發(fā)布時(shí)間：2018-01-03 17:08

  本文關(guān)鍵詞：BACE-1在大鼠嗅球內(nèi)分布、活性和調(diào)節(jié)的初步研究　出處：《中南大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： BACE-1 AD SD大鼠 嗅覺剝奪 Aβ

【摘要】： 目的:通過研究β-位點(diǎn)-淀粉樣前體蛋白剪切酶-1(β-site-APP cleavageenzyme-1,BACE-1)在成年健康SD大鼠嗅球內(nèi)的分布、活性以及嗅覺剝奪(模擬神經(jīng)元功能下降/抑制)對(duì)BACE-1表達(dá)和活性的調(diào)節(jié),初步探討B(tài)ACE-1在大腦/神經(jīng)元功能/代謝下降中的生物學(xué)作用,為AD早期治療提供新的思路。 方法: 第一部分:通過Western blot對(duì)成年健康SD大鼠腦組織中含有不同數(shù)量糖基的BACE-1進(jìn)行檢測(cè);通過BACE-1脫糖基實(shí)驗(yàn)結(jié)合Western blot和BACE-1活性檢測(cè)等方法對(duì)帶有不同數(shù)量糖基的BACE-1活性進(jìn)行分析;采用BACE-1基因野生型小鼠腦組織作為陽性對(duì)照,BACE-1基因敲除小鼠腦組織作為陰性對(duì)照對(duì)BACE-1抗體特異性進(jìn)行檢測(cè)。 第二部分:通過多種BACE-1抗體用免疫組織化學(xué)染色方法結(jié)合嗅球尼氏染色以及免疫熒光化學(xué)多重標(biāo)記染色方法檢測(cè)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大鼠單側(cè)嗅覺剝奪模型,84只動(dòng)物隨機(jī)分成左側(cè)嗅覺剝奪組56只、假手術(shù)組7只、正常對(duì)照組21只。每組又根據(jù)不同的存活時(shí)間(4、7、14、21、30、45或60天)將動(dòng)物隨機(jī)分成7個(gè)組。左側(cè)嗅覺剝奪組大鼠右側(cè)鼻孔保持通暢作為自身對(duì)照。另外8只動(dòng)物(嗅覺剝奪組4只、假手術(shù)組4只)在存活30天后處死進(jìn)行生物化學(xué)研究。通過免疫組織化學(xué)染色方法檢測(cè)成年嬌y"D大鼠單側(cè)嗅覺剝奪后雙側(cè)嗅球BACE-1表達(dá);通過免疫熒光染色方法檢測(cè)雙側(cè)嗅球的BACE-1與tyrosine hydroxylase(TH),olfactorymarker protein(OMP),synaptophysin,amyloid precursor protein(APP)和presenilin-1(PS-1)等共定位表達(dá);通過免疫組織化學(xué)染色結(jié)合cytochrome oxidase/succidate dehydrogenase(CO/SDH)組織化學(xué)染色方法研究成年健康SD大鼠同一個(gè)嗅小球以及單側(cè)嗅覺剝奪后雙側(cè)嗅球的BACE-1表達(dá)與CO/SDH活性進(jìn)行相關(guān)性研究;Western blot檢測(cè)雙側(cè)嗅球BACE-1與其剪切產(chǎn)物(β-CTF)蛋白量;通過BACE-1和γ-secretases活性檢測(cè)等方法對(duì)雙側(cè)嗅球進(jìn)行BACE-1以及γ-secretases活性檢測(cè):通過ELISA檢測(cè)成年健康SD大鼠單側(cè)嗅覺剝奪后雙側(cè)嗅球APP降解通路終產(chǎn)物(Aβ)量。 結(jié)果: 第一部分:成年健康SD大鼠腦組織中Western blot顯示出多條不同分子量的BACE-1形式,不同分子量的BACE-1在脫糖基處理后表現(xiàn)出相同的酶剪切活性。 第二部分:嗅球內(nèi)有大量BACE-1免疫陽性產(chǎn)物,免疫陽性產(chǎn)物主要分布于嗅神經(jīng)纖維和嗅小球?qū)?單個(gè)嗅小球之間的BACE-1表達(dá)程度不同。嗅球中BACE-1和synaptophysin與OMP共表達(dá)。 第三部分:成年健康SD大鼠單個(gè)嗅球BACE-1表達(dá)與其CO/SDH活性相反;嗅覺剝奪后嗅球CO/SDH活性下降,BACE-1表達(dá)和活性顯著上調(diào);β-CTF和Aβ產(chǎn)生顯著增加;APP和PS-1未見明顯變化。 結(jié)論:成年健康SD大鼠腦組織同時(shí)存在帶有多個(gè)糖基的BACE-1形式,并且BACE-1的去糖基化可能沒有明顯改變其活性;成年健康SD大鼠嗅球內(nèi)有大量BACE-1表達(dá),BACE-1主要定位于嗅神經(jīng)纖維和嗅小球中的嗅神經(jīng)軸突終末;成年健康SD大鼠嗅覺剝奪后引起嗅球的BACE-1表達(dá)和活性顯著上調(diào),而且伴隨有其酶切產(chǎn)物(β-CTF和Aβ)的顯著增加,雙側(cè)嗅球APP和γ-secretases/PS-1表達(dá)和活性未見明顯差異,提示嗅覺剝奪模擬的神經(jīng)元活性/代謝降低狀態(tài)可能通過上調(diào)BACE-1表達(dá)和活性途徑而不是γ-secretases途徑來引起Aβ過度產(chǎn)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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R322

【共引文獻(xiàn)】

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