation/reoxygenation,OGD/R)模型。使用Western blot法檢測缺血后小鼠大腦皮層和原代皮層神經(jīng)元中TIGAR和p53蛋白的表達;在缺血前0.5 h給予p53抑制劑pifithrin-alpha(PFT-α,30μM),Western blot法檢測PFT-α對原代皮層神經(jīng)元中TIGAR以及p53靶蛋白Bax表達的影響;構建p53慢病毒感染原代皮層神經(jīng)元細胞敲除p53蛋白,檢測細胞內(nèi)TIGAR、p53、Bax蛋白的水平。使用強生血糖儀檢測小鼠缺血前后的血糖水平,Western blo法檢測胰島素(insulin,1 U/kg)對小鼠缺血皮層區(qū)TIGAR表達的影響。小鼠尾靜脈注射50%的葡萄糖溶液(500 g/kg),檢測血糖水平和大腦皮層區(qū)TIGAR的表達。采用不同濃度的葡萄糖溶液(0-150μM)刺激HT22細胞2 h,Western blot法檢測細胞內(nèi)TIGAR的水平,CCK8檢測細胞的活性以及在熒光顯微鏡下觀察HT22細胞的形態(tài)。小鼠側腦室注射腎上腺素(0.12mg/kg)、尾靜脈注射氫化可的松(50,100,200,400 mg/kg)以及胰高血糖素(0.1,0.5,1,5,10 mg/kg),Western-blot法檢測大腦皮層區(qū)TIGAR的水平;采用不同濃度的腎上腺素(0.1,1,10,50μM)、氫化可的松(0.01,0.1,1,10,100μM)、胰高血糖素(0.001,0.01,0.1,1,10μM)以及胰島素(0.0625,0.125,0.25,0.5,1μM)刺激HT22細胞3 h,檢測細胞內(nèi)TIGAR的水平。體外培養(yǎng)原代皮層神經(jīng)元細胞,采用過氧化氫(H2O2,10,30,90μM)共同培養(yǎng)(3,6,12 h)造氧化應激模型,CCK8檢測細胞存活率,GSH/GSSG試劑盒檢測細胞內(nèi)的GSH水平,DHE探針標記檢測細胞內(nèi)ROS變化以及Western-blot法檢測細胞內(nèi)TIGAR的水平。提前4 h給予抗氧化劑NADPH(10μM),檢測細胞內(nèi)TIGAR和ROS的水平。使用Western blot法檢測缺血后小鼠大腦皮層和原代皮層神經(jīng)元中SP1蛋白的表達;在缺血前24 h給予SP1抑制劑MIT(mithramycin A,300 n M),Western blot法檢測MIT對原代皮層神經(jīng)元中TIGAR表達的影響;構建SP1慢病毒感染原代皮層神經(jīng)元敲除SP1蛋白,檢測細胞內(nèi)TIGAR和SP1的蛋白水平。結果:小鼠腦缺血再灌注后,TIGAR水平明顯升高,在3 h達到峰值(P0.001)。原代皮層神經(jīng)元在OGD/R后,TIGAR和p53蛋白水平明顯升高,分別在3 h和6 h達到峰值(P0.01,P0.001)。Western blot結果顯示:使用p53抑制劑PFT-α后對缺血再灌注誘導的TIGAR的上升沒有影響,但是能夠明顯抑制p53靶蛋白Bax的水平(P0.001)。構建的p53慢病毒能夠顯著抑制原代皮層神經(jīng)元細胞中p53和Bax的蛋白水平,而對TIGAR的表達沒有明顯影響。小鼠腦缺血再灌注后,血糖水平明顯上升,在0.5 h達到峰值(P0.001),使用胰島素控制血糖水平后(P0.001),TIGAR的誘導表達被部分抑制了(P0.01)。給小鼠尾靜脈注射50%的葡萄糖溶液,血糖水平明顯上升,但是TIGAR的水平?jīng)]有明顯變化。體外培養(yǎng)海馬神經(jīng)元細胞系HT22細胞,加入葡萄糖繼續(xù)培養(yǎng)2 h,對TIGAR的表達、細胞活力以及形態(tài)均未造成明顯影響。HT22細胞在無糖預培養(yǎng)4 h后加入葡萄糖繼續(xù)培養(yǎng)2 h,Western blot結果顯示TIGAR的水平?jīng)]有變化。在體內(nèi)實驗中,Western blot結果顯示:腎上腺素能夠明顯誘導TIGAR蛋白(P0.001,P0.001,P0.001);3 h時氫化可的松能誘導TIGAR蛋白表達,且呈現(xiàn)劑量依賴性,50 mg/kg的氫化可的松在給藥后1 h和2 h時明顯激活TIGAR表達(P0.01,P0.01);0.5和1 mg/kg的胰高血糖素在給藥后2 h時能夠誘導TIGAR的表達(P0.05,P0.05)。體外實驗中,HT22細胞加入激素后繼續(xù)培養(yǎng)3 h,Western blot結果顯示:1μM的腎上腺素、0.1μM的氫化可的松和1μM的胰高血糖素能夠增加TIGAR的表達(P0.01,P0.001,P0.01),同時,低劑量的胰島素在一定低濃度范圍內(nèi)抑制TIGAR蛋白的表達而高劑量的胰島素能激活TIGAR(P0.01,P0.001)。培養(yǎng)原代皮層神經(jīng)元細胞,加入H2O2繼續(xù)培養(yǎng)造氧化應激模型,CCK-8、GSH、DHE和Western blot結果顯示:30和90μM的H2O2有效抑制了神經(jīng)元細胞的活力(P0.001,P0.001),降低了細胞內(nèi)的GSH水平(P0.05,P0.001),增加了細胞內(nèi)的ROS水平(P0.01,P0.01),同時在3 h時H2O2能夠明顯激活TIGAR的表達,且呈現(xiàn)劑量依賴性。提前4 h加入抗氧化劑NADPH,Western blot和DHE結果顯示:NADPH能夠部分抑制TIGAR的上調(diào)以及ROS的增加(P0.001,P0.05)。Western blot結果顯示:腦缺血再灌注后,小鼠大腦皮層區(qū)和原代皮層神經(jīng)元細胞中SP1蛋白明顯被激活,分別在0.5 h和1 h達到峰值(P0.001,P0.001),使用SP1抑制劑MIT后能夠明顯抑制缺血再灌注誘導的TIGAR的上升(P0.001),構建的SP1慢病毒能夠明顯抑制原代皮層神經(jīng)元細胞中SP1蛋白的表達(P0.001),同時能夠明顯抑制缺血再灌注誘導的TIGAR的上升(P0.001)。結論:腦缺血再灌注激活TIGAR和p53,并且TIGAR的變化不依賴于p53蛋白。腦缺血再灌注誘導血糖上升,控制血糖水平能夠部分抑制TIGAR的表達;葡萄糖不能直接調(diào)控TIGAR;升血糖激素(腎上腺素、氫化可的松、胰高血糖素)能夠誘導TIGAR,而降血糖激素胰島素在一定劑量范圍內(nèi)抑制TIGAR的表達。ROS能夠誘導TIGAR的表達,抑制ROS則抑制TIGAR的水平。腦缺血再灌注誘導SP1的表達,體外實驗證實,TIGAR受SP1的調(diào)節(jié)。
[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.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R743.3

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