本文選題：烏司他丁 + 全腦缺血再灌注損傷　； 參考：《復(fù)旦大學(xué)》2014年碩士論文

【摘要】：目的：通過觀察烏司他丁對全腦缺血15min再灌注6h、24h、48h后大鼠血腦屏障通透性的變化及對小窩蛋白-1(Caveolin-1, CAV-1)、閉合小環(huán)蛋白-1 (Zonula Occludens-1, ZO-1)蛋白表達(dá)的改變,探討烏司他丁對全腦缺血再灌注損傷大鼠的腦保護(hù)作用。方法：清潔級健康雄性Wistar大鼠108只,體重240g-280g,隨機(jī)分為3組(n=12)：假手術(shù)組(Sham Group, S組)、全腦缺血再灌注模型組(Global Cerebral Ischemia/Reperfusion Injury Model Group, GCI/R組)、10,000U/Kg烏司他丁干預(yù)組(Ulinastatin Intervention Group,U組)。采用Pulsinelli四血管法(4-vessel occluding,4-VO)建立大鼠全腦缺血再灌注模型。在全腦缺血15min再灌注6h、24h、48h三個時間點,分別采用伊文思藍(lán)(Evans Blue, EB)染色法觀測全腦缺血再灌注損傷后血腦屏障(Blood Brain Barrier, BBB)的完整性；核磁共振(Magnetic resonance imaging, MRI)方法觀測全腦缺血再灌注后血腦屏障通透性的變化；應(yīng)用免疫組織化學(xué)法(Immunohistochemistry, IHC)和蛋白質(zhì)印跡法(Western Blotting, WB)觀測海馬區(qū)CAV-1和ZO-1的表達(dá)變化。結(jié)果：1、本實驗成功復(fù)制全腦缺血再灌注大鼠模型：大鼠在夾閉雙側(cè)頸總動脈后60s內(nèi)出現(xiàn)意識喪失；雙側(cè)瞳孔散大、角膜反射消失；四肢上舉；翻正反射消失。2、EB染色顯示全腦缺血再灌注后,大鼠血腦屏障完整性受到破環(huán)：S組在全腦缺血再灌注6h、24h、48h時均未見明顯藍(lán)染區(qū),測定腦組織內(nèi)EB含量少；GCI/R組再灌注6h、24h、48h時均可見明顯藍(lán)染區(qū),測定腦組織內(nèi)EB含量發(fā)現(xiàn),再灌注后6h時EB含量開始增多,再灌注24h時EB含量達(dá)高峰,然后開始下降,但與S組相比有顯著性差異(P0.05)；U組EB染色區(qū)域較GCI/R組小,測定腦組織內(nèi)EB含量發(fā)現(xiàn)全腦缺血再灌注后6h、24h、48h時與GCI/R相比有顯著性降低(P0.05),但仍高于S組。3、MRI顯示全腦缺血再灌注損傷后大鼠血腦屏障出現(xiàn)滲漏。S組在全腦缺血再灌注后6h、24h、48h三個時間點觀察到,海馬CA1區(qū)、側(cè)腦室、第三腦室及皮層區(qū)等區(qū)域在給予釓噴酸葡胺注射液(Gadopentetic Acid Dimeglumine Salt Injection, Gd-DTPA)前后信號變化差異不大；GCI/R組在全腦缺血再灌注后6h、24h、48h三個時間點觀察到,海馬CA1區(qū)、側(cè)腦室、第三腦室及皮層區(qū)等區(qū)域通過Gd-DTPA增強(qiáng)后,顯現(xiàn)顯著的強(qiáng)化信號,而且強(qiáng)化信號強(qiáng)度明顯高于S組,統(tǒng)計學(xué)有顯著性差異(P0.05)；U組在全腦缺血再灌注6h、24h、48h三個時間點經(jīng)Gd-DTPA增強(qiáng)后,顯現(xiàn)的強(qiáng)化信號均高于S組,但顯著低于GCI/R組,統(tǒng)計學(xué)有顯著性差異(P0.05)。4、在經(jīng)過全腦缺血再灌注損傷后海馬區(qū)CAV-1蛋白的表達(dá)出現(xiàn)變化。免疫組織化學(xué)顯示：在全腦缺血再灌注后6h、24h、48h海馬區(qū)均顯現(xiàn)S組CAV-1陽性細(xì)胞數(shù)目表達(dá)較多；GCI/R組CAV-1陽性細(xì)胞數(shù)目顯著下降,明顯低于S組水平,統(tǒng)計學(xué)有顯著性差異(P0.05)；U組在再灌注各時間點的CAV-1陽性細(xì)胞數(shù)目低于S組水平,但顯著高于GCI/R組,統(tǒng)計學(xué)有顯著性差異(P0.05)。同樣經(jīng)蛋白質(zhì)印跡進(jìn)行海馬區(qū)CAV-1蛋白的測定也顯示,全腦缺血再灌注后6h、24h、48h,GCI/R組大鼠海馬區(qū)內(nèi)CAV-1蛋白表達(dá)水平顯著低于S組和U組,統(tǒng)計學(xué)有顯著性差異(P0.05)；而U組的CAV-1蛋白表達(dá)水平低于S組。5、在經(jīng)過全腦缺血再灌注損傷后ZO-1蛋白的表達(dá)出現(xiàn)變化。免疫組織化學(xué)顯示：在全腦缺血再灌注后6h、24h、48h海馬區(qū)均顯現(xiàn)S組ZO-1陽性細(xì)胞數(shù)目表達(dá)較多；而GCI/R組ZO-1陽性細(xì)胞數(shù)目顯著下降,明顯低于S組水平,統(tǒng)計學(xué)有顯著性差異(P0.05)；而U組在再灌注后6h、24h、48h時間點的ZO-1陽性細(xì)胞數(shù)目低于S組水平,但顯著高于S組,統(tǒng)計學(xué)有顯著性差異(P0.05)。經(jīng)蛋白質(zhì)印跡進(jìn)行海馬區(qū)ZO-1蛋白的測定也顯示,全腦缺血再灌注后6h、24h、48h三時間點,GCI/R組大鼠海馬區(qū)內(nèi)ZO-1蛋白表達(dá)水平顯著低于S組和U組,統(tǒng)計學(xué)有顯著性差異(P0.05)；而U組的ZO-1蛋白表達(dá)水平低于S組。小結(jié)：1、全腦缺血再灌注損傷大鼠血腦屏障的完整性受到破環(huán),通透性增大,出現(xiàn)滲漏。2、全腦缺血再灌注損傷大鼠,在海馬區(qū)觀測到與血腦屏障構(gòu)成相關(guān)的CAV-1及ZO-1蛋白均表達(dá)下降。3、烏司他丁干預(yù)后全腦缺血再灌注損傷大鼠,全腦缺血再灌注損傷大鼠血腦屏障的通透性明顯改善。4、烏司他丁明顯改善全腦缺血再灌注損傷大鼠血腦屏障的通透性,可能與上調(diào)大鼠CAV-1及ZO-1蛋白水平的表達(dá)有關(guān)。
[Abstract]:Objective: To observe the changes in the permeability of blood brain barrier (BBB) in rats after 15min reperfusion of 6h, 24h, 48h and the changes in the expression of protein -1 (Caveolin-1, CAV-1) and closed small cyclic protein -1 (Zonula Occludens-1, ZO-1) in rats with 6h, 24h and 48h, and to explore the protective effect of ulinastatin on cerebral ischemia reperfusion injury in rats. 108 healthy male Wistar rats of clean grade and weight 240g-280g were randomly divided into 3 groups (n=12): the sham operation group (Sham Group, S group), the whole brain ischemia reperfusion model group (Global Cerebral Ischemia/Reperfusion Injury Model Group, group). The whole brain ischemia reperfusion model was established by four 4-vessel occluding (4-VO). At the three time points of 6h, 24h and 48h in the whole brain ischemia, the integrity of the blood brain barrier (Blood Brain) after cerebral ischemia-reperfusion injury was observed by the Evans blue (Evans Blue, EB) staining. The changes of blood brain barrier permeability after whole cerebral ischemia and reperfusion were observed by CE imaging, MRI). The expression changes of CAV-1 and ZO-1 in hippocampus were observed by immunohistochemistry (Immunohistochemistry, IHC) and protein blotting (Western Blotting, WB). Results: 1, the rat model of whole brain ischemia reperfusion was successfully replicated in this experiment: Rats The loss of consciousness occurred in 60s after occlusion of the bilateral common carotid artery; bilateral pupil was large and corneal reflex disappeared; the extremities were lifted in the extremities; the positive reflex disappeared.2, and the EB staining showed that after the whole brain ischemia reperfusion, the blood brain barrier integrity was broken in the rat: the S group had no obvious blue staining area at 6h, 24h and 48h in the whole brain, and the determination of E in the brain tissue The content of B was less; in group GCI/R, 6h, 24h and 48h were all obvious blue staining areas, and the content of EB in brain tissue was found. The content of EB began to increase when 6h was reperfusion, and the EB content reached the peak when reperfusion 24h, and then began to fall, but there was a significant difference compared with S group (P0.05). 6h, 24h, 48h were significantly lower than GCI/R (P0.05), but it was still higher than.3 in S group, but it was still higher than S group.3. MRI showed that the blood brain barrier in rats after total cerebral ischemia reperfusion injury was in.S group in 6h, 24h, 48h three time points after whole cerebral ischemia reperfusion, and the region of hippocampus, lateral ventricle, ventricle and cortex area were given. There was little difference between the signal changes before and after the Dimeglumine Gadopentetic Acid Injection (Gadopentetic Acid Dimeglumine Salt Injection, Gd-DTPA), and the GCI/R group observed three time points of 6h, 24h, 48h after whole cerebral ischemia and reperfusion, and the significant intensification signals were displayed in the CA1 region of the hippocampus, the lateral ventricle, the third ventricle and the cortex region. The intensification of signal intensity was significantly higher than that of the S group (P0.05). In group U, the intensification signals were higher than those in the S group at the three time points of 6h, 24h, 48h, but significantly lower than those in the GCI/R group, and there was a significant difference (P0.05).4, in the hippocampus after the whole brain ischemia reperfusion injury. The expression of white was changed. Immunohistochemical staining showed that the number of CAV-1 positive cells in group S was more expressed in 6h, 24h, and 48h hippocampus after cerebral ischemia and reperfusion, and the number of CAV-1 positive cells in group GCI/R decreased significantly, obviously lower than that in S group, and there was a significant difference (P0.05); U group was CAV-1 positive in each time point of reperfusion. The number of cells was lower than that of the S group, but it was significantly higher than that in the GCI/R group, and there was a significant difference (P0.05). The determination of CAV-1 protein in hippocampus of the hippocampus also showed that the expression of CAV-1 protein in the hippocampus of 6h, 24h, 48h, GCI/R group was significantly lower than that of S and U groups after cerebral ischemia and reperfusion, and there was a significant difference between the group of S and U (P0.05). The expression of CAV-1 protein in group U was lower than that of group S.5, and the expression of ZO-1 protein was changed after whole cerebral ischemia reperfusion injury. Immunohistochemistry showed that the number of ZO-1 positive cells in S group showed more expression in 6h, 24h and 48h hippocampus after whole cerebral ischemia and reperfusion, and the number of ZO-1 positive cells in GCI/R group decreased significantly, significantly lower than that of those in the GCI/R group. There was significant difference in group level and Statistics (P0.05), while the number of ZO-1 positive cells in group U was lower than that in group S after reperfusion at 6h, 24h and 48h, but significantly higher than that in group S (P0.05). The determination of ZO-1 protein in hippocampal region by Western blot also showed 6h, 24h, three time points after cerebral ischemia-reperfusion. The expression level of ZO-1 protein in the hippocampus of the rats was significantly lower than that in the S group and the U group, and there was a significant difference (P0.05), while the ZO-1 protein expression level in group U was lower than that in the S group. 1, the integrity of the blood brain barrier in the rats with whole cerebral ischemia reperfusion injury was damaged, the permeability increased, the leakage of.2, the whole brain ischemia-reperfusion injury rats, in the sea, in the sea. The expression of CAV-1 and ZO-1 protein related to the structure of blood brain barrier was decreased by.3. The permeability of blood brain barrier in rats with whole brain ischemia reperfusion injury was significantly improved by Ulinastatin, and the permeability of blood brain barrier in rats with whole brain ischemia reperfusion injury obviously improved the permeability of blood brain barrier in rats with cerebral ischemia-reperfusion injury. The expression of CAV-1 and ZO-1 protein was up-regulated in rats.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林惠文;;烏司他丁對心肺復(fù)蘇患者腦復(fù)蘇的作用及其預(yù)后的研究[J];中國實用醫(yī)藥;2012年24期

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本文編號：2079789