本文選題：TREM2 + PDTC��； 參考：《廣東藥科大學(xué)》2017年碩士論文

【摘要】：阿爾茨海默病(Alzheimer’s Disease,AD),是一種進(jìn)行性神經(jīng)退行性疾病,其特征為細(xì)胞外淀粉樣蛋白沉積和細(xì)胞內(nèi)神經(jīng)原纖維纏結(jié)。其臨床癥狀主要表現(xiàn)為認(rèn)知功能障礙和記憶減退,并伴有一系列精神和行為改變。AD發(fā)生的病因迄今不明,Aβ學(xué)說被公認(rèn)為是AD發(fā)生的重要機(jī)制。該學(xué)說認(rèn)為,由于代謝異常,導(dǎo)致腦內(nèi)相關(guān)酶活性失調(diào),產(chǎn)生有毒性作用的蛋白片段。該片段不能被及時(shí)清除,使其在腦內(nèi)特定區(qū)域堆積,形成β淀粉樣沉淀,進(jìn)一步發(fā)展為老年斑,最終引起神經(jīng)元凋亡,誘發(fā)阿爾茨海默病。β淀粉樣蛋白(Aβ)是由39-43個(gè)氨基酸組成的構(gòu)成老年斑的主要成分。Aβ40和Aβ42片段是淀粉樣蛋白前體(APP)經(jīng)蛋白水解酶水解后產(chǎn)生的最常見的β淀粉樣蛋白片段,其中Aβ42片段含量較少,但是其更容易聚集形成淀粉樣蛋白沉積,因此具有更強(qiáng)的毒性。小膠質(zhì)細(xì)胞是腦內(nèi)常駐免疫細(xì)胞,是中樞神經(jīng)系統(tǒng)(CNS)免疫防線重要的構(gòu)成細(xì)胞,在各種神經(jīng)系統(tǒng)疾病以及炎癥反應(yīng)中具有極其重要的作用。生理狀態(tài)下,活化的小膠質(zhì)細(xì)胞在維持中樞神經(jīng)系統(tǒng)正常功能中發(fā)揮重要的作用。II型髓系細(xì)胞觸發(fā)型受體(TREM2)是小膠質(zhì)細(xì)胞的表面受體,是小膠質(zhì)細(xì)胞高度表達(dá)的與自身活化相關(guān)的蛋白,參與吞噬Aβ、修復(fù)受損神經(jīng)元和清除細(xì)胞碎片的過程。近年來,基因組學(xué)研究發(fā)現(xiàn),罕見的TREM2突變可顯著增加阿爾茨海默病的發(fā)病機(jī)率。研究表明,TREM2缺失或單劑量不足都可導(dǎo)致AD模型鼠清除Aβ的能力明顯下降。盡管基因突變可增加AD的發(fā)病機(jī)率,但是研究表明,年齡因素仍是AD發(fā)病的最主要因素。多種疾病研究發(fā)現(xiàn),TREM2的表達(dá)受核轉(zhuǎn)錄因子NF-κB的調(diào)控。NF-κB是一類關(guān)鍵的核轉(zhuǎn)錄因子,幾乎在所有類型細(xì)胞的胞質(zhì)中都有以異源或同源二聚體非活性形式存在的NF-κB復(fù)合物,與機(jī)體代謝、基因轉(zhuǎn)錄翻譯和調(diào)控蛋白質(zhì)表達(dá)等生命活動(dòng)密切相關(guān)。NF-κB作為細(xì)胞信號轉(zhuǎn)導(dǎo)途徑的樞紐,具有廣泛的生物學(xué)活性,能夠在基因水平上參與炎癥、腫瘤、免疫、細(xì)胞增殖和細(xì)胞凋亡等多種生理、病理過程的調(diào)控。吡咯烷二硫代甲酸(PDTC)是一種可以透過細(xì)胞膜的特異性的NF-κB活化抑制劑,可以在多種細(xì)胞中抑制NF-κB的激活,產(chǎn)生相應(yīng)的生物學(xué)活性。目的:本實(shí)驗(yàn)通過提高類AD模型小鼠腦內(nèi)TREM2的表達(dá),促進(jìn)小膠質(zhì)細(xì)胞的增殖活化,進(jìn)一步檢測活化的小膠質(zhì)細(xì)胞對AD模型鼠病理過程的影響及其產(chǎn)生作用的機(jī)制。方法:1.腦立體定位注射Aβ1-42到昆明小鼠單側(cè)海馬CA1區(qū)制備類AD小鼠動(dòng)物模型。對側(cè)側(cè)腦室注射不同濃度的PDTC。實(shí)驗(yàn)小鼠分為:空白組、Aβ組、PDTC處理組(6 mg/L、8 mg/L、600 mg/L、800 mg/L),注射后第7天取材。2.常規(guī)蘇木素-伊紅(HE)染色觀察不同組中海馬區(qū)損傷部位組織形態(tài)變化。3.免疫組織化學(xué)檢測不同組海馬內(nèi)Aβ的沉積,免疫組織化學(xué)和Western Blot檢測海馬區(qū)TREM2、CD11B、BDNF和DCX的表達(dá)。4.免疫組織熒光技術(shù)檢測海馬區(qū)Aβ和小膠質(zhì)細(xì)胞共表達(dá)、小膠質(zhì)細(xì)胞和TREM2共表達(dá)。結(jié)果:1.HE染色結(jié)果顯示:與空白組相比,注射Aβ各組小鼠海馬區(qū)出現(xiàn)不同程度的損傷;Aβ組小鼠海馬損傷區(qū)域最為明顯,與Aβ組相比,給予PDTC處理后,PDTC各組海馬區(qū)損傷明顯減輕。2.Aβ免疫組織化學(xué)檢測結(jié)果顯示:空白組海馬區(qū)沒有Aβ沉積,注射Aβ后各組模型鼠海馬區(qū)均有Aβ沉積;其中,Aβ組海馬區(qū)Aβ沉積最多,PDTC處理后海馬區(qū)Aβ沉積明顯減少,結(jié)果具有顯著性差異(P0.01)。3.TREM2免疫組織化學(xué)檢測結(jié)果顯示:與空白組相比,Aβ組海馬區(qū)TREM2的表達(dá)降低,結(jié)果具有顯著性差異(P0.01);PDTC處理后,TREM2表達(dá)增加,與Aβ相比,差異具有顯著性(P0.01)。且在一定范圍內(nèi),TREM2的表達(dá)與PDTC劑量呈正相關(guān),;CD11B免疫組織化學(xué)結(jié)果顯示,空白組海馬區(qū)小膠質(zhì)細(xì)胞均勻分布,無聚集現(xiàn)象;Aβ組和PDTC各組海馬區(qū)小膠質(zhì)細(xì)胞在Aβ周圍集中分布;與Aβ組相比,PDTC各組小膠質(zhì)細(xì)胞表達(dá)增加,結(jié)果具有顯著性差異(P0.01)。在一定劑量范圍內(nèi)與PDTC給藥劑量呈正相關(guān)。4.免疫組織熒光共定位檢測結(jié)果顯示:Aβ周圍小膠質(zhì)細(xì)胞大量聚集活化,Aβ沉積內(nèi)有小膠質(zhì)細(xì)胞的浸潤;TREM2與小膠質(zhì)細(xì)胞表達(dá)位置相關(guān),具有共表達(dá)關(guān)系。5.BDNF、DCX免疫組織化學(xué)檢測結(jié)果顯示:與空白組相比,Aβ組BDNF和DCX的表達(dá)均有下降,PDTC各組BDNF和DCX的表達(dá)上升,差異具有顯著性(P0.01)。在一定范圍內(nèi),BDNF和DCX的表達(dá)與PDTC劑量具有依賴關(guān)系。結(jié)論:1.PDTC干預(yù)調(diào)節(jié)小鼠海馬內(nèi)TREM2的表達(dá)。2.TREM2表達(dá)增加,可激活小膠質(zhì)細(xì)胞并維持特定小膠質(zhì)細(xì)胞表型,從而清除Aβ和抑制炎癥反應(yīng)。3.活化的小膠質(zhì)細(xì)胞可能分泌與神經(jīng)再生相關(guān)的神經(jīng)營養(yǎng)因子如BDNF,促進(jìn)神經(jīng)元再生,改善AD病理狀態(tài)。
[Abstract]:Alzheimer 's Disease (AD) is an progressive neurodegenerative disease characterized by extracellular amyloid protein deposition and intracellular neurofibrillary tangles. Its clinical symptoms are cognitive dysfunction and memory impairment, accompanied by a series of mental and behavioral changes in.AD to date, A beta The theory is recognized as an important mechanism for the occurrence of AD. It is believed that the protein fragment of the brain related enzyme activity is caused by abnormal metabolism, which can not be removed in time to accumulate in certain regions of the brain, form beta amyloid precipitation, develop into senile plaques, eventually induce neuronal apoptosis and induce apoptosis. Beta amyloid (A beta) is the most common beta amyloid fragment produced by the hydrolysis of amyloid precursor (APP) by 39-43 amino acids, which is the main component of the aged plaque,.A beta 40 and A beta 42. The content of A beta 42 is less, but it is more easily aggregated to form amyloid eggs. White deposits are more toxic. Microglia, a resident immune cell in the brain, is an important constituent of the central nervous system (CNS) immune defense line. It plays an important role in various nervous system diseases and inflammatory reactions. Under physiological condition, activated microglia can maintain the normal function of the central nervous system. The important role of.II type myeloid cell contact cell receptor (TREM2) is the surface receptor of microglia, which is highly expressed by microglia and the proteins associated with self activation. It participates in the process of phagocytosis of A beta, repairing damaged neurons and scavenging cell debris. In recent years, the study of genomics has found that rare TREM2 mutations can be significantly increased. The incidence of Alzheimer's disease. Studies have shown that TREM2 deletion or single dose deficiency can lead to a significant decrease in the ability of AD model mice to clear A beta. Although genetic mutations can increase the incidence of AD, studies have shown that age is still the most important factor in the pathogenesis of AD. A variety of disease studies have found that the expression of TREM2 is subject to the nuclear transcription factor NF-. The regulation of kappa B.NF- kappa B is a key nuclear transcription factor. There are almost all types of NF- kappa B complex in the cytoplasm of all types of cells, which exist in the form of heterogeneous or homologous two polymer, and closely related to the body metabolism, gene transcription translation and the regulation of protein expression, which are closely related to.NF- kappa B as the hub of cell signal transduction pathway. PDTC is a specific NF- kappa B activation inhibitor that can inhibit the activation of NF- kappa B in a variety of cells and produces phase in a variety of cells. Objective: Objective: to improve the proliferation and activation of microglia by improving the expression of TREM2 in the brain of AD model mice, the effect of activated microglia on the pathological process of AD model rats and the mechanism of its effect were further detected. Method: 1. stereotactic stereotaxic injection of A beta 1-42 to the unilateral hippocampal CA1 area of Kunming mice AD mice were prepared by injecting different concentrations of PDTC. in the lateral ventricle. The mice were divided into blank group, A beta group, PDTC treatment group (6 mg/L, 8 mg/L, 600 mg/L, 800 mg/L), and seventh days after injection,.2. routine hematoxylin eosin (HE) staining was used to observe the changes of tissue morphology of the injured part of the CNOOC region. The deposition of A beta in the same group, immunohistochemistry and Western Blot for the detection of TREM2, CD11B, BDNF and DCX in the hippocampus, the expression of.4. immunofluorescence technique was used to detect the co expression of A beta and microglia in the hippocampus, and the co expression of microglia and TREM2. The damage area of the hippocampus in A beta group was most obvious. Compared with the A beta group, after PDTC treatment, the hippocampal damage in each group of PDTC was significantly reduced by.2.A beta immunochemistry test results showed that there was no A beta deposition in the hippocampus of the blank group and A beta deposition in the hippocampus of each model rat after the injection of A beta, and A beta deposition in the hippocampus of A beta group. At most, the A beta deposition in hippocampus decreased significantly after PDTC treatment. The results showed significant difference (P0.01). The results of.3.TREM2 immunohistochemical staining showed that the expression of TREM2 in the hippocampus of A beta group was significantly lower than that in the blank group (P0.01), and the expression of TREM2 increased after PDTC treatment, and the difference was significant (P0.01) compared with A beta. In a certain range, the expression of TREM2 was positively correlated with the dose of PDTC, and the results of CD11B immunohistochemical staining showed that the microglia in the hippocampus of the blank group was distributed uniformly and without aggregation, and the microglia in the hippocampus of A beta and PDTC groups was distributed around A beta, and the microglia expression in PDTC groups increased significantly compared with the A beta group, and the results were significant. Sex difference (P0.01). The results of positive correlation of.4. immunofluorescence with the dose of PDTC in a certain dose range showed that the microglia around A beta was activated in large quantities and there were microglia infiltration in A beta deposition; TREM2 was associated with the position of microglia expression, and there was a co expression relationship between.5.BDNF and DCX immuno histochemical examination. Compared with the blank group, the expression of BDNF and DCX in A beta group decreased, the expression of BDNF and DCX in PDTC groups increased, and the difference was significant (P0.01). In a certain range, the expression of BDNF and DCX was dependent on the PDTC dose. Conclusion: 1.PDTC intervention to regulate the expression of TREM2 in the hippocampus of mice increased and could activate small glue. The cells also maintain a specific microglia phenotype, thus removing A beta and inhibiting the activation of.3. activated microglia may secrete neurotrophic factors related to nerve regeneration, such as BDNF, promote neuron regeneration and improve the pathological state of AD.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16

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