乌司他丁对全脑缺血再灌注损伤大鼠血脑屏障通透性和Caveolin-1蛋白表达的影响

发布时间：2018-06-28 23:20

本文选题：乌司他丁 + 全脑缺血再灌注损伤　；参考：《复旦大学》2014年硕士论文

【摘要】：目的：通过观察乌司他丁对全脑缺血15min再灌注6h、24h、48h后大鼠血脑屏障通透性的变化及对小窝蛋白-1(Caveolin-1, CAV-1)、闭合小环蛋白-1 (Zonula Occludens-1, ZO-1)蛋白表达的改变,探讨乌司他丁对全脑缺血再灌注损伤大鼠的脑保护作用。方法：清洁级健康雄性Wistar大鼠108只,体重240g-280g,随机分为3组(n=12)：假手术组(Sham Group, S组)、全脑缺血再灌注模型组(Global Cerebral Ischemia/Reperfusion Injury Model Group, GCI/R组)、10,000U/Kg乌司他丁干预组(Ulinastatin Intervention Group,U组)。采用Pulsinelli四血管法(4-vessel occluding,4-VO)建立大鼠全脑缺血再灌注模型。在全脑缺血15min再灌注6h、24h、48h三个时间点,分别采用伊文思蓝(Evans Blue, EB)染色法观测全脑缺血再灌注损伤后血脑屏障(Blood Brain Barrier, BBB)的完整性；核磁共振(Magnetic resonance imaging, MRI)方法观测全脑缺血再灌注后血脑屏障通透性的变化；应用免疫组织化学法(Immunohistochemistry, IHC)和蛋白质印迹法(Western Blotting, WB)观测海马区CAV-1和ZO-1的表达变化。结果：1、本实验成功复制全脑缺血再灌注大鼠模型：大鼠在夹闭双侧颈总动脉后60s内出现意识丧失；双侧瞳孔散大、角膜反射消失；四肢上举；翻正反射消失。2、EB染色显示全脑缺血再灌注后,大鼠血脑屏障完整性受到破环：S组在全脑缺血再灌注6h、24h、48h时均未见明显蓝染区,测定脑组织内EB含量少；GCI/R组再灌注6h、24h、48h时均可见明显蓝染区,测定脑组织内EB含量发现,再灌注后6h时EB含量开始增多,再灌注24h时EB含量达高峰,然后开始下降,但与S组相比有显著性差异(P0.05)；U组EB染色区域较GCI/R组小,测定脑组织内EB含量发现全脑缺血再灌注后6h、24h、48h时与GCI/R相比有显著性降低(P0.05),但仍高于S组。3、MRI显示全脑缺血再灌注损伤后大鼠血脑屏障出现渗漏。S组在全脑缺血再灌注后6h、24h、48h三个时间点观察到,海马CA1区、侧脑室、第三脑室及皮层区等区域在给予钆喷酸葡胺注射液(Gadopentetic Acid Dimeglumine Salt Injection, Gd-DTPA)前后信号变化差异不大；GCI/R组在全脑缺血再灌注后6h、24h、48h三个时间点观察到,海马CA1区、侧脑室、第三脑室及皮层区等区域通过Gd-DTPA增强后,显现显著的强化信号,而且强化信号强度明显高于S组,统计学有显著性差异(P0.05)；U组在全脑缺血再灌注6h、24h、48h三个时间点经Gd-DTPA增强后,显现的强化信号均高于S组,但显著低于GCI/R组,统计学有显著性差异(P0.05)。4、在经过全脑缺血再灌注损伤后海马区CAV-1蛋白的表达出现变化。免疫组织化学显示：在全脑缺血再灌注后6h、24h、48h海马区均显现S组CAV-1阳性细胞数目表达较多；GCI/R组CAV-1阳性细胞数目显著下降,明显低于S组水平,统计学有显著性差异(P0.05)；U组在再灌注各时间点的CAV-1阳性细胞数目低于S组水平,但显著高于GCI/R组,统计学有显著性差异(P0.05)。同样经蛋白质印迹进行海马区CAV-1蛋白的测定也显示,全脑缺血再灌注后6h、24h、48h,GCI/R组大鼠海马区内CAV-1蛋白表达水平显著低于S组和U组,统计学有显著性差异(P0.05)；而U组的CAV-1蛋白表达水平低于S组。5、在经过全脑缺血再灌注损伤后ZO-1蛋白的表达出现变化。免疫组织化学显示：在全脑缺血再灌注后6h、24h、48h海马区均显现S组ZO-1阳性细胞数目表达较多；而GCI/R组ZO-1阳性细胞数目显著下降,明显低于S组水平,统计学有显著性差异(P0.05)；而U组在再灌注后6h、24h、48h时间点的ZO-1阳性细胞数目低于S组水平,但显著高于S组,统计学有显著性差异(P0.05)。经蛋白质印迹进行海马区ZO-1蛋白的测定也显示,全脑缺血再灌注后6h、24h、48h三时间点,GCI/R组大鼠海马区内ZO-1蛋白表达水平显著低于S组和U组,统计学有显著性差异(P0.05)；而U组的ZO-1蛋白表达水平低于S组。小结：1、全脑缺血再灌注损伤大鼠血脑屏障的完整性受到破环,通透性增大,出现渗漏。2、全脑缺血再灌注损伤大鼠,在海马区观测到与血脑屏障构成相关的CAV-1及ZO-1蛋白均表达下降。3、乌司他丁干预后全脑缺血再灌注损伤大鼠,全脑缺血再灌注损伤大鼠血脑屏障的通透性明显改善。4、乌司他丁明显改善全脑缺血再灌注损伤大鼠血脑屏障的通透性,可能与上调大鼠CAV-1及ZO-1蛋白水平的表达有关。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743

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