脑缺血预处理对炎症相关因子表达的研究

发布时间：2018-06-29 21:34

本文选题：缺血预处理 + 缺血再灌注损伤　；参考：《河北北方学院》2017年硕士论文

【摘要】：随着人口老龄化的趋势加快,脑血管病已经成为我国第一位致残和死亡原因,对该病的研究一直是科研和临床研究的热点。脑缺血预处理(cerebral ischemic preconditioning,CIP)是指对脑组织采用缺血处理,如一次或多次短暂性、非致死性脑缺血再灌注(ischemia-reperfusion,I/R)刺激,启动脑组织产生内源性保护机制,将对致死性的缺血产生显著的耐受,从而减弱或阻止脑缺血缺氧引起的瀑布反应。炎性反应在缺血再灌注损伤的机制中占重要地位,包括一系列炎症细胞和炎症因子的参与,还涉及到固有免疫系统和适应性免疫系统的激活,起到了免疫调节和趋化作用,因此减轻炎症反应是治疗缺血性脑血管病的重要环节。炎症过程中,高迁移率族蛋白B1(high mobility group protein,HMGB1)主动分泌至胞外,与晚期糖基化终末产物受体(receptor for advanced glycation end-products,RAGE)和Toll样受体4(Toll-like receptor 4,TLR4)结合,最终激活核因子κB(Nuclear factor-kappa B,NF-κB),从而介导炎性反应。本研究通过建立大鼠局灶性CIP模型,深入研究RAGE和TLR4在CIP的生物学作用及可能机制,为再灌注损伤的临床防治提供新的靶点。将198只Sprague Dawley成年雄性健康大鼠随机分为假手术组(Sham,n=66)、缺血预处理组(CIP,n=66)和缺血再灌注组(I/R,n=66),采用改良的Zea-Longa方法制备大鼠大脑中动脉栓塞(MCAO)模型,缺血再灌注组给予缺血2小时后恢复血流,缺血预处理组采用二次线栓法,缺血10分钟为预处理措施,72小时后给予缺血2小时再恢复脑部血流,假手术组仅分离颈总动脉,不给于栓塞处理。随机分为再灌注后0.5 d,1 d,2 d,3 d,7 d共5个时间点,于再灌注1d时每组随机选取6只采用TTC染色法用于测定脑梗死体积,各个时间点采用Zea Longa评分标准进行神经功能评分,通过免疫组织化学染色法测定RAGE和TLR4蛋白水平的表达变化,定量即时聚合酶链锁反应(Real-time Quantitative Polymerase Chain Reaction,RT-qPCR)法测定RAGE和TLR4 mRNA表达。本研究发现,Sham组无梗死灶,CIP组在1 d时脑梗死体积为明显低于I/R组(P0.05);Sham组无神经功能缺损,CIP组大鼠在0.5 d、1 d、2 d、3 d、7 d的神经功能行为缺陷评分与I/R组大鼠比较明显减低,有显著性差异(P0.05);与sham组相比,CIP组与IR组缺血半暗带区RAGE阳性细胞数显著升高(P0.05),CIP组较I/R组的表达明显降低(P0.05),TLR4阳性细胞数也在缺血半暗带区明显升高,CIP组与IR组均显著高于sham组,CIP组低于I/R组,差异显著(P0.05)。CIP组和I/R组RAGE、TLR4 mRNA表达均显著高于sham组(P0.05),均于1 d时达高峰,随再灌注时间的延长两者表达逐渐下降,CIP组较I/R组的表达均明显降低(P0.05)。因此,脑缺血再灌注损伤可激活炎症介质,产生级联反应,CIP可使大脑产生缺血耐受,明显减轻炎症反应,并减小脑梗死体积,RAGE、TLR4在缺血损伤过程中表达上调,脑缺血耐受机制可能与下调缺血再灌注损伤时RAGE和TLR4的表达有关。
[Abstract]:With the aging of the population, cerebrovascular disease has become the first cause of disability and death in China, the research on the disease has been a hot spot in scientific research and clinical research. Cerebral ischemic preconditioning (cerebral ischemic preconditioning) refers to the use of ischemic treatment in brain tissues, such as once or more transient, non-fatal cerebral ischemia-reperfusion (I- / R) stimulation to activate endogenous protective mechanisms in brain tissue, which will produce significant tolerance to fatal ischemia. This weakens or blocks the waterfall response caused by cerebral ischemia and hypoxia. Inflammatory response plays an important role in the mechanism of ischemia-reperfusion injury, including the involvement of a series of inflammatory cells and inflammatory factors, as well as the activation of the innate and adaptive immune system, which plays an important role in immune regulation and chemotaxis. Therefore, reducing inflammatory reaction is an important link in the treatment of ischemic cerebrovascular disease. In the process of inflammation, the high mobility group protein B1 (high mobility group protein HMGB1 is secreted into the extracellular cells, which binds to the late glycation end product receptor (receptor for advanced glycation end-products rage and Toll-like receptor 4 (TLR4), and finally activates nuclear factor-kappa BNF- 魏 B, which mediates the inflammatory response. In order to provide a new target for clinical prevention and treatment of reperfusion injury, the biological function and possible mechanism of rage and TLR4 in CIP were studied by establishing focal CIP model in rats. 198 Sprague Dawley adult male healthy rats were randomly divided into three groups: sham operation group (Shambun 66), ischemic preconditioning group (CIP) and ischemia reperfusion group (I / R). The model of middle cerebral artery embolization (MCAO) was established by modified Zea-Longa method. In the ischemia reperfusion group, the blood flow was recovered after 2 hours of ischemia, and in the ischemic preconditioning group, the cerebral blood flow was recovered after 72 hours of ischemia for 72 hours, and the common carotid artery was only separated in the sham operation group. No embolization. The rats were randomly divided into 5 time points at 0.5 d ~ 1 d ~ 2 d ~ 3 d ~ 7 d after reperfusion. 6 rats in each group were randomly selected to measure the volume of cerebral infarction by TTC staining on the 1st day after reperfusion. The neurological function was evaluated by Zea Longa scoring standard at each time point. The expression of rage and TLR4 protein was detected by immunohistochemical staining, and the expression of rage and TLR4 mRNA was detected by Real-time quantitative Polymerase chain reaction (RT-PCR). It was found that the cerebral infarct volume in the CIP group without infarction was significantly lower than that in the I / R group at 1 day (P0.05). The neurological behavioral impairment scores in the CIP group were significantly lower than those in the I / R group at 0.5 d, 1 d, 2 d, 3 d and 7 d, respectively, compared with those in the I / R group. Compared with sham group and IR group, the number of rage positive cells in ischemic penumbra increased significantly (P0.05) the expression of rage positive cells in CIP group was significantly lower than that in I / R group (P0.05), and the number of TLR4 positive cells in ischemic penumbra was also significantly increased in sham group and IR group. Higher than the sham group, lower than the I / R group, The expression of RAGEN TLR4 mRNA in CIP group and I / R group was significantly higher than that in sham group (P0.05), and reached its peak at 1 day. The expression of RAGETLR4 mRNA in CIP group decreased gradually with the prolongation of reperfusion time (P0.05), and the expression of RAGETLR4 mRNA in CIP group was significantly lower than that in I / R group (P0.05). Therefore, cerebral ischemia-reperfusion injury can activate inflammatory mediators, produce cascade reaction CIP can induce cerebral ischemic tolerance, significantly reduce inflammatory response, and reduce the volume of cerebral infarction and up regulate the expression of RAGEN TLR4 during the process of ischemic injury. The mechanism of cerebral ischemic tolerance may be related to down-regulating the expression of rage and TLR4 in ischemic reperfusion injury.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

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