肝脏X受体激活对野生型小鼠和AD小鼠海马放射状胶质细胞的影响及机制研究

发布时间：2018-05-04 03:05

本文选题：肝脏X受体 + TO901317　；参考：《第三军医大学》2012年硕士论文

【摘要】：阿尔茨海默氏病(Alzheimer`s disease，AD)是一种以记忆减退、认知、语言障碍及人格改变为主要症状的退行性神经系统疾病，其特征性病理学改变是大脑皮层和海马等结构细胞外大量β-淀粉样斑块(β amyloid peptide, Aβ)沉积、细胞内神经元纤维缠结(neuofibrillary tangle，NFT)，并最终导致皮层和海马等部位神经元的变性和丢失，临床表现为进行性的认知功能障碍。成年后哺乳动物和人类海马齿回颗粒下区(Dentategyrus-subgranular zone, DG-SGZ)存在可分化为新生神经元的神经干细胞，其增殖、存活、迁移和分化的过程称为神经发生。研究显示来源于成年海马的新生神经元可以整合入海马环路并参与学习记忆形成。在成年海马内，神经干细胞的增殖和分化命运是由这些干细胞所处的壁龛(Niche)所决定，Niche是由海马DG-SGZ内与神经干细胞相邻的颗粒细胞、星形胶质细胞及其细胞外基质组成的组织构筑和其分泌的可溶性分子环境所构成。海马DG-SGZ星形胶质细胞保留有神经干细胞的形态特征，并保留有放射状长突起，能够分化为神经元，也是海马内重要的神经干细胞库。海马中保留有胚胎期放射状突起的星形胶质细胞是海马神经发生过程中新生神经元迁移和突起成熟的关键结构。同时海马齿回中的Krüppel样因子9(Krüppel like-factor9, Klf-9)是海马Niche中维持神经干细胞自我更新和新生神经元后期成熟所必须的关键因子。本实验室前期研究发现，APPswe/PS1ΔE9双转基因AD小鼠海马神经发生显著下降，BrdU标记增殖的神经干细胞相对于同龄的野生型鼠显著减少。众所周知， AD发生时，海马微环境中发生的最大改变就是进行性的Aβ沉积造成的神经系统紊乱。因此，如何改善海马微环境，，调节和促进AD海马神经发生可能是未来治疗AD的有效策略。肝脏X受体(liver X receptor，LXR)属于核受体超家族中的一类转录因子，分为LXRα和LXRβ两种亚型。LXRα与LXRβ在体内分布有较大差别，其中LXRα主要分布在肝脏等与脂类代谢相关的器官，而LXRβ在全身各组织均有广泛表达，在中枢神经系统分布较为丰富，参与脑内胆固醇的代谢。三磷酸腺苷结合盒亚家族A1和G1(ATP binding cassette transporters A1and G1，ABCA1和ABCG1)和载脂蛋白E(apolipoprotein E，apoE)是LXR的靶基因，LXR主要通过对上述基因的调控而参与细胞胆固醇的转运。研究发现LXR可通过调节Aβ的生成而影响AD的病理进程，APP/PS1双转基因AD小鼠敲除LXRα或LXRβ可导致Aβ生成显著增加，而给予LXR激动剂TO901317后可显著改善AD小鼠的学习记忆行为，但这一作用与Aβ水平变化无显著相关性。我们前期研究发现，LXR参与调控大脑皮质发育过程中放射状胶质细胞(radial neuroglia cell，RGCs)向星形胶质细胞的转化， LXRβ基因敲除小鼠RGCs长突起断裂并提前转化为星形胶质细胞，从而导致新生神经元不能迁移到靶区。LXR是否通过直接调节AD小鼠海马DG-SGZ区Niche中RGCs的结构和功能而影响新生神经元的迁移和成熟，其机制如何？目前未见报道。本实验通过LXR激动剂TO901317灌胃，采用免疫组织化学观察3月龄野生型鼠海马DG区星形胶质细胞和放射状胶质细胞形态及数量的变化，RT-PCR检测LXR信号及影响神经发生相关分子表达。通过观察和比较不同月龄APP/PS1双转基因AD小鼠和野生型小鼠海马DG-SGZ区中的RGCs和星形胶质细胞的差异及LXR靶基因和新生神经元成熟相关因子的表达变化，阐明LXR对AD小鼠海马DG-SGZ中放射状胶质细胞转化和对新生神经元成熟的影响及其机制。主要研究结果如下： 1.TO901317对野生型成年小鼠海马LXR受体及相关分子表达的影响与DMSO对照组相比，TO901317处理使3月龄野生型鼠海马LXRβ表达显著下降，而LXRα表达变化不明显。TO901317处理组海马中ABCA1、ABCG1和apoE表达均有显著上调。另外，与DMSO对照组相比，TO901317处理组海马中Klf-9表达显著升高。 2.TO901317对野生型小鼠海马DG区星形胶质细胞和放射状胶质细胞的作用与DMSO对照组相比，TO901317处理使3月龄野生型小鼠海马DG区GFAP阳性的星形胶质细胞数量显著减少，GFAP阳性细胞的胞体显著减小，突起数量显著减少，而海马其他区域和其他脑区GFAP阳性细胞数量变化不明显。TO901317处理导致DG-SGZ区GFAP阳性的放射状长突起和BLBP阳性的放射状胶质细胞数量显著增多。 3.TO901317对AD小鼠海马DG区星形胶质细胞和放射状胶质细胞的作用与同龄的野生型小鼠相比， AD小鼠海马DG-SGZ区GFAP阳性星形胶质细胞显著增生， TO901317处理可以显著降低DG-SGZ区GFAP阳性的星形胶质细胞数量。野生型小鼠TO901317处理后也可显著降低海马DG-SGZ星形胶质细胞增生。6月、9月和13月龄野生型小鼠TO901317激动剂处理后BLBP阳性细胞数量均显著升高。同样，6月、9月和13月龄AD小鼠TO901317激动剂处理后海马齿回BLBP阳性细胞数量显著增加。与同龄的野生型小鼠相比，AD小鼠海马DG区BLBP阳性细胞数显著下降。 4.TO901317对AD小鼠海马LXR靶基因和Klf-9表达的影响与同龄的野生鼠小鼠相比，6月龄、10月龄、13月龄APP/PS1双转基因AD小鼠海马ABCA1mRNA水平无明显变化。采用TO901317处理后,6月龄、10月龄、13月龄AD小鼠海马ABCA1mRNA较DMSO对照组AD小鼠相比显著升高。6月龄、10月龄、13月龄AD小鼠与同龄的野生鼠小鼠相比海马Klf-9mRNA水平无明显变化。TO901317处理6月龄、10月龄、13月龄AD小鼠海马Klf-9mRNA表达显著升高。结论 1.TO901317可显著增加野生型和AD小鼠海马DG-SGZ区域GFAP阳性放射状突起的数量，可显著增加该区BLBP阳性细胞数量，提示LXR通过参与调节海马DG-SGZ的放射状胶质细胞的转化而调控海马神经发生。 2.TO901317可调节AD小鼠海马LXR受体相关靶基因ABCA1, ABCG1, aPOE和Klf-9的表达，提示LXR可能通过该信号通路而参与成年海马神经发生的调节。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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