BACE-1在大鼠嗅球内分布、活性和调节的初步研究

发布时间：2018-01-03 17:08

本文关键词：BACE-1在大鼠嗅球内分布、活性和调节的初步研究　出处：《中南大学》2007年博士论文　论文类型：学位论文

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【摘要】： 目的:通过研究β-位点-淀粉样前体蛋白剪切酶-1(β-site-APP cleavageenzyme-1,BACE-1)在成年健康SD大鼠嗅球内的分布、活性以及嗅觉剥夺(模拟神经元功能下降/抑制)对BACE-1表达和活性的调节,初步探讨BACE-1在大脑/神经元功能/代谢下降中的生物学作用,为AD早期治疗提供新的思路。方法: 第一部分:通过Western blot对成年健康SD大鼠脑组织中含有不同数量糖基的BACE-1进行检测;通过BACE-1脱糖基实验结合Western blot和BACE-1活性检测等方法对带有不同数量糖基的BACE-1活性进行分析;采用BACE-1基因野生型小鼠脑组织作为阳性对照,BACE-1基因敲除小鼠脑组织作为阴性对照对BACE-1抗体特异性进行检测。第二部分:通过多种BACE-1抗体用免疫组织化学染色方法结合嗅球尼氏染色以及免疫荧光化学多重标记染色方法检测成年健康SD大鼠嗅球BACE-1定位分布。第三部分:制作成年健康SD大鼠单侧嗅觉剥夺模型,84只动物随机分成左侧嗅觉剥夺组56只、假手术组7只、正常对照组21只。每组又根据不同的存活时间(4、7、14、21、30、45或60天)将动物随机分成7个组。左侧嗅觉剥夺组大鼠右侧鼻孔保持通畅作为自身对照。另外8只动物(嗅觉剥夺组4只、假手术组4只)在存活30天后处死进行生物化学研究。通过免疫组织化学染色方法检测成年娇y"D大鼠单侧嗅觉剥夺后双侧嗅球BACE-1表达;通过免疫荧光染色方法检测双侧嗅球的BACE-1与tyrosine hydroxylase(TH),olfactorymarker protein(OMP),synaptophysin,amyloid precursor protein(APP)和presenilin-1(PS-1)等共定位表达;通过免疫组织化学染色结合cytochrome oxidase/succidate dehydrogenase(CO/SDH)组织化学染色方法研究成年健康SD大鼠同一个嗅小球以及单侧嗅觉剥夺后双侧嗅球的BACE-1表达与CO/SDH活性进行相关性研究;Western blot检测双侧嗅球BACE-1与其剪切产物(β-CTF)蛋白量;通过BACE-1和γ-secretases活性检测等方法对双侧嗅球进行BACE-1以及γ-secretases活性检测:通过ELISA检测成年健康SD大鼠单侧嗅觉剥夺后双侧嗅球APP降解通路终产物(Aβ)量。结果: 第一部分:成年健康SD大鼠脑组织中Western blot显示出多条不同分子量的BACE-1形式,不同分子量的BACE-1在脱糖基处理后表现出相同的酶剪切活性。第二部分:嗅球内有大量BACE-1免疫阳性产物,免疫阳性产物主要分布于嗅神经纤维和嗅小球层,单个嗅小球之间的BACE-1表达程度不同。嗅球中BACE-1和synaptophysin与OMP共表达。第三部分:成年健康SD大鼠单个嗅球BACE-1表达与其CO/SDH活性相反;嗅觉剥夺后嗅球CO/SDH活性下降,BACE-1表达和活性显著上调;β-CTF和Aβ产生显著增加;APP和PS-1未见明显变化。结论:成年健康SD大鼠脑组织同时存在带有多个糖基的BACE-1形式,并且BACE-1的去糖基化可能没有明显改变其活性;成年健康SD大鼠嗅球内有大量BACE-1表达,BACE-1主要定位于嗅神经纤维和嗅小球中的嗅神经轴突终末;成年健康SD大鼠嗅觉剥夺后引起嗅球的BACE-1表达和活性显著上调,而且伴随有其酶切产物(β-CTF和Aβ)的显著增加,双侧嗅球APP和γ-secretases/PS-1表达和活性未见明显差异,提示嗅觉剥夺模拟的神经元活性/代谢降低状态可能通过上调BACE-1表达和活性途径而不是γ-secretases途径来引起Aβ过度产生。
[Abstract]:Objective: To study the beta site amyloid precursor protein cleaving enzyme (beta -site-APP cleavageenzyme-1, -1 BACE-1) distributed in the olfactory bulb of adult healthy SD rats, and the activity of olfactory deprivation (analog neuron function decline / inhibition) to regulate the expression and activity of BACE-1, a preliminary exploration of the biological effect of BACE-1 during brain / neuronal function / metabolism decline, and provide a new way for the early treatment of AD.
Method:
The first part: through the Western blot of adult SD rat brain tissue containing varying amounts of sugar residues were detected by BACE-1; BACE-1 desugarization based experiment combined with Western blot and BACE-1 activity detection methods with different number of sugar based BACE-1 activity was analyzed; the brain tissue BACE-1 gene in wild-type mice as a positive control. BACE-1 gene knockout mice brain tissue as negative control were used to detect BACE-1 antibody specificity.
The second part: the distribution of BACE-1 in the olfactory bulb of adult healthy SD rats was detected by immunohistochemical staining combined with olfactory bulb Nissl staining and immunofluorescence chemical multiple labeling method. The results were as follows: BACE-1.
The third part: the production of healthy adult SD rats with unilateral olfactory deprivation model, 84 rats were randomly divided into left animal olfactory deprivation group 56 rats, 7 rats in the sham operation group, normal control group 21 rats in each group. According to the different survival time (4,7,14,21,30,45 or 60 days) of the animal were randomly divided into 7 groups. The left olfactory deprivation group in the right nostril to maintain patency as own control. The other 8 animal (olfactory deprivation group 4 rats, 4 rats in sham group) in survival after 30 days of biochemistry. By immunohistochemical staining method to detect adult y D rats Johnson "unilateral olfactory stripping of bilateral olfactory bulb BACE-1 expression from BACE-1 and tyrosine; hydroxylase method for detection of bilateral olfactory bulb staining by immunofluorescence (TH), olfactorymarker protein (OMP), synaptophysin, amyloid precursor protein (APP) and presenilin-1 (PS-1) as co expression by immunohistochemical staining; Combined with cytochrome oxidase/succidate dehydrogenase (CO/SDH) histochemical staining method of healthy adult SD rats of the same glomerulus and unilateral olfactory deprivation after bilateral olfactory bulb BACE-1 expression and CO/SDH activity relationship study; Western blot detection of bilateral olfactory bulb BACE-1 and its cleavage products (beta -CTF) protein by BACE-1 and gamma; -secretases activity detection the method of bilateral olfactory bulb were detected BACE-1 and -secretases gamma activity: healthy adult SD rats with unilateral olfactory deprivation after bilateral olfactory bulb APP degradation pathway end products detected by ELISA (A 3).
Result:
Part one: in adult healthy SD rats, Western blot showed multiple BACE-1 forms with different molecular weights. BACE-1 with different molecular weight showed the same enzyme shearing activity after deglycosylation.
The second part: there are a large number of BACE-1 immunoreactive products in the olfactory bulb, and the immunoreactive products are mainly distributed in the olfactory nerve fiber and olfactory glomerulus. The expression level of BACE-1 between single olfactory bulbs is different. The expression of BACE-1 and synaptophysin in the olfactory bulb is co expressed with OMP.
The third part: BACE-1 expression in single olfactory bulb of adult healthy SD rats is opposite to CO/SDH activity. After olfactory deprivation, CO/SDH activity of olfactory bulb decreases, BACE-1 expression and activity are upregulated significantly, beta -CTF and A beta increase significantly, APP and PS-1 have no obvious change.
Conclusion: the rat brain tissue of healthy adult SD exist at the same time with multiple glycosylated form of BACE-1, and the deglycosylation of BACE-1 may not significantly alter their activity; a large number of BACE-1 expression in the olfactory bulb of adult healthy SD rats, BACE-1 was mainly located in the olfactory nerve fibers and glomeruli in the olfactory axon terminals; healthy adult SD rats of olfactory deprivation caused by the expression and activity of BACE-1 was significantly up-regulated in the olfactory bulb, but also its digestion products (beta -CTF and A beta) increased significantly, bilateral olfactory bulb APP and gamma -secretases/PS-1 expression and activity showed no significant difference, indicating neuronal activity / olfactory deprivation metabolic state may reduce the simulation by up regulating the expression and activity of BACE-1 pathway but not -secretases pathway caused by A beta gamma overproduction.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R322

【共引文献】