TRPC通道在Aβ誘導(dǎo)的阿爾茨海默病（AD）小鼠的蛋白表達研究
發(fā)布時間：2019-07-04 15:22
【摘要】：阿爾茨海默病(AD)是一種發(fā)生在老年人中的神經(jīng)退行性疾病,臨床上主要以進行性認知功能減退為主要表現(xiàn),其典型的病理特征為β淀粉樣蛋白(Aβ)為核心的老年斑、神經(jīng)纖維纏結(jié)以及神經(jīng)元死亡。目前公認的Aβ沉積是AD形成的最終通路,而鈣穩(wěn)態(tài)失調(diào)能夠?qū)е律窠?jīng)元結(jié)構(gòu)以及功能發(fā)生改變,甚至導(dǎo)致神經(jīng)元的不可逆性損傷。近年來的研究發(fā)現(xiàn)經(jīng)典瞬時受體電位(TRPC)通道是一類位于細胞膜上的非選擇性鈣離子通道,在大腦神經(jīng)元中高度表達,并且研究表明TRPC通道在神經(jīng)系統(tǒng)中主要參與增殖、分化、凋亡、退行性變和突觸可塑性等方面的功能。但是,在Aβ誘導(dǎo)的AD小鼠模型中,TRPC通道蛋白的表達情況以及功能機制目前還尚不清楚。目的探討經(jīng)典瞬時受體電位(TRPC)通道蛋白在Aβ1-42誘導(dǎo)的阿爾茨海默病(AD)小鼠海馬區(qū)中的表達情況。方法1.雄性5周齡ICR小鼠36只,適應(yīng)性喂養(yǎng)5天后,將其隨機分為AD組和對照組,每組18只。2.應(yīng)用Aβ1-42側(cè)腦室注射制作AD小鼠模型,側(cè)腦室注射第十天后進行Morris水迷宮定位航行和空間探索實驗測定小鼠學(xué)習(xí)記憶能力,行為學(xué)檢測后處死動物,采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)方法檢測海馬區(qū)TRPC1～TRPC7mRNA的表達情況,運用免疫熒光和Western blot等實驗技術(shù)檢測小鼠海馬區(qū)的TRPC4通道蛋白的表達。結(jié)果1.Morris水迷宮行為學(xué)檢測結(jié)果顯示,在定位航行實驗中,從第三天開始,與對照組相比,AD組小鼠水迷宮測試的逃避潛伏期延長(P0.05),尋找平臺游泳路程較長(P0.05),在空間探索實驗中,與對照組相比,AD組在目標象限停留時間明顯縮短(P0.01),穿越平臺次數(shù)明顯減少(P0.01)。2.RT PCR檢測結(jié)果顯示,在對照組及AD組中,TRPC1～TRPC7通道m(xù)RNA均有不同程度的表達,與對照組相比,AD組TRPC4mRNA表達明顯增高(P0.01)。3.免疫熒光結(jié)果顯示TRPC4通道蛋白廣泛表達在海馬區(qū)的神經(jīng)細胞膜上,且與對照組相比,AD組TRPC4熒光強度增強。4.Western blot結(jié)果顯示,與對照組相比,AD組TRPC4通道蛋白表達增高(P0.05)。結(jié)論側(cè)腦室注射Aβ1-42寡聚體后,小鼠學(xué)習(xí)記憶功能減退,海馬區(qū)TRPC4通道m(xù)RNA及蛋白表達含量均增加,表明TRPC通道參與到因Aβ沉積而導(dǎo)致的AD發(fā)病機制中。
[Abstract]:Alzheimer's disease (AD) is a neurodegenerative disease in the elderly, which is mainly characterized by progressive cognitive impairment. The typical pathological features of Alzheimer's disease are Alzheimer's disease (A 尾), neurofibrillar tangles and neuronal death. At present, A 尾 deposition is recognized as the final pathway of AD formation, and calcium homeostasis disorder can lead to changes in the structure and function of neurons, and even lead to irreversible damage of neurons. In recent years, it has been found that the classical transient receptor potential (TRPC) channel is a kind of non-selective calcium channel located on the cell membrane, which is highly expressed in brain neurons, and studies have shown that TRPC channel is mainly involved in proliferation, differentiation, apoptosis, degeneration and synaptic plasticity in the nervous system. However, the expression and functional mechanism of TRPC channel protein in AD mice induced by A 尾 are not clear at present. Objective to investigate the expression of classical transient receptor potential (TRPC) channel protein in hippocampus of (AD) mice with Alzheimer's disease induced by A 尾 1-42. Method 1. Thirty-six male 5-week-old ICR mice were randomly divided into AD group (n = 18) and control group (n = 18) after 5 days of adaptive feeding. The AD mouse model was established by intraventricular injection of A 尾 1-42. The learning and memory ability of the mice was measured by Morris water maze localization navigation and space exploration test 10 days after lateral ventricle injection. The animals were killed after behavioral examination. The expression of TRPC1~TRPC7mRNA in hippocampus was detected by reverse transcription polymerase chain reaction (RT-PCR), and the expression of TRPC4 channel protein in hippocampus was detected by immunofluorescence and Western blot. Results the results of 1.Morris water maze behavior test showed that from the third day, compared with the control group, the escape latency of the water maze test in the AD group was prolonged (P 0.05), and the swimming distance was longer (P 0.05). In the space exploration experiment, compared with the control group, the residence time in the target quadrant of the AD group was significantly shortened (P 0.01). The number of crossing the platform was significantly decreased (P 0.01). The results of 2.RT PCR showed that TRPC1~TRPC7 channel mRNA was expressed in different degrees in the control group and AD group, and the expression of TRPC4mRNA in the AD group was significantly higher than that in the control group (P 0.01). The results of immunofluorescence showed that TRPC4 channel protein was widely expressed in hippocampal nerve cell membrane, and the fluorescence intensity of TRPC4 in AD group was higher than that in control group. 4. Western blot results showed that the expression of TRPC4 channel protein in AD group was higher than that in control group (P 0.05). Conclusion after intracerebroventricular injection of A 尾 1 鈮，
本文編號：2510014
[Abstract]:Alzheimer's disease (AD) is a neurodegenerative disease in the elderly, which is mainly characterized by progressive cognitive impairment. The typical pathological features of Alzheimer's disease are Alzheimer's disease (A 尾), neurofibrillar tangles and neuronal death. At present, A 尾 deposition is recognized as the final pathway of AD formation, and calcium homeostasis disorder can lead to changes in the structure and function of neurons, and even lead to irreversible damage of neurons. In recent years, it has been found that the classical transient receptor potential (TRPC) channel is a kind of non-selective calcium channel located on the cell membrane, which is highly expressed in brain neurons, and studies have shown that TRPC channel is mainly involved in proliferation, differentiation, apoptosis, degeneration and synaptic plasticity in the nervous system. However, the expression and functional mechanism of TRPC channel protein in AD mice induced by A 尾 are not clear at present. Objective to investigate the expression of classical transient receptor potential (TRPC) channel protein in hippocampus of (AD) mice with Alzheimer's disease induced by A 尾 1-42. Method 1. Thirty-six male 5-week-old ICR mice were randomly divided into AD group (n = 18) and control group (n = 18) after 5 days of adaptive feeding. The AD mouse model was established by intraventricular injection of A 尾 1-42. The learning and memory ability of the mice was measured by Morris water maze localization navigation and space exploration test 10 days after lateral ventricle injection. The animals were killed after behavioral examination. The expression of TRPC1~TRPC7mRNA in hippocampus was detected by reverse transcription polymerase chain reaction (RT-PCR), and the expression of TRPC4 channel protein in hippocampus was detected by immunofluorescence and Western blot. Results the results of 1.Morris water maze behavior test showed that from the third day, compared with the control group, the escape latency of the water maze test in the AD group was prolonged (P 0.05), and the swimming distance was longer (P 0.05). In the space exploration experiment, compared with the control group, the residence time in the target quadrant of the AD group was significantly shortened (P 0.01). The number of crossing the platform was significantly decreased (P 0.01). The results of 2.RT PCR showed that TRPC1~TRPC7 channel mRNA was expressed in different degrees in the control group and AD group, and the expression of TRPC4mRNA in the AD group was significantly higher than that in the control group (P 0.01). The results of immunofluorescence showed that TRPC4 channel protein was widely expressed in hippocampal nerve cell membrane, and the fluorescence intensity of TRPC4 in AD group was higher than that in control group. 4. Western blot results showed that the expression of TRPC4 channel protein in AD group was higher than that in control group (P 0.05). Conclusion after intracerebroventricular injection of A 尾 1 鈮，
本文編號：2510014
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