本文選題：肝臟X受體 + TO901317　； 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：阿爾茨海默氏病(Alzheimer`s disease，AD)是一種以記憶減退、認(rèn)知、語(yǔ)言障礙及人格改變?yōu)橹饕Y狀的退行性神經(jīng)系統(tǒng)疾病，其特征性病理學(xué)改變是大腦皮層和海馬等結(jié)構(gòu)細(xì)胞外大量β-淀粉樣斑塊(β amyloid peptide, Aβ)沉積、細(xì)胞內(nèi)神經(jīng)元纖維纏結(jié)(neuofibrillary tangle，NFT)，并最終導(dǎo)致皮層和海馬等部位神經(jīng)元的變性和丟失，臨床表現(xiàn)為進(jìn)行性的認(rèn)知功能障礙。成年后哺乳動(dòng)物和人類海馬齒回顆粒下區(qū)(Dentategyrus-subgranular zone, DG-SGZ)存在可分化為新生神經(jīng)元的神經(jīng)干細(xì)胞，其增殖、存活、遷移和分化的過(guò)程稱為神經(jīng)發(fā)生。研究顯示來(lái)源于成年海馬的新生神經(jīng)元可以整合入海馬環(huán)路并參與學(xué)習(xí)記憶形成。在成年海馬內(nèi)，神經(jīng)干細(xì)胞的增殖和分化命運(yùn)是由這些干細(xì)胞所處的壁龕(Niche)所決定，Niche是由海馬DG-SGZ內(nèi)與神經(jīng)干細(xì)胞相鄰的顆粒細(xì)胞、星形膠質(zhì)細(xì)胞及其細(xì)胞外基質(zhì)組成的組織構(gòu)筑和其分泌的可溶性分子環(huán)境所構(gòu)成。海馬DG-SGZ星形膠質(zhì)細(xì)胞保留有神經(jīng)干細(xì)胞的形態(tài)特征，并保留有放射狀長(zhǎng)突起，能夠分化為神經(jīng)元，也是海馬內(nèi)重要的神經(jīng)干細(xì)胞庫(kù)。海馬中保留有胚胎期放射狀突起的星形膠質(zhì)細(xì)胞是海馬神經(jīng)發(fā)生過(guò)程中新生神經(jīng)元遷移和突起成熟的關(guān)鍵結(jié)構(gòu)。同時(shí)海馬齒回中的Krüppel樣因子9(Krüppel like-factor9, Klf-9)是海馬Niche中維持神經(jīng)干細(xì)胞自我更新和新生神經(jīng)元后期成熟所必須的關(guān)鍵因子。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)，APPswe/PS1ΔE9雙轉(zhuǎn)基因AD小鼠海馬神經(jīng)發(fā)生顯著下降，BrdU標(biāo)記增殖的神經(jīng)干細(xì)胞相對(duì)于同齡的野生型鼠顯著減少。眾所周知， AD發(fā)生時(shí)，海馬微環(huán)境中發(fā)生的最大改變就是進(jìn)行性的Aβ沉積造成的神經(jīng)系統(tǒng)紊亂。因此，如何改善海馬微環(huán)境，，調(diào)節(jié)和促進(jìn)AD海馬神經(jīng)發(fā)生可能是未來(lái)治療AD的有效策略。 肝臟X受體(liver X receptor，LXR)屬于核受體超家族中的一類轉(zhuǎn)錄因子，分為L(zhǎng)XRα和LXRβ兩種亞型。LXRα與LXRβ在體內(nèi)分布有較大差別，其中LXRα主要分布在肝臟等與脂類代謝相關(guān)的器官，而LXRβ在全身各組織均有廣泛表達(dá)，在中樞神經(jīng)系統(tǒng)分布較為豐富，參與腦內(nèi)膽固醇的代謝。三磷酸腺苷結(jié)合盒亞家族A1和G1(ATP binding cassette transporters A1and G1，ABCA1和ABCG1)和載脂蛋白E(apolipoprotein E，apoE)是LXR的靶基因，LXR主要通過(guò)對(duì)上述基因的調(diào)控而參與細(xì)胞膽固醇的轉(zhuǎn)運(yùn)。研究發(fā)現(xiàn)LXR可通過(guò)調(diào)節(jié)Aβ的生成而影響AD的病理進(jìn)程，APP/PS1雙轉(zhuǎn)基因AD小鼠敲除LXRα或LXRβ可導(dǎo)致Aβ生成顯著增加，而給予LXR激動(dòng)劑TO901317后可顯著改善AD小鼠的學(xué)習(xí)記憶行為，但這一作用與Aβ水平變化無(wú)顯著相關(guān)性。我們前期研究發(fā)現(xiàn)，LXR參與調(diào)控大腦皮質(zhì)發(fā)育過(guò)程中放射狀膠質(zhì)細(xì)胞(radial neuroglia cell，RGCs)向星形膠質(zhì)細(xì)胞的轉(zhuǎn)化， LXRβ基因敲除小鼠RGCs長(zhǎng)突起斷裂并提前轉(zhuǎn)化為星形膠質(zhì)細(xì)胞，從而導(dǎo)致新生神經(jīng)元不能遷移到靶區(qū)。LXR是否通過(guò)直接調(diào)節(jié)AD小鼠海馬DG-SGZ區(qū)Niche中RGCs的結(jié)構(gòu)和功能而影響新生神經(jīng)元的遷移和成熟，其機(jī)制如何？目前未見(jiàn)報(bào)道。 本實(shí)驗(yàn)通過(guò)LXR激動(dòng)劑TO901317灌胃，采用免疫組織化學(xué)觀察3月齡野生型鼠海馬DG區(qū)星形膠質(zhì)細(xì)胞和放射狀膠質(zhì)細(xì)胞形態(tài)及數(shù)量的變化，RT-PCR檢測(cè)LXR信號(hào)及影響神經(jīng)發(fā)生相關(guān)分子表達(dá)。通過(guò)觀察和比較不同月齡APP/PS1雙轉(zhuǎn)基因AD小鼠和野生型小鼠海馬DG-SGZ區(qū)中的RGCs和星形膠質(zhì)細(xì)胞的差異及LXR靶基因和新生神經(jīng)元成熟相關(guān)因子的表達(dá)變化，闡明LXR對(duì)AD小鼠海馬DG-SGZ中放射狀膠質(zhì)細(xì)胞轉(zhuǎn)化和對(duì)新生神經(jīng)元成熟的影響及其機(jī)制。主要研究結(jié)果如下： 1.TO901317對(duì)野生型成年小鼠海馬LXR受體及相關(guān)分子表達(dá)的影響 與DMSO對(duì)照組相比，TO901317處理使3月齡野生型鼠海馬LXRβ表達(dá)顯著下降，而LXRα表達(dá)變化不明顯。TO901317處理組海馬中ABCA1、ABCG1和apoE表達(dá)均有顯著上調(diào)。另外，與DMSO對(duì)照組相比，TO901317處理組海馬中Klf-9表達(dá)顯著升高。 2.TO901317對(duì)野生型小鼠海馬DG區(qū)星形膠質(zhì)細(xì)胞和放射狀膠質(zhì)細(xì)胞的作用 與DMSO對(duì)照組相比，TO901317處理使3月齡野生型小鼠海馬DG區(qū)GFAP陽(yáng)性的星形膠質(zhì)細(xì)胞數(shù)量顯著減少，GFAP陽(yáng)性細(xì)胞的胞體顯著減小，突起數(shù)量顯著減少，而海馬其他區(qū)域和其他腦區(qū)GFAP陽(yáng)性細(xì)胞數(shù)量變化不明顯。TO901317處理導(dǎo)致DG-SGZ區(qū)GFAP陽(yáng)性的放射狀長(zhǎng)突起和BLBP陽(yáng)性的放射狀膠質(zhì)細(xì)胞數(shù)量顯著增多。 3.TO901317對(duì)AD小鼠海馬DG區(qū)星形膠質(zhì)細(xì)胞和放射狀膠質(zhì)細(xì)胞的作用 與同齡的野生型小鼠相比， AD小鼠海馬DG-SGZ區(qū)GFAP陽(yáng)性星形膠質(zhì)細(xì)胞顯著增生， TO901317處理可以顯著降低DG-SGZ區(qū)GFAP陽(yáng)性的星形膠質(zhì)細(xì)胞數(shù)量。野生型小鼠TO901317處理后也可顯著降低海馬DG-SGZ星形膠質(zhì)細(xì)胞增生。6月、9月和13月齡野生型小鼠TO901317激動(dòng)劑處理后BLBP陽(yáng)性細(xì)胞數(shù)量均顯著升高。同樣，6月、9月和13月齡AD小鼠TO901317激動(dòng)劑處理后海馬齒回BLBP陽(yáng)性細(xì)胞數(shù)量顯著增加。與同齡的野生型小鼠相比，AD小鼠海馬DG區(qū)BLBP陽(yáng)性細(xì)胞數(shù)顯著下降。 4.TO901317對(duì)AD小鼠海馬LXR靶基因和Klf-9表達(dá)的影響 與同齡的野生鼠小鼠相比，6月齡、10月齡、13月齡APP/PS1雙轉(zhuǎn)基因AD小鼠海馬ABCA1mRNA水平無(wú)明顯變化。采用TO901317處理后,6月齡、10月齡、13月齡AD小鼠海馬ABCA1mRNA較DMSO對(duì)照組AD小鼠相比顯著升高。6月齡、10月齡、13月齡AD小鼠與同齡的野生鼠小鼠相比海馬Klf-9mRNA水平無(wú)明顯變化。TO901317處理6月齡、10月齡、13月齡AD小鼠海馬Klf-9mRNA表達(dá)顯著升高。 結(jié)論 1.TO901317可顯著增加野生型和AD小鼠海馬DG-SGZ區(qū)域GFAP陽(yáng)性放射狀突起的數(shù)量，可顯著增加該區(qū)BLBP陽(yáng)性細(xì)胞數(shù)量，提示LXR通過(guò)參與調(diào)節(jié)海馬DG-SGZ的放射狀膠質(zhì)細(xì)胞的轉(zhuǎn)化而調(diào)控海馬神經(jīng)發(fā)生。 2.TO901317可調(diào)節(jié)AD小鼠海馬LXR受體相關(guān)靶基因ABCA1, ABCG1, aPOE和Klf-9的表達(dá)，提示LXR可能通過(guò)該信號(hào)通路而參與成年海馬神經(jīng)發(fā)生的調(diào)節(jié)。
[Abstract]:Alzheimer`s disease (AD) is a degenerative neurologic disease characterized by memory impairment, cognition, language disorder, and personality change. Its characteristic pathological changes are a large number of beta amyloid plaques (beta amyloid peptide, A beta) deposited outside the cortical and hippocampal structures of the cerebral cortex and neurons. Neuofibrillary tangle (NFT) and eventually lead to degeneration and loss of neurons in the cortex and hippocampus. The clinical manifestation is progressive cognitive dysfunction. There is a neural stem cell that can differentiate into newborn neurons in the subregion of adult mammalian and human hippocampal dentine gyrus (Dentategyrus-subgranular zone, DG-SGZ). The process of colonization, survival, migration and differentiation is called neurogenesis. Studies show that newborn neurons from the adult hippocampus can be integrated into the hippocampal loop and participate in learning and memory formation. In the adult hippocampus, the proliferation and differentiation of neural stem cells is determined by the Niche of these stem cells, and Niche is within the hippocampus DG-SGZ. The granular cells adjacent to the neural stem cells, the tissue construction of astrocytes and their extracellular matrix and their secreted soluble molecular environment. The hippocampal DG-SGZ astrocytes retain the morphological characteristics of neural stem cells and retain long radiate protrusions, which can differentiate into neurons, and are also important gods in the hippocampus. Kr u ppel like factor 9 (Kr u ppel like-factor9, Klf-9) in hippocampal gyrus is the key to the self-renewal and new nerve of neural stem cells in hippocampus Niche. The key factors that must be made in the late maturing of the yuan. The previous study in our laboratory found that the hippocampal neurogenesis of APPswe/PS1 Delta E9 double transgenic AD mice decreased significantly, and the BrdU labeled neural stem cells were significantly reduced relative to the same age wild mice. As we all know, the biggest change in the hippocampus microenvironment was the progressive A beta in the occurrence of AD. Therefore, how to improve the hippocampal microenvironment, regulate and promote AD hippocampal neurogenesis may be an effective strategy for the treatment of AD in the future.
The liver X receptor (liver X receptor, LXR) belongs to a class of transcription factors in the nuclear receptor superfamily. It is divided into LXR A and LXR beta two subtypes,.LXR A and LXR beta in the body, and LXR A is mainly distributed in the liver and other organs related to lipid metabolism, while LXR beta is widely expressed in all tissues of the whole body and is distributed in the central nervous system. A1 and G1 (ATP binding cassette transporters A1and G1, ABCA1 and ABCG1) and apolipoprotein E (apolipoprotein) are the target genes, which are mainly involved in the transport of cholesterol by the regulation of these genes. Regulating the formation of A beta and affecting the pathological process of AD, APP/PS1 double transgenic AD mice knockout LXR alpha or LXR beta can lead to a significant increase in A beta generation, while the LXR agonist TO901317 can significantly improve the learning and memory behavior of AD mice, but this effect is not significantly related to the changes in A beta level. Our previous study found that LXR participate in the regulation of the brain. The transformation of radial neuroglia cell (RGCs) to astrocytes during the development of cortex, LXR beta gene knockout mouse RGCs long protruded and astrocytes in advance, which leads to the failure of newborn neurons to migrate to the target area.LXR by directly regulating RGCs node in Niche of AD mice's DG-SGZ region Niche. Structure and function affect the migration and maturation of neonatal neurons. What is the mechanism?
In this experiment, the morphologic and quantitative changes of astrocytes and radiated glial cells in the hippocampal DG region of 3 month old wild type rats were observed by LXR agonist TO901317, and RT-PCR was used to detect the LXR signal and to affect the expression of the neurogenesis related molecules. By observing and comparing the APP/PS1 double transgenic AD mice and the wild of different months of age, and compared with the wild. The difference of RGCs and astrocytes in the hippocampal DG-SGZ area and the changes in the expression of the LXR target gene and the maturation related factors of the newborn neurons in the hippocampus of type mice, and to elucidate the effect and the mechanism of LXR on the transformation of radial glial cells and the maturation of the newborn neurons in the DG-SGZ of the hippocampus of AD mice and its mechanism. The main results are as follows:
Effect of 1.TO901317 on expression of LXR receptor and related molecules in hippocampus of wild type adult mice
Compared with the DMSO control group, TO901317 treatment significantly decreased the expression of LXR beta in the hippocampus of 3 month old wild rats, while the expression of LXR alpha was not significantly increased in the hippocampus ABCA1, ABCG1 and apoE in the.TO901317 treatment group. In addition, the Klf-9 expression in the hippocampus was significantly higher in the TO901317 treatment group than in the DMSO control group.
Effects of 2.TO901317 on astrocytes and radial glial cells in DG region of wild type mice
Compared with the DMSO control group, TO901317 treatment reduced the number of GFAP positive astrocytes in the hippocampal DG region of 3 month old wild type mice significantly, the cell body of GFAP positive cells decreased significantly, the number of protuberances decreased significantly, while the number of GFAP positive cells in other hippocampus and other brain regions changed no obvious.TO901317 processing leading to the GFAP positive in DG-SGZ region. The number of radial long projections and BLBP positive radial glial cells increased significantly.
Effects of 3.TO901317 on astrocytes and radial glial cells in hippocampal DG region of AD mice
Compared with the wild type mice of the same age, the GFAP positive astrocytes in the hippocampal DG-SGZ region of AD mice were significantly proliferated, and TO901317 treatment could significantly reduce the number of astrocytes of GFAP positive in DG-SGZ region. TO901317 treatment in the wild type mice could significantly reduce the.6 month of the hippocampal DG-SGZ astrocyte proliferation, in September and 13 month old in the wild type. The number of BLBP positive cells increased significantly after the treatment of rat TO901317 agonists. Similarly, the number of BLBP positive cells in the Houhai dentate cycle increased significantly in June, September and 13 month old AD mice treated with TO901317 agonists. Compared with the same age wild type mice, the number of BLBP positive cells in the hippocampal DG region of AD mice decreased significantly.
Effects of 4.TO901317 on LXR target gene and Klf-9 expression in hippocampus of AD mice
Compared with the same aged mice, the hippocampal ABCA1mRNA level of 6 month old, 10 month old, and 13 month old APP/PS1 double transgenic AD mice was not significantly changed. After TO901317 treatment, 6 month old, 10 month old, 13 month old AD mice were significantly higher than the DMSO control group AD mice to increase the.6 month, 10 month old, 13 month old AD mice and the same age of wild mice mice. Compared with hippocampal Klf-9mRNA level, there was no significant change in the level of Klf-9mRNA in hippocampus of.TO901317 treated 6 month old, 10 month old and 13 month old AD mice.
conclusion
1.TO901317 can significantly increase the number of GFAP positive radial projections in the DG-SGZ region of the wild and AD mice, which can significantly increase the number of BLBP positive cells in this area, suggesting that LXR regulates the hippocampal neurogenesis by regulating the transformation of radial glial cells that regulate the hippocampal DG-SGZ.
2.TO901317 can regulate the expression of LXR receptor related target genes ABCA1, ABCG1, aPOE and Klf-9 in the hippocampus of AD mice, suggesting that LXR may be involved in the regulation of adult hippocampal neurogenesis through this signaling pathway.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16

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相關(guān)期刊論文 前3條

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