本文選題：阿爾茨海默癥 + 小膠質(zhì)細(xì)胞��； 參考：《河北師范大學(xué)》2012年博士論文

【摘要】：阿爾茨海默癥(Alzheimer's disease, AD)是一種不可逆的神經(jīng)退行性疾病,其特征表現(xiàn)為記憶和認(rèn)知功能的逐漸喪失。阿爾茨海默癥通�？梢苑譃閮煞N類型：一是家族性AD,二是散發(fā)型AD,大多數(shù)患者為后者。后者通常在60歲之后才會出現(xiàn)明顯的AD病征。因此,散發(fā)型AD是與衰老相關(guān)的一種神經(jīng)退行性疾病。目前已經(jīng)明確AD的病理特征為神經(jīng)元內(nèi)由于tau蛋白過度磷酸化形成的神經(jīng)纏節(jié)、細(xì)胞間β-淀粉樣蛋白(Aβ)沉積形成的老年斑。由小膠質(zhì)細(xì)胞(microglia, MG)和星形膠質(zhì)細(xì)胞(astrocytes, AS)過度激活引發(fā)的系列炎癥反應(yīng)被認(rèn)為是AD的第三個病理特征和發(fā)病原因之一。神經(jīng)炎癥反應(yīng)機制為：不溶性Aβ的聚集和纖維化激活了MG。激活的MG通過產(chǎn)生活性氧、補體成分、促炎癥細(xì)胞因子、趨化因子等引發(fā)一系列的炎癥反應(yīng),形成神經(jīng)毒性,造成神經(jīng)元死亡；產(chǎn)生的促炎癥細(xì)胞因子又可激活A(yù)S,從而共同參與腦局部炎癥反應(yīng),最終形成AD。但隨著研究發(fā)現(xiàn),在正常的老年人腦內(nèi)也存在彌散的Ap聚集物。Aβ的沉積是腦老化的產(chǎn)物。還有研究發(fā)現(xiàn)MG的形態(tài)與年齡有關(guān)。隨著老齡化,MG的形態(tài)變?yōu)闋I養(yǎng)不良狀態(tài)。在tau-蛋白磷酸化位點和Aβ斑塊周圍同樣也發(fā)現(xiàn)MG的營養(yǎng)不良狀態(tài)。因此認(rèn)為漸行性的MG老化和MG保護功能的丟失是AD發(fā)病的原因之一。AS在AD發(fā)病中的作用也有不同的觀點。在慢性神經(jīng)退行性疾病中,眾多的研究表明AS可以做為具有固有免疫能力的細(xì)胞參與免疫識別、免疫調(diào)節(jié)和炎癥過程。但也有研究發(fā)現(xiàn)AS也存在著營養(yǎng)不良狀態(tài)。在轉(zhuǎn)基因鼠中隨著鼠齡的增加,遠(yuǎn)離Aβ斑塊的AS表現(xiàn)出脫分枝,GFAP表達(dá)降低,呈現(xiàn)出營養(yǎng)不良狀態(tài)。因此提出AD發(fā)病的AS老化假說,即圍繞Aβ斑塊的AS表現(xiàn)為膠質(zhì)化,而遠(yuǎn)離Aβ斑塊的AS為營養(yǎng)不良狀態(tài)。因此,針對MG和AS在AD發(fā)病中的作用有兩種截然不同的假說。一是MG和AS過度激活和膠質(zhì)化引發(fā)的炎癥反應(yīng)是AD發(fā)病的原因之一,表現(xiàn)為腦部促炎癥因子的增加和MG及AS的過度激活；二是MG和AS老化及保護功能喪失是AD發(fā)病的原因之一,表現(xiàn)為營養(yǎng)不良的MG和AS細(xì)胞形態(tài)。本研究針對上述兩種假說,利用不形成老年斑的AD模型——加速衰老模型(accelerated senescence-prone mouse8, SAMP8)小鼠作為研究材料,利用水迷宮、免疫組織化學(xué)染色、PT-PCR、Western blo、luminex等技術(shù)和方法研究了SAMP8小鼠在AD發(fā)病進程中認(rèn)知能力的改變,海馬和皮層中MG和AS的形態(tài)、數(shù)量以及功能的變化,并研究了不同性別之間的差異。在此基礎(chǔ)上研究了不同年齡階段的鍛煉對學(xué)習(xí)和記憶能力的干預(yù)作用及免疫機制。本研究結(jié)果表明,2、5月齡的雄、雌SAMP8小鼠認(rèn)知能力均沒有顯著變化,但8月齡的小鼠認(rèn)知能力顯著下降,雌鼠認(rèn)知損傷更為嚴(yán)重。2、5月齡小鼠海馬和皮層中,MG和AS的形態(tài)為胞體肥大、分支較多,處于激活狀態(tài)；8月齡小鼠的MG和AS的形態(tài)發(fā)生了顯著的改變。MG周圍組織開始出現(xiàn)空泡,胞體開始萎縮,分支出現(xiàn)斷裂；有些AS胞體模糊,沒有完整的胞體形態(tài),分支減少；而且AS完整細(xì)胞的數(shù)量顯著降低,在雌性小鼠海馬和皮層中,AS數(shù)量降低更為明顯。MG表面標(biāo)記CDllb和AS表面標(biāo)記GFAP表達(dá)也顯著降低。本研究從營養(yǎng)功能、吞噬作用及促炎癥因子和趨化因子分泌三個方面研究了MG和AS功能。第一,隨著月齡增加,營養(yǎng)功能降低。8月齡的小鼠海馬和皮層中的BDNF和GDNF表達(dá)顯著降低,GDNF的蛋白水平顯著降低。其中GDNF的表達(dá)和蛋白水平從5月齡就開始顯著降低,而且雌性小鼠海馬中GDNF的降低幅度比雄性小鼠更大。第二,隨著月齡增加,吞噬功能降低。TLR2、TLR4和MyD88基因的表達(dá)在2、5月齡時沒有顯著性差異,但在8月齡時顯著性降低。性別之間沒有顯著性差異。第三,隨著月齡增加,促炎癥因子和趨化因子水平增加。海馬和皮層中的L-1β、TNF-α在2、5月齡時沒有顯著性差異,在8月齡時顯著增加,而IFN-γ則沒有顯著性差異。趨化因子MCP-1的水平在5月齡時開始顯著增加。MG是中樞神經(jīng)系統(tǒng)(CNS)的免疫細(xì)胞,AS是支持細(xì)胞,其激活首先表現(xiàn)的是免疫修復(fù)和營養(yǎng)支持功能。正常的修復(fù)過程可能包括兩個階段。第一階段是炎癥反應(yīng)階段。MG和AS表現(xiàn)為激活狀態(tài),通過吞作用,產(chǎn)生促炎癥因子,清理造成微損傷的異物或者細(xì)胞碎片,表現(xiàn)為炎癥反應(yīng)；隨后進入第二階段,修復(fù)階段。此階段,營養(yǎng)因子水平增加,炎癥因子水平降低,損傷的神經(jīng)元得到修復(fù)。SAMP8小鼠3月齡出現(xiàn)氧化應(yīng)激,5月齡出現(xiàn)tau蛋白過度磷酸化,這些都會對腦部造成微損傷,從而激活MG和AS。我們的研究結(jié)果也表明2、5月齡的MG和AS為激活狀態(tài)。但此時的MG和AS功能處于正常水平,所以能夠完成自我修復(fù)的過程,所以不會造成促炎癥因子的積累,同時在認(rèn)知水平上也不會有顯著的減低,我們的研究結(jié)果說明了這一點。隨著小鼠老齡化,MG和AS開始老化,其吞噬功能降低,自我修復(fù)功能停留在第一階段,炎癥反應(yīng)階段,造成促炎癥因子的積累；其次營養(yǎng)功能降低,分泌的營養(yǎng)因子水平降低,不能很好完成后續(xù)的修復(fù),從而造成損傷的持續(xù)和擴大,最終造成神經(jīng)元的死傷。同時趨化因子分泌的增加,進一步招募外周的吞噬細(xì)胞進入CNS,進一步加重了炎癥反應(yīng)。因此,根據(jù)上述研究結(jié)果, 我們認(rèn)為在小鼠老化過程中,MG和AS表現(xiàn)出相應(yīng)的老化,其保護功能相應(yīng)降低。到目前為止還沒有有效的藥物治療AD,而運動鍛煉提供了唯一不使用藥物的干預(yù)措施。我們的研究結(jié)果表明,小鼠不同年齡階段的鍛煉產(chǎn)生的效果不同。病理特征產(chǎn)生之前就開始的全程有規(guī)律的鍛煉(2月齡到8月齡)和早期有規(guī)律的鍛煉(2月齡到4月齡)對認(rèn)知功能的提高有顯著的作用,而病理出現(xiàn)之后的鍛煉(6月齡到8月齡)只有延緩和改善作用。研究結(jié)果還表明鍛煉對雌性小鼠的影響更顯著。通過我們的研究發(fā)現(xiàn)鍛煉預(yù)防AD的免疫機制為：鍛煉延緩了MG和AS的衰老,阻止了AS數(shù)量的減少,從而保證了MG和AS的功能。具體表現(xiàn)為：增加了BDNF和GDNF的表達(dá)和GDNF的蛋白水平；增加了TLR2及Myd88的表達(dá),提高了吞噬功能；提高了TNF-γ的水平,降低了促炎癥細(xì)胞因子的水平。因此,我們認(rèn)為體育鍛煉延緩了神經(jīng)膠質(zhì)細(xì)胞的老化,保證了神經(jīng)膠質(zhì)細(xì)胞的功能。
[Abstract]:Blzheimer (Alzheimer's disease, AD) is an irreversible neurodegenerative disease characterized by a gradual loss of memory and cognitive function. Alzheimer's disease is usually divided into two types: one is familial AD, two is a scattered AD, and most patients are the latter. The latter usually occurs after 60 years of age. AD symptoms. Therefore, the diffuse AD is a neurodegenerative disease associated with senescence. It is now clear that the pathological features of AD are the neural tangle formed by the excessive phosphorylation of tau protein in neurons, and the senile plaques formed by the deposition of beta amyloid protein (A beta) between cells. Microglia (MG) and astrocytes (astroc). Ytes, AS) a series of inflammatory reactions induced by excessive activation is considered to be one of the third pathological features and causes of AD. The mechanism of neuroinflammatory reaction is that the aggregation and fibrosis of insoluble A beta activates MG. activated MG by producing a series of inflammatory reactions by producing living oxygen, complement components, and promoting inflammatory cell causes, chemokines, and so on. The formation of neurotoxicity causes neuronal death, and the resulting pro-inflammatory cytokines can activate AS to participate in the local inflammatory response of the brain and eventually form AD., but as the study found that the deposition of the dispersed Ap aggregation.A beta in the normal human brain is a product of brain aging. And the study found that the form of MG and the age of the MG have been found. With aging, the form of MG becomes malnutrition. The malnutrition of MG is also found around the tau- protein phosphorylation site and the A beta plaque. Therefore, the gradual MG aging and the loss of MG protection function are one of the reasons for the pathogenesis of AD. The role of.AS in the pathogenesis of AD is also different. In chronic neurodegenerative disease In the disease, many studies have shown that AS can be used as a cell with inherent immune ability to participate in immune recognition, immunomodulation and inflammatory processes. But there are also studies found that AS also has malnutrition. In transgenic mice, with the increase of the age of the rat, the AS, which is far away from the A beta plaque, shows debranching, the expression of GFAP is reduced, and a malnutrition form is present. Therefore, the AS aging hypothesis of AD is proposed, that is, the AS performance around A beta plaques is glial, while AS far away from A beta plaque is malnutrition. Therefore, there are two distinct hypotheses on the role of MG and AS in AD pathogenesis. The increase of pro-inflammatory factors and excessive activation of MG and AS; two is the aging of MG and AS and the loss of protective function is one of the reasons for the pathogenesis of AD, which shows the MG and AS cell morphology of malnutrition. This study aims at the above two hypotheses, using the AD model that does not form the senile plaque, the accelerated aging model (accelerated senescence-prone mouse8, SAMP8) as a research material, the mice used water maze, immunohistochemical staining, PT-PCR, Western blo, Luminex and other techniques and methods to study the changes in the cognitive ability of SAMP8 mice during the pathogenesis of AD, the morphology, quantity and function of MG and AS in the hippocampus and cortex, and study the differences between different sexes. The results of this study showed that the cognitive ability of 2,5 months old male and female SAMP8 mice was not significantly changed, but the cognitive ability of 8 month old mice decreased significantly, and the cognitive impairment of female mice was more serious in the hippocampus and the cortex of.2,5 month old mice, MG and AS. In 8 month old mice, the morphology of MG and AS had a significant change in the morphology of MG and AS, and the cell body began to appear vacuoles, the body began to atrophy and the branch broke; some AS cell bodies were blurred, there was no complete cell morphology, and the branch decreased significantly, and the number of AS intact cells decreased significantly in female, in female In the hippocampus and cortex of mice, the number of AS decreased more obviously than the.MG surface marker CDllb and AS surface labeling GFAP expression. This study studied the function of MG and AS from three aspects: nutritional function, phagocytosis, proinflammatory factors and chemokine secretion. First, with the increase of month age, the nutrition function reduced the hippocampus and skin of mice aged.8 months. The expression of BDNF and GDNF in the layer decreased significantly, and the protein level of GDNF decreased significantly. The expression of GDNF and protein level began to decrease significantly from 5 month old, and the decrease of GDNF in the hippocampus of female mice was greater than that of male mice. Second, the phagocytic function decreased as the month of age increased, and the expression of TLR4 and MyD88 genes was at the age of 2,5. There was no significant difference, but there was no significant difference at 8 month old. There was no significant difference between sex. Third, the levels of pro-inflammatory and chemokines increased with the increase of month's age. There was no significant difference between L-1 beta and TNF- alpha in the hippocampus and cortex at the age of 2,5, and there was a significant increase at 8 month old, while IFN- gamma had no significant difference. Chemokines were not. The level of MCP-1 increases significantly at 5 month old when.MG is an immune cell of the central nervous system (CNS), and AS is a supporting cell. Its activation is the first expression of immune repair and nutritional support. The normal repair process may include two stages. The first stage is the activation state of the inflammatory response order.MG and AS, through the swallowing, and production. To promote inflammatory factors, clean up the foreign bodies or cell fragments that cause microdamage, and appear to be inflammatory reactions; then enter the second stage, repair stage. In this stage, the level of nutrition factor increases, the level of the inflammatory factors is reduced, the injured neurons are repaired.SAMP8 mice 3 month old out of oxidative stress, and 5 month old of the tau protein overphosphorylation, Some of the brain causes slight damage to the brain, thus activating MG and AS.. Our results also indicate that MG and AS are activated at 2,5 months, but the MG and AS functions at this time are at normal level, so it is possible to complete the process of self repair, so that there is no accumulation of pro-inflammatory factors and no significant reduction in cognitive levels, The results of our study show that as the aging of mice, the aging of MG and AS, its phagocytosis, the function of self repair stay in the first stage, the inflammatory reaction stage, the accumulation of pro-inflammatory factors; secondly, the decrease of nutritional function and the level of secreted nutrient factors, which can not complete the subsequent repair, thus making a good result. The persistence and enlargement of damage eventually causes the death and injury of neurons, and the increase of chemokine secretion and further recruitment of the peripheral phagocytes into CNS, which further aggravates the inflammatory response. Therefore, according to the results of these studies, we believe that in the aging process of mice, MG and AS show corresponding aging and their protective functions are correspondingly reduced. So far, there is no effective drug treatment for AD, and exercise provides the only intervention that does not use drugs. Our results show that the effects of exercise at different stages of the mice are different. Regular exercise (2 month old to 8 month old) and early regular exercise started before the pathological features were produced. (2 month old to 4 month old) had a significant effect on the improvement of cognitive function, and the effect of exercise (6 month old to 8 month old) after pathological appearance (6 month old to 8 month old) was only delayed and improved. The results also showed that exercise had more significant effects on female mice. Through our study, we found that exercise prevention of AD's immune mechanism was that exercise delayed the aging of MG and AS. The decrease in the number of AS has been stopped, which ensures the function of MG and AS, which is characterized by increasing the expression of BDNF and GDNF and the protein level of GDNF, increasing the expression of TLR2 and Myd88, improving the phagocytosis, improving the level of TNF- gamma, and reducing the level of pro-inflammatory cytokines. Therefore, we think that physical exercise has delayed the neuroglue. The aging of the mass cells ensures the function of the glial cells.

【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R749.16

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