Presenilins條件性雙基因敲除小鼠超微結(jié)構(gòu)的增齡性變化及運(yùn)動(dòng)能力的研究
發(fā)布時(shí)間：2018-12-17 21:38
【摘要】：阿爾茨海默癥(Alzheimer's disease, AD)是最常見的中樞神經(jīng)系統(tǒng)退行性疾病。隨著年齡的增加,AD的發(fā)病率幾乎呈指數(shù)增長(zhǎng),嚴(yán)重影響了老年人的健康和生活品質(zhì)。大多數(shù)AD呈散發(fā)型,少數(shù)患者具有家族性(Familial Alzheimer's disease, FAD),為常染色體顯性遺傳。研究發(fā)現(xiàn),分別位于14號(hào)及1號(hào)染色體上的Presenilinl(PS1)及Presenilin2(PS2)基因的突變是導(dǎo)致早發(fā)性FAD最主要的原因。用前腦特異性敲除PS1的小鼠與全身性敲除PS2的小鼠繁育出的PS1/2條件性雙基因敲除(dKO)小鼠表現(xiàn)出：神經(jīng)元大量凋亡、Tau蛋白的高度磷酸化、劇烈的中樞神經(jīng)系統(tǒng)和外周神經(jīng)系統(tǒng)的炎癥反應(yīng)和認(rèn)知功能障礙等一系列類似AD的癥狀。 有研究證據(jù)表明PS1參與了突觸的形成并且具有調(diào)節(jié)突觸功能的作用。大腦記憶功能的實(shí)現(xiàn)是通過增強(qiáng)神經(jīng)元之間的聯(lián)系而產(chǎn)生的。細(xì)胞核和線粒體參與細(xì)胞凋亡途徑。PS1基因敲除會(huì)阻礙細(xì)胞內(nèi)v-ATPaseV0al亞基從內(nèi)質(zhì)網(wǎng)運(yùn)輸?shù)饺苊阁w,影響了v型ATP酶在溶酶體膜上的組裝,進(jìn)而阻礙了溶酶體的降解功能。 AD的病理學(xué)特征主要表現(xiàn)為神經(jīng)元大量凋亡、老年斑的增加和神經(jīng)纖維纏結(jié)。越來越多的證據(jù)表明,AD有一個(gè)較長(zhǎng)的臨床前階段。在臨床前階段,病人的病理學(xué)特征增加,機(jī)體功能下降。但是這些特征不足以確診為癡呆。實(shí)際上導(dǎo)致AD的病理生理過程在AD臨床確診前幾年甚至幾十年已經(jīng)漸漸發(fā)生。一些非認(rèn)知相關(guān)的行為,如運(yùn)動(dòng)功能障礙,可提示隨后的AD病程的發(fā)展。因此,這類運(yùn)動(dòng)功能障礙的行為可作為AD臨床前階段的表現(xiàn)特征。研究結(jié)果顯示了,AD病人在臨床前階段或者臨床后期均表現(xiàn)出運(yùn)動(dòng)行為異常。 本文主要研究Presenilins條件性雙基因敲除對(duì)于小鼠海馬與皮層超微結(jié)構(gòu)的增齡性影響以及對(duì)運(yùn)動(dòng)平衡能力的影響。 1. Presenilins條件性雙基因敲除對(duì)于小鼠海馬與皮層超微結(jié)構(gòu)的影響 選用3月齡、6月齡及12月齡Presenilins1/Presenilins2雙敲除小鼠(dKO)和對(duì)照小鼠(CON),運(yùn)用透射電子顯微鏡技術(shù),分別觀察海馬與皮層突觸、細(xì)胞核、線粒體、溶酶體超微結(jié)構(gòu)的改變。結(jié)果顯示3月齡dK0小鼠海馬與皮層溶酶體已向具有消化能力的次級(jí)溶酶體轉(zhuǎn)化。6月齡dKO小鼠海馬與皮層的突觸后致密物厚度減小；皮層細(xì)胞核膜內(nèi)陷,核不規(guī)則；海馬與皮層線粒體出現(xiàn)腫脹、脊變形或消失；出現(xiàn)高電子密度的次級(jí)溶酶體,并伴有脂褐素小體出現(xiàn)。12月齡dKO小鼠海馬與皮層突觸后致密物厚度減��；海馬與皮層核膜內(nèi)陷,染色質(zhì)固縮沿核膜分布；線粒體嚴(yán)重受損,脊大范圍溶解；出現(xiàn)較多次級(jí)溶酶體和脂褐素小體。 Presenilins條件性雙基因敲除對(duì)小鼠海馬與皮層有增齡性的病理影響,這些病理改變將為相關(guān)的藥物評(píng)價(jià)和對(duì)阿爾茨海默病的深入研究提供形態(tài)學(xué)依據(jù)。 2. Presenilins條件性雙基因敲除對(duì)于小鼠運(yùn)動(dòng)平衡能力的影響 選用2月齡、6月齡及12月齡Presenilins1/Presenilins2雙敲除小鼠(dKO)和對(duì)照小鼠(CON),以行為學(xué)實(shí)驗(yàn)為手段,分別從曠場(chǎng)實(shí)驗(yàn)、轉(zhuǎn)棒實(shí)驗(yàn)、平衡木實(shí)驗(yàn)和衣架實(shí)驗(yàn)來全面的觀察dKO小鼠的運(yùn)動(dòng)平衡能力。結(jié)果顯示自2月齡起dKO小鼠出現(xiàn)輕微的運(yùn)動(dòng)功能障礙。這也從另一個(gè)方面證實(shí)dKO小鼠是較好的AD模型。同時(shí)對(duì)dKO小鼠全面的運(yùn)動(dòng)功能研究也為了更深入的研究其認(rèn)知或非認(rèn)知行為奠定了基礎(chǔ)。 3. Presenilins條件性雙基因敲除對(duì)于小鼠運(yùn)動(dòng)平衡能力的影響的分子機(jī)制研究 選用2月齡、6月齡及12月齡Presenilins1/Presenilins2雙敲除小鼠(dKO)和對(duì)照小鼠(CON),使用熒光定量PCR技術(shù)檢測(cè)了皮層、小腦和海馬的calbindinD28k mRNA表達(dá)水平；使用Western blot技術(shù)檢測(cè)檢測(cè)了dKO小鼠的皮層、小腦和海馬的calbindinD28k蛋白表達(dá)水平。結(jié)果顯示dK0小鼠的皮層、小腦和海馬的calbindinD28k mRNA和蛋白水平與CON小鼠相比均無顯著性差異。同時(shí)dK0小鼠和CON小鼠的calbindinD28k mRNA和蛋白在小腦部位的表達(dá)量顯著高于皮層和海馬部位。 該結(jié)果表明dK0小鼠出現(xiàn)運(yùn)動(dòng)功能障礙并不是通過calbindinD28k起到調(diào)節(jié)作用的,且calbindinD28k在小鼠小腦部位有高表達(dá)水平。
[Abstract]:Alzheimer's disease (AD) is the most common degenerative disease of the central nervous system. With the increase of age, the incidence of AD is almost exponential, which seriously affects the health and quality of life of the elderly. Most of the AD is sporadic, and a few patients have familial Alzheimer's disease (FAD), which is autosomal dominant inheritance. It was found that the mutation of Presenilinl (PS1) and Presenilin2 (PS2) gene on chromosome 14 and chromosome 1 was the main cause of early FAD. The PS1/ 2-conditioned two-gene knockout (dKO) mice bred with the mouse of PS1-specific knockout PS1 and the mouse of the systemic knockout PS2 showed a significant amount of neuronal apoptosis, the high phosphorylation of the Tau protein, Severe central nervous system and peripheral nervous system inflammation and cognitive dysfunction, a series of symptoms like AD. There is evidence that PS1 is involved in the formation of synapses and has the function of regulating synapses Effect. The brain memory function is realized by enhancing the contact between the neurons. The nuclear and the mitochondria are involved in the apoptosis of the cells. pathway. PS1 knockout will block the transport of the intracellular v-CaseVal subunit from the endoplasmic reticulum to the lysosome, which affects the assembly of the v-type ATP enzyme on the lysosome membrane, thus preventing the degradation of lysosomes. The pathology of AD is mainly characterized by a large number of neurons, an increase of senile plaque and a god. There is a growing number of evidence that there is a longer AD in AD Preclinical stage. At the pre-clinical stage, the pathology features of the patient are increased, and the machine The body function is down. But these features are not sufficient The diagnosis of dementia. In fact, the pathophysiological process of AD has been in the past few years or even decades prior to the diagnosis of AD. It is gradually taking place. Some non-cognitive-related behaviors, such as motion dysfunction, can prompt the subsequent AD The development of the course of the disease is, therefore, the behavior of this type of motor dysfunction can be used as the pre-clinical stage of AD The results of the study showed that the AD patients were in the pre-clinical stage or in the post-clinical stage. In this paper, the effects of Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse and the effect of the Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse are studied in this paper. The effect of dynamic balance capacity. 1. Presenilins conditional double gene knockout in mice The effects of the ultrastructure of the hippocampus and the cortex were 3 months, 6 months and 12 months of Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON). the synapse and the nucleus of the horse and the cortex, The changes of the ultrastructure of the mitochondria and lysosomes in the hippocampus and cortex of dK0 mice at the age of 3 months have been transformed into secondary lysosomes with digestive ability. The thickness of the postsynaptic density of the hippocampus and the cortex of the dKO mice at 6 months of age is reduced, and the nuclear membrane of the cortex and the nucleus do not have the rules. in that hippocampus and cortex of the 12-month-old dKO mice, the thickness of the dense matter in the hippocampus and the cortex of the cortex of the 12-month-old dKO mice was reduced, and the nuclei of the hippocampus and the cortex of the cortex and the chromatin condensation were distributed along the nuclear membrane; The mitochondria are severely damaged, and the ridge is dissolved in a large extent; it is shown that There are many levels of lysosomes and lipofuscin. Presenilins conditional double gene knock-out has a pathological effect on the aging of the hippocampus and the cortex of the mouse, and these pathological changes will be relevant drug evaluation and control. The in-depth study of Alzheimer's disease provides a morphological basis. 2. Presenilins Two-month, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON) were selected for the effects of conditional double gene knockout on the exercise balance of mice The experiment of behavior experiment, the experiment of rotating rod, the experiment of the balance wood and the experiment of the balance wood were carried out. and the exercise balance ability of the dKO mice is comprehensively observed by the experiment of the clothes rack and the clothes hanger. Mild exercise dysfunction in dKO mice from 2 months of age was shown. This also demonstrated that dKO mice were better AD models from another aspect, while the overall exercise function of dKO mice was studied. It also laid the foundation for a more in-depth study of its cognitive or non-cognitive behavior. 3. Presenilins The molecular mechanism of the effect of the two-gene knockout on the balance of mice's motion was selected from 2-month-old, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double-knockout mice (dKO) and control mice (CON), and the skin was detected using the fluorescence quantitative PCR technique. The expression level of calbindin D28k in the layer, cerebellum and hippocampus was detected by Western blot. calbindin D28k protein expression level in the cortex, cerebellum, and hippocampus. The results showed that calbini in the cortex, cerebellum and hippocampus of dK0 mice nD28k mRNA and protein levels were not significantly different from that of CON mice, while calbindinD2 of dK0 mice and CON mice The expression of 8k mRNA and protein in the cerebellum was significantly higher than that of the cortex and the hippocampus. The results showed that the dK0 mice had a motor dysfunction and not adjusted by calbindin D28k.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16
本文編號(hào)：2384832
[Abstract]:Alzheimer's disease (AD) is the most common degenerative disease of the central nervous system. With the increase of age, the incidence of AD is almost exponential, which seriously affects the health and quality of life of the elderly. Most of the AD is sporadic, and a few patients have familial Alzheimer's disease (FAD), which is autosomal dominant inheritance. It was found that the mutation of Presenilinl (PS1) and Presenilin2 (PS2) gene on chromosome 14 and chromosome 1 was the main cause of early FAD. The PS1/ 2-conditioned two-gene knockout (dKO) mice bred with the mouse of PS1-specific knockout PS1 and the mouse of the systemic knockout PS2 showed a significant amount of neuronal apoptosis, the high phosphorylation of the Tau protein, Severe central nervous system and peripheral nervous system inflammation and cognitive dysfunction, a series of symptoms like AD. There is evidence that PS1 is involved in the formation of synapses and has the function of regulating synapses Effect. The brain memory function is realized by enhancing the contact between the neurons. The nuclear and the mitochondria are involved in the apoptosis of the cells. pathway. PS1 knockout will block the transport of the intracellular v-CaseVal subunit from the endoplasmic reticulum to the lysosome, which affects the assembly of the v-type ATP enzyme on the lysosome membrane, thus preventing the degradation of lysosomes. The pathology of AD is mainly characterized by a large number of neurons, an increase of senile plaque and a god. There is a growing number of evidence that there is a longer AD in AD Preclinical stage. At the pre-clinical stage, the pathology features of the patient are increased, and the machine The body function is down. But these features are not sufficient The diagnosis of dementia. In fact, the pathophysiological process of AD has been in the past few years or even decades prior to the diagnosis of AD. It is gradually taking place. Some non-cognitive-related behaviors, such as motion dysfunction, can prompt the subsequent AD The development of the course of the disease is, therefore, the behavior of this type of motor dysfunction can be used as the pre-clinical stage of AD The results of the study showed that the AD patients were in the pre-clinical stage or in the post-clinical stage. In this paper, the effects of Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse and the effect of the Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse are studied in this paper. The effect of dynamic balance capacity. 1. Presenilins conditional double gene knockout in mice The effects of the ultrastructure of the hippocampus and the cortex were 3 months, 6 months and 12 months of Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON). the synapse and the nucleus of the horse and the cortex, The changes of the ultrastructure of the mitochondria and lysosomes in the hippocampus and cortex of dK0 mice at the age of 3 months have been transformed into secondary lysosomes with digestive ability. The thickness of the postsynaptic density of the hippocampus and the cortex of the dKO mice at 6 months of age is reduced, and the nuclear membrane of the cortex and the nucleus do not have the rules. in that hippocampus and cortex of the 12-month-old dKO mice, the thickness of the dense matter in the hippocampus and the cortex of the cortex of the 12-month-old dKO mice was reduced, and the nuclei of the hippocampus and the cortex of the cortex and the chromatin condensation were distributed along the nuclear membrane; The mitochondria are severely damaged, and the ridge is dissolved in a large extent; it is shown that There are many levels of lysosomes and lipofuscin. Presenilins conditional double gene knock-out has a pathological effect on the aging of the hippocampus and the cortex of the mouse, and these pathological changes will be relevant drug evaluation and control. The in-depth study of Alzheimer's disease provides a morphological basis. 2. Presenilins Two-month, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON) were selected for the effects of conditional double gene knockout on the exercise balance of mice The experiment of behavior experiment, the experiment of rotating rod, the experiment of the balance wood and the experiment of the balance wood were carried out. and the exercise balance ability of the dKO mice is comprehensively observed by the experiment of the clothes rack and the clothes hanger. Mild exercise dysfunction in dKO mice from 2 months of age was shown. This also demonstrated that dKO mice were better AD models from another aspect, while the overall exercise function of dKO mice was studied. It also laid the foundation for a more in-depth study of its cognitive or non-cognitive behavior. 3. Presenilins The molecular mechanism of the effect of the two-gene knockout on the balance of mice's motion was selected from 2-month-old, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double-knockout mice (dKO) and control mice (CON), and the skin was detected using the fluorescence quantitative PCR technique. The expression level of calbindin D28k in the layer, cerebellum and hippocampus was detected by Western blot. calbindin D28k protein expression level in the cortex, cerebellum, and hippocampus. The results showed that calbini in the cortex, cerebellum and hippocampus of dK0 mice nD28k mRNA and protein levels were not significantly different from that of CON mice, while calbindinD2 of dK0 mice and CON mice The expression of 8k mRNA and protein in the cerebellum was significantly higher than that of the cortex and the hippocampus. The results showed that the dK0 mice had a motor dysfunction and not adjusted by calbindin D28k.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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2 ;Prolonged Alzheimer-like Tau Hyperphosphorylation Induced by Simultaneous Inhibition of Phosphoinositol-3 Kinase and Protein Kinase C in N2a cells[J];Acta Biochimica et Biophysica Sinica;2005年05期
3 吳馥梅，杜紅燕，，章子貴;突觸界面曲率及其生理意義[J];神經(jīng)解剖學(xué)雜志;1994年01期
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