泛素羧基末端水解酶L1对缺氧缺血性脑损伤作用及其机制探讨

发布时间：2018-08-07 11:30

【摘要】：研究背景新生儿缺氧缺血性脑病(Hypoxic-ischemic encephalopathy,HIE)通常是由于围产期窒息后血流、氧气中断导致的脑损伤,其发病机制是一个复杂的病理过程,目前脑损伤的严重程度,持续时间,产前损伤的时间点难以准确界定。HIE是新生儿围产期最大的威胁之一[1-2]。近年来,新生儿危重症急救虽然取得了巨大发展,但HIE仍有较高的发病率,患儿遗留不同程度的神经系统后遗症甚至死亡[3],给家庭和社会造成沉重负。目前对于新生儿HIE的治疗缺乏有效手段。HIE的发病机制并不十分明确。炎症因子(细胞因子和趋化因子)参与了HIE的病理过程,在HIE发病机制中起到了十分重要的作用。由于目前HIE仍缺乏有效的治疗方法,因此,探讨HIE的发病机制及其可能的治疗方法尤为必要。泛素羧基末端水解酶L1(Ubiquitin carboxy-terminal hydrolase L1,UCHL1)对于缺血缺氧后的神经元存活起到了重要的作用。HIE的病理过程中炎症反应起到了至关重要的作用,但目前关于UCHL1参与调节脑损伤后的炎症反应的相关的相关文献报道较少,且缺氧缺血性脑损伤(Hypoxic-Ischemic Brain Damage,HIBD)时UCHL1对于炎症因子表达的影响并不十分明确。因此,本研究的目的是探讨HIBD时UCHL1蛋白对神经元的作用以及UCHL1蛋白对炎症因子(白介素-6,IL-6;白介素-10,IL-10;肿瘤坏死因子-α,TNF-α)表达的影响。目的探讨UCHL1蛋白在缺氧缺血性脑损伤(HIBD)中的作用以及UCHL1蛋白对炎症因子(白介素-6,IL-6;白介素-10,IL-10;肿瘤坏死因子-α,TNF-α)表达的影响。材料与方法7日龄SD大鼠(40只)随机分为正常对照组(20只)和缺氧缺血(HI)组(20只),其中HI组给予缺氧缺血处理,建立HIBD动物模型,空白对照组不予处理。PC12细胞用于建立氧糖剥夺(OGD)细胞模型。PC12细胞分为:正常对照组,OGD组,LDN(即LDN-57444,UCHL1蛋白抑制剂)+OGD组,UCHL1+OGD组。各组细胞处理因素:正常对照组:正常条件培养;OGD组:氧糖剥夺处理细胞,不予UCHL1蛋白或LDN预处理细胞;LDN+OGD组:LDN预先加入细胞培养基中作用2h,然后给予OGD处理4h;UCHL1+OGD组:UCHL1预先加入细胞培养基中作用2h,然后给予OGD处理4h。6h和24h用免疫组化法检测UCHL1在幼鼠脑组织中的表达情况。24h,48h,72h用Hoeschst33258染色法检测PC12细胞凋亡情况,ELISA试剂盒检测PC12细胞上清液中IL-6、IL-10、TNF-α、UCHL1浓度。SPSS17.0统计学软件进行数据处理。结果UCHL1蛋白在HI组幼鼠中表达较正常对照组升高;在同一时间点,各组PC12细胞凋亡情况各不相同;同正常对照组相比,OGD组和LDN+OGD组TNF-α表达水平在24h,48h,72h均升高(P0.05);同OGD组相比,LDN+OGD组TNF-α表达水平在24h,48h,72h均升高(P0.05),UCHL1+OGD组TNF-α表达水平在24h,48h,72h均降低(P0.05)。同正常对照组相比,OGD组IL-6表达水平在72h升高(P0.05),LDN+OGD组IL-6表达水平在24h,48h,72h均升高(P0.05);同OGD组相比,LDN+OGD组IL-6表达水平在24h,48h,72h均升高(P0.05);同OGD组相比,UCHL1+OGD组IL-6表达水平在24h,48h降低(P0.05)。同正常对照组相比,OGD组IL-10表达水平在24h,48h升高(P0.05),LDN+OGD组IL-10表达水平在24h,48h,72h均升高(P0.05);同OGD组相比,LDN+OGD组IL-10表达水平在24h,48h,72h均降低(P0.05);同OGD组相比,UCHL1+OGD组IL-10表达水平在24h,48h升高(P0.05)。同正常对照组相比,OGD组UCHL1表达水平在24h,48h,72h均升高(P0.05);同OGD组相比,LDN+OGD组UCHL1表达水平在24h,48h升高(P0.05)。结论HIBD后UCHL1蛋白对于神经元细胞的存活至关重要,UCHL1蛋白对于HI时神经元细胞具有保护作用,UCHL1蛋白的脑保护作用机制可能是通过下调TNF-α和IL-6的表达以及上调IL-10的表达发挥作用。
[Abstract]:Background neonatal hypoxic and ischemic encephalopathy (Hypoxic-ischemic encephalopathy, HIE) is usually due to cerebral injury caused by blood flow and oxygen interruption after perinatal asphyxia. The pathogenesis is a complicated pathological process. The severity, duration, and time points of the brain injury are difficult to accurately define the.HIE as the newborn. One of the greatest perinatal threats [1-2]. in recent years, although great progress has been made in neonatal critical care, but HIE still has a high incidence, the children left with different degrees of nervous system sequelae and even death [3], causing heavy burden to the family and society. At present, the treatment of neonatal HIE is lack of the effective means of.HIE. It is not very clear that inflammatory factors (cytokines and chemokines) are involved in the pathological process of HIE and play a very important role in the pathogenesis of HIE. As HIE is still lacking in effective treatment, it is particularly necessary to explore the pathogenesis of HIE and its possible treatment methods. The ubiquitin carboxyl terminal hydrolase L1 (Ubiquitin CA) Rboxy-terminal hydrolase L1, UCHL1) plays an important role in the pathological process of.HIE, which plays an important role in the pathological process of neuronal survival after ischemic anoxia, but there are few related literature related to the involvement of UCHL1 in regulating the inflammatory response after brain injury (Hypoxic-Ische), and hypoxic-ischemic brain damage (Hypoxic-Ische). The effect of UCHL1 on the expression of inflammatory factors is not very clear when mic Brain Damage, HIBD). Therefore, the purpose of this study is to explore the effect of UCHL1 protein on the neuron and the effect of UCHL1 protein on the expression of inflammatory factors (il--6, IL-6; -10, IL-10; alpha, alpha). Effects of oxygen ischemic brain injury (HIBD) and the effect of UCHL1 protein on the expression of inflammatory factors (IL -6, IL-6; interleukin -10, IL-10; tumor necrosis factor - alpha, TNF- alpha). Materials and methods 7 day old SD rats (40 rats) were randomly divided into normal control group (20 rats) and hypoxic-ischemic (HI) group (20 rats), of which the HI group was given hypoxia ischemia treatment, and HIB was established. HIB D animal model, blank control group did not treat.PC12 cells to establish oxygen sugar deprivation (OGD) cell model.PC12 cells divided into normal control group, OGD group, LDN (LDN-57444, UCHL1 protein inhibitor) +OGD group, UCHL1+OGD group. The cell processing factors in each group: normal control group: normal condition culture; OGD group: oxygen sugar deprivation treatment cells, no UCHL1 eggs White or LDN pretreated cells; group LDN+OGD: LDN was pre added to the cell culture medium to act 2h, and then treated with OGD to treat 4H; UCHL1+OGD group: UCHL1 was pre added to the cell medium to act as 2H. Then OGD processing 4h.6h and 24h immunohistochemistry were used to detect the expression in the brain tissue of young mice. PC12 cell apoptosis, ELISA kit detection of PC12 cell supernatant IL-6, IL-10, TNF- alpha, UCHL1 concentration.SPSS17.0 statistics software for data processing. Results UCHL1 protein expression in the HI group is higher than the normal control group; at the same time point, PC12 cell apoptosis in each group is not the same; and the normal control group compared with the OGD group and the The expression level of TNF- alpha in +OGD group increased in 24h, 48h and 72h (P0.05). Compared with the OGD group, the expression level of TNF- alpha in LDN+OGD group was increased in 24h, 48h, and 72h all increased. Compared with the OGD group, the expression level of IL-6 in LDN+OGD group increased in 24h, 48h and 72h (P0.05). Compared with the OGD group, IL-6 expression level in the UCHL1+OGD group was lower than that of the normal control group. The expression level of IL-10 in LDN+OGD group decreased in 24h, 48h and 72h (P0.05). Compared with the OGD group, the IL-10 expression level in UCHL1+OGD group was higher in 24h and 48h (P0.05) than in the normal control group. Protein is very important for the survival of neuron cells. UCHL1 protein has protective effect on HI neurons. The mechanism of UCHL1 protein's brain protection may play a role by downregulating the expression of TNF- alpha and IL-6 and up regulation of the expression of IL-10.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742

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