Sestrin2沉默后加重大鼠蛛网膜下腔出血后早期脑损伤的作用研究

发布时间：2019-05-17 18:48

【摘要】：背景蛛网膜下腔出血(subarachnoid hemorrhage,SAH)具有极高的致残率及致死率,是一类在神经外科中较为常见的出血性脑血管疾病。临床发现大部分蛛网膜下腔出血导致的死亡都发生于72小时内,因此,有学者提出了“早期脑损伤”的概念,即蛛网膜下腔出血后72小时内,多因素造成整个脑组织的直接损伤,并认为蛛网膜下腔出血后早期脑损伤可能是导致患者预后不良的主要原因。蛛网膜下腔出血后脑损伤涉及一系列病理生理过程,其中氧化应激损伤在神经细胞死亡、血-脑屏障破坏及其他脑损伤中具有重要作用。Sestrin2(Sesn2)是一种诱导型应激蛋白,氧化应激能诱导其表达,通过抑制细胞内活性氧生成而发挥抗氧化作用,已有文献报道,Sesn2在神经退行性疾病中具有神经保护作用,我们前期研究也表明其在脑缺血再灌注损伤中具有一定的脑保护作用,但在蛛网膜下腔出血后诱发的早期脑损伤(early brain injury,EBI)中的作用机制仍然不太清楚。目的研究大鼠蛛网膜下腔出血后诱发的早期脑损伤中,Sesn2是否具有脑保护作用。方法1.通过视交叉前池注射法来建立大鼠蛛网膜下腔出血(sah)的模型。2.构建一条controlsirna及三条特异性sesn2sirna(sesn2-1、sesn2-2、sesn2-3),将sirna注射至大鼠左侧侧脑室,24小时后建立大鼠sah模型,用westernblot及real-timeqpcr方法检测其干扰效率,筛选其中最有效的sesn2sirna干扰片段。3.将实验动物随机分为假手术组(sham)、蛛网膜下腔出血组(sah)、scramblesirna+sah组、sesn2sirna+sah组。每组大鼠在sah后24小时处死。观察干扰sesn2基因后,大鼠神经功能损伤、脑水肿情况,测量脑组织evansblue含量评估血-脑屏障通透性,he染色观察神经元形态学改变,测定谷胱甘肽(gsh)和氧化型谷胱甘肽(gssg)浓度对氧化应激损伤进行评价。结果1.与sah组相比,三条特异性片段中sesn2-2干扰效果最佳,controlsirna无干扰效果。2.与sah组相比,干扰sesn2后,神经功能损伤加重,脑组织水含量明显增多,血-脑屏障通透性明显加强,神经元损伤加重,氧化应激明显增强。结论对sesn2进行干扰后,可加重蛛网膜下腔出血后早期脑损伤,从而说明sesn2具有一定的脑保护作用。
[Abstract]:Background subarachnoid hemorrhage (subarachnoid hemorrhage,SAH) is a common hemorrhagic cerebrovascular disease in neurosurgery because of its high disability rate and mortality. It has been found that most of the deaths caused by subarachnoid hemorrhage occur within 72 hours. Therefore, some scholars have put forward the concept of "early brain injury", that is, within 72 hours after subarachnoid hemorrhage. Multiple factors cause the direct injury of the whole brain tissue, and it is considered that the early brain injury after subarachnoid hemorrhage may be the main cause of poor prognosis. Brain injury after subarachnoid hemorrhage involves a series of pathophysiological processes, in which oxidative stress injury plays an important role in nerve cell death, blood-brain barrier destruction and other brain injuries. Sestrin 2 (Sesn2) is an induced stress protein. Oxidative stress can induce its expression and play an antioxidant role by inhibiting the production of reactive oxygen species in cells. It has been reported that Sesn2 plays a neuroprotective role in neurodegenerative diseases. Our previous studies have also shown that it has a certain cerebral protective effect in cerebral ischemia-reperfusion injury, but the mechanism of its role in early brain injury induced by subarachnoid hemorrhage (early brain injury,EBI) is still unclear. Objective to study whether Sesn2 has cerebral protective effect in early brain injury induced by subarachnoid hemorrhage in rats. Method 1. The (sah) model of subarachnoid hemorrhage in rats was established by injection of anterior optic chiasma cistern. 2. One controlsirna and three specific sesn2sirna (sesn2-1,sesn2-2,sesn2-3) were constructed and sirna was injected into the left lateral ventricle of rats. 24 hours later, the rat sah model was established. The interference efficiency was detected by westernblot and real-timeqpcr. Screening the most effective sesn2sirna interference fragments. 3. The experimental animals were randomly divided into (sham), subarachnoid hemorrhage group (sah), scramblesirna sah group and sesn2sirna sah group. The rats in each group were killed 24 hours after sah. After interfering with sesn2 gene, the nerve function injury and brain edema were observed, the evansblue content in brain tissue was measured to evaluate the permeability of blood-brain barrier, and the morphological changes of neurons were observed by he staining. The oxidative stress damage was evaluated by measuring the concentration of Glutathion (gsh) and oxidized Glutathion (gssg). Result 1. Compared with sah group, sesn2-2 interference effect was the best among the three specific fragments, and controlsirna had no interference effect. 2. Compared with sah group, after interfering with sesn2, the damage of nerve function was aggravated, the content of water in brain tissue was significantly increased, the permeability of blood-brain barrier was significantly enhanced, the damage of neurons was aggravated, and the oxidative stress was significantly enhanced. Conclusion interference with sesn2 can aggravate the early brain injury after subarachnoid hemorrhage, which indicates that sesn2 has certain cerebral protective effect.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.35

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