發(fā)布時(shí)間：2018-08-07 11:30

【摘要】：研究背景新生兒缺氧缺血性腦病(Hypoxic-ischemic encephalopathy,HIE)通常是由于圍產(chǎn)期窒息后血流、氧氣中斷導(dǎo)致的腦損傷,其發(fā)病機(jī)制是一個(gè)復(fù)雜的病理過(guò)程,目前腦損傷的嚴(yán)重程度,持續(xù)時(shí)間,產(chǎn)前損傷的時(shí)間點(diǎn)難以準(zhǔn)確界定。HIE是新生兒圍產(chǎn)期最大的威脅之一[1-2]。近年來(lái),新生兒危重癥急救雖然取得了巨大發(fā)展,但HIE仍有較高的發(fā)病率,患兒遺留不同程度的神經(jīng)系統(tǒng)后遺癥甚至死亡[3],給家庭和社會(huì)造成沉重負(fù)。目前對(duì)于新生兒HIE的治療缺乏有效手段。HIE的發(fā)病機(jī)制并不十分明確。炎癥因子(細(xì)胞因子和趨化因子)參與了HIE的病理過(guò)程,在HIE發(fā)病機(jī)制中起到了十分重要的作用。由于目前HIE仍缺乏有效的治療方法,因此,探討HIE的發(fā)病機(jī)制及其可能的治療方法尤為必要。泛素羧基末端水解酶L1(Ubiquitin carboxy-terminal hydrolase L1,UCHL1)對(duì)于缺血缺氧后的神經(jīng)元存活起到了重要的作用。HIE的病理過(guò)程中炎癥反應(yīng)起到了至關(guān)重要的作用,但目前關(guān)于UCHL1參與調(diào)節(jié)腦損傷后的炎癥反應(yīng)的相關(guān)的相關(guān)文獻(xiàn)報(bào)道較少,且缺氧缺血性腦損傷(Hypoxic-Ischemic Brain Damage,HIBD)時(shí)UCHL1對(duì)于炎癥因子表達(dá)的影響并不十分明確。因此,本研究的目的是探討HIBD時(shí)UCHL1蛋白對(duì)神經(jīng)元的作用以及UCHL1蛋白對(duì)炎癥因子(白介素-6,IL-6;白介素-10,IL-10;腫瘤壞死因子-α,TNF-α)表達(dá)的影響。目的探討UCHL1蛋白在缺氧缺血性腦損傷(HIBD)中的作用以及UCHL1蛋白對(duì)炎癥因子(白介素-6,IL-6;白介素-10,IL-10;腫瘤壞死因子-α,TNF-α)表達(dá)的影響。材料與方法7日齡SD大鼠(40只)隨機(jī)分為正常對(duì)照組(20只)和缺氧缺血(HI)組(20只),其中HI組給予缺氧缺血處理,建立HIBD動(dòng)物模型,空白對(duì)照組不予處理。PC12細(xì)胞用于建立氧糖剝奪(OGD)細(xì)胞模型。PC12細(xì)胞分為:正常對(duì)照組,OGD組,LDN(即LDN-57444,UCHL1蛋白抑制劑)+OGD組,UCHL1+OGD組。各組細(xì)胞處理因素:正常對(duì)照組:正常條件培養(yǎng);OGD組:氧糖剝奪處理細(xì)胞,不予UCHL1蛋白或LDN預(yù)處理細(xì)胞;LDN+OGD組:LDN預(yù)先加入細(xì)胞培養(yǎng)基中作用2h,然后給予OGD處理4h;UCHL1+OGD組:UCHL1預(yù)先加入細(xì)胞培養(yǎng)基中作用2h,然后給予OGD處理4h。6h和24h用免疫組化法檢測(cè)UCHL1在幼鼠腦組織中的表達(dá)情況。24h,48h,72h用Hoeschst33258染色法檢測(cè)PC12細(xì)胞凋亡情況,ELISA試劑盒檢測(cè)PC12細(xì)胞上清液中IL-6、IL-10、TNF-α、UCHL1濃度。SPSS17.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)處理。結(jié)果UCHL1蛋白在HI組幼鼠中表達(dá)較正常對(duì)照組升高;在同一時(shí)間點(diǎn),各組PC12細(xì)胞凋亡情況各不相同;同正常對(duì)照組相比,OGD組和LDN+OGD組TNF-α表達(dá)水平在24h,48h,72h均升高(P0.05);同OGD組相比,LDN+OGD組TNF-α表達(dá)水平在24h,48h,72h均升高(P0.05),UCHL1+OGD組TNF-α表達(dá)水平在24h,48h,72h均降低(P0.05)。同正常對(duì)照組相比,OGD組IL-6表達(dá)水平在72h升高(P0.05),LDN+OGD組IL-6表達(dá)水平在24h,48h,72h均升高(P0.05);同OGD組相比,LDN+OGD組IL-6表達(dá)水平在24h,48h,72h均升高(P0.05);同OGD組相比,UCHL1+OGD組IL-6表達(dá)水平在24h,48h降低(P0.05)。同正常對(duì)照組相比,OGD組IL-10表達(dá)水平在24h,48h升高(P0.05),LDN+OGD組IL-10表達(dá)水平在24h,48h,72h均升高(P0.05);同OGD組相比,LDN+OGD組IL-10表達(dá)水平在24h,48h,72h均降低(P0.05);同OGD組相比,UCHL1+OGD組IL-10表達(dá)水平在24h,48h升高(P0.05)。同正常對(duì)照組相比,OGD組UCHL1表達(dá)水平在24h,48h,72h均升高(P0.05);同OGD組相比,LDN+OGD組UCHL1表達(dá)水平在24h,48h升高(P0.05)。結(jié)論HIBD后UCHL1蛋白對(duì)于神經(jīng)元細(xì)胞的存活至關(guān)重要,UCHL1蛋白對(duì)于HI時(shí)神經(jīng)元細(xì)胞具有保護(hù)作用,UCHL1蛋白的腦保護(hù)作用機(jī)制可能是通過(guò)下調(diào)TNF-α和IL-6的表達(dá)以及上調(diào)IL-10的表達(dá)發(fā)揮作用。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R742

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