双转基因小鼠模型中microRNA-153对RTN4的表达调控

目的探讨mir-153在阿尔茨海默病发病机制中的作用。方法通过microRNA芯片及Real-timePCR检测APPswe/PSΔE9双转基因小鼠脑内mir-153的表达;构建高表达mir-153的稳转细胞系,通过western-blot检测稳转细胞系内RTN4的蛋白表达;构建野生型及突变型RTN4的3’UTR荧光素酶报告载体,分别将其与mir-153表达载体或microRNA阴性对照载体共转染入293T细胞内,检测海肾荧光素酶的相对活性以验证mir-153对RTN4 mRNA的作用靶点。结果 microRNA芯片及Real-time PCR检测均证实3月龄APPswe/PSΔE9双转基因小鼠脑内mir-153的表达较同龄野生对照显著降低;在高表达mir-153的稳转细胞系内RTN4的蛋白水平明显降低;共转染野生型RTN4的3’UTR/mir-153表达载体可使海肾荧光素酶相对活性较共转染野生型RTN4的3’UTR/miRNA阴性对照质粒组显著降低（P〈0.05）,而共转染突变型RTN4的3’UTR/mir-153表达载体组则较之无明显差异。结论在3月龄APPswe/PSΔE9双转基因小鼠脑内存在mir-153的异常表达;mir-153可调控RTN4的蛋白表达;mir-153对RTN4的表达调控是通过结合RTN4 3’UTR 839-845碱基处的作用位点而实现的。     

Mir-106b对阿尔茨海默病昼夜节律调控的初步研究

目的探讨mir-106b在阿尔茨海默病发病中的作用。方法取6月龄APPswe/PSΔE9小鼠脑组织,进行microRNA芯片的检测;利用real-time PCR对芯片检测结果进行验证;构建mir-106b表达载体,将其转染至SH-SY5Y细胞中构建mir-106b稳定转染的稳转细胞系。用Targetsan、Pictar、miRanda等靶基因预测软件,对mir-106b的靶基因进行预测,根据靶基因的功能选择可能与AD发病相关的靶基因,并用Western blot对所选择的靶基因在稳转细胞中的表达进行验证。用双荧光素酶报告检测系统检测mir-106b与其靶基因的结合位点。结果芯片结果显示,与野生型小鼠相比,6月龄APPswe/PSΔE9小鼠脑组织中mir-106b的表达下降,经real-time PCR验证,mir-106b的表达在APPswe/PSΔE9小鼠脑组织中的表达较野生型小鼠升高,差别具有统计学意义（P=0.03）。mir-106b稳转细胞系较对照mir-106b的表达升高2～5倍。神经PAS结构域蛋白2（neuronal PAS domain protein2,NPAS2）在mir-106b稳转细胞中的表达明显下降。双荧光素酶报告实验证实：与对照相比,带有NPAS2的3＇UTR的荧光素酶报告载体与mir-106b表达载体共转染,海肾荧光素酶/萤火虫荧光素酶的相对活性降低;荧光素酶报告载体的NPAS2-3＇UTR突变后与mir-106b表达载体共转染,海肾荧光素酶/萤火虫荧光素酶的相对活性升高。结论mir-106b可能通过调节机体生物钟基因NPAS2的表达,影响阿尔茨海默病人的生活节律,参与阿尔茨海默病的发生。     

miR-106b在阿尔茨海默病中的作用机制

目的 探讨miR-106b在阿尔茨海默病(Alzheimer's disease,AD)发病中的作用.方法 取3月龄和6月龄APPswe/PSΔE9小鼠脑组织,进行microRNA芯片的检测;利用real-time PCR检测3、6、9月龄APPswe/PSΔE9小鼠脑组织中miR-106b的表达,对芯片检测结果进行验证;通过构建miR-106b稳定转染细胞系和miR-106b knockdown研究miR-106b与TGFBR2表达之间的关系; 构建TGFBR2 3'UTR-荧光素酶报告载体,验证miR-106b是否可以直接调控TGFBR2蛋白的表达;采用Western blot的方法检测APPswe/ΔPSΔE9小鼠和对照小鼠脑组织中TGFBR2蛋白的表达情况.结果 miR-106b在3月龄和6月龄AD模型小鼠脑组织中表达升高,在9月龄模型小鼠脑组织中表达降低;通过体外实验,我们发现miR-106b与TGFBR2蛋白的表达呈负相关;荧光素酶报告实验表明TGFBR2 3'UTR序列中包含miR-106b的结合位点;TGFBR2蛋白在3、6、9、12月龄AD模型小鼠脑组织中表达均降低.结论 miR-106b可能通过调控TGFBR2蛋白的表达影响TGF-β信号通路,从而参与AD的发病.     

阿尔茨海默病中miR-29c对APP表达调控的初步研究

目的探讨microRNA29c（miR-29c）在阿尔茨海默病中的作用。方法采用了microarray芯片检测3月龄、6月龄APPswe/PSΔE9双转基因阿尔茨海默病小鼠大脑中microRNA表达情况并利用实时定量PCR验证结果可靠性,通过microRNA靶基因数据库预测选出与阿尔茨海默病相关的靶基因,构建miR-29c表达载体,将其转染SH-SY5Y及HEK-293T细胞,在高表达miR-29c的SH-SY5Y及HEK-293T细胞系中验证miR-29c对靶基因的调控作用。将靶基因APP mRNA的野生及突变3’UTR序列克隆到双荧光素酶报告基因载体,用双荧光素酶报告检测系统检测miR-29c与靶基因的结合位点。结果根据microRNA芯片结果筛选出在3、6月龄APPSWE/PS1ΔE9双转基因小鼠大脑表达均有差异的miR-29c,通过实时定量PCR证实miR-29c在3月、6月、9月龄小鼠中表达明显升高。通过microRNA靶基因数据库预测miR-29c可以调控阿尔茨海默病的靶基因APP,利用Western blot检测到高表达miR-29c的SH-SY5Y细胞及HEK-293T细胞中APP蛋白表达减少。将miR-29c表达载体与带有野生及突变APPmRNA的3’UTR的双荧光素酶报告载体共转染HEK-293T细胞,通过双荧光素酶检测系统检测未找到miR-29c与APP mRNA 3’UTR的结合位点。结论 miR-29c对APP表达的具有负向调控作用,但其调控位点可能不位于其3’UTR区域。     

阿尔茨海默病中mir-222作用机制的初步研究

目的探讨mir-222在阿尔茨海默病发病机制中的作用。方法取6月龄APPswe／PSAE9小鼠脑组织,进行microRNA芯片的检测;利用real—timePCR对芯片检测结果进行验证;构建mir-222表达载体,将其转染至SH-SY5Y细胞中,转染后48小时提取蛋白检测p27kipl的表达情况。结果芯片结果显示,与野生型C57BL／6J小鼠相比,6月龄APPswe／PSAE9小鼠脑组织中mir-222表达明显下降,经real-timePCR验证,差别具有统计学意义（P=O．012）;将mir-222表达载体转染至SH—SY5Y细胞后,p27kipl表达减少。结论mir-222可能通过调节细胞周期抑制因子p27kipl的表达参与阿尔茨海默病的发生。     

川芎嗪对痴呆小鼠模型学习记忆能力的影响

目的研究川芎嗪对痴呆小鼠学习记忆的影响。方法对APPswe/PSΔE9双转基因小鼠在3月龄开始食物中连续给川芎嗪（100 mg/kg.d）进行治疗,停药后立即进行水迷宫实验,并与未给药APPswe/PSΔE9双转基因小鼠和及野生鼠进行比较。结果治疗组与APPswe/PSΔE9双转基因组比较,平台寻找潜伏期缩短,穿过原平台区的频次增加。结论川芎嗪能改善APPswe/PSΔE9痴呆模型小鼠的学习记忆能力。     

12种中药单体对APPswe/PSΔE9转基因痴呆模型小鼠学习记忆作用的比较

目的利用APPswe/PSΔE9转基因阿尔茨海默病小鼠模型探索治疗痴呆有效的中药单体。方法根据目前的研究资料,选定12种中药单体,加入鼠粮中对APPswe/PSΔE9双转基因小鼠在5月龄开始进行治疗,以安理申作为阳性药对照,4周后进行水迷宫实验,并与安慰剂组小鼠进行比较。结果在水迷宫实验中,与安慰剂组相比,吴茱萸碱能够显著缩短寻找平台的潜伏期,显著增加穿过原平台区的次数。结论吴茱萸碱能够显著改善APPswe/PSΔE9痴呆模型小鼠的学习记忆能力。     

柚皮苷及其与吴茱萸碱联合用药对APP~（swe）/PS~（ΔE9）转基因痴呆模型小鼠学习记忆能力的影响

目的探讨柚皮苷及柚皮苷与吴茱萸碱联合用药对APPswe/PSΔE9转基因痴呆模型小鼠学习记忆能力的影响,研究柚皮苷与吴茱萸碱联合使用是否具有协同或叠加作用。方法 APPswe/PSΔE9转基因痴呆模型小鼠随机分组,每组12只,雌雄各半,分别选择在3月龄和5月龄两个时间段开始进行治疗,各给药组按设定剂量分别单独或联合加入鼠粮中。3月龄给药16周、5月龄给药4周后进行水迷宫实验,以安理申作为阳性药对照,并与安慰剂组小鼠进行比较。Thioflavin-S荧光染色检测单独使用柚皮苷治疗转基因小鼠脑组织老年斑的形成。结果单独使用柚皮苷4周和16周,缩短寻找平台的潜伏期分别为22%、32%;单独使用吴茱萸碱4周和16周,缩短寻找平台的潜伏期分别为33%、10%。柚皮苷治疗16周减少海马老年斑26%。柚皮苷与吴茱萸碱联合应用能够显著缩短寻找平台的潜伏期47%。结论单独使用柚皮苷或吴茱萸碱均能改善痴呆模型小鼠的学习记忆能力,柚皮苷长期的使用优于短期的治疗效果,但是吴茱萸碱短期有效,长期作用反而下降;柚皮苷与吴茱萸碱联合应用能够显著改善APPswe/PSΔE9痴呆模型小鼠的学习记忆能力,但联合用药组并未表现出强于单独给药组的协同保护效应。     

胃癌相关mir-148a靶向调控胃泌素受体CCKBR

目的：研究胃癌相关miR-148a与胃泌素受体CCKBR的调控关系,并分析其调控结合位点。方法生物信息学预测人CCKBR 3’ UTR上miR-148a 的结合位点;利用 PCR扩增 miR-148a 前体构建真核表达载体;Northern Blot检测miR-148a真核表达载体的表达;构建CCKBR 3’UTR野生型和突变型荧光素酶报告载体,并利用双荧光素酶活性分析检测分析miR-148a对CCKBR基因表达的调控和结合位点;Western Blot检测miR-148a过表达对CCKBR蛋白表达的作用。结果在人CCKBR 3’UTR上找到3个miR-148a的潜在结合位点;miR-148a真核表达载体构建成功,转染胃癌细胞后可显著过表达;miR-148a通过人CCKBR 3’UTR上423bp处的结合位点抑制CCKBR的基因表达;miR-148a过表达显著抑制胃癌细胞中CCKBR的蛋白表达。结论 CCKBR是胃癌相关miR-148a的靶基因,miR-148a通过其3’UTR上的结合位点抑制CCKBR的基因表达和蛋白合成,提示miR-148a可能通过调控CCKBR参与胃癌的发生发展。     

阿尔茨海默病模型小鼠自主活动能力的分析

目的研究阿尔茨海默病APP/PS双转基因小鼠自主行为的改变和作为阿尔茨海默病行为特征的可行性。方法对APPswe/PSΔE9双转基因小鼠和野生鼠分别在4月龄、6月龄、8月龄进行旷场自主行为实验,并与Morris水迷宫分析进行比较,利用SPSS16.0软件统计分析。结果在4月龄、6月龄转基因小鼠的学习记忆能力、以及自主性探究性行为与野生鼠比较有明显的差异。表现为：模型鼠学习记忆能力减弱,兴奋性增高,自主活动增加。这些表现与临床患者的症状有许多相似性,8月龄时,差异减小。结论APPswe/PSΔE9双转基因小鼠的自主行为学表现为烦躁不安,自主活动增加,与阿尔茨海默病患者的临床表现有许多相似之处,可作为小鼠模型判别的行为标志之一,在阿尔茨海默病的病因病机研究和药物研发等方面具有一定的应用价值。     

TREM2 Overexpression has No Improvement on Neuropathology and Cognitive Impairment in Aging APPswe/PS1dE9 Mice

Previously, we showed that overexpression of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific immune receptor, in the brain of a middle-aged (7 months old) APPswe/PS1dE9 mice could ameliorate Alzheimer’s disease (AD)-related neuropathology by enhancement of microglial amyloid-β (Aβ) phagocytosis. Since AD is an age-related neurodegenerative disorder, it is critical to assess the efficacy of TREM2 overexpression in aging animals with an advanced disease stage. In vivo, we employed a lentiviral strategy to overexpress TREM2 in the brain of aging (18 months old) APPswe/PS1dE9 mice, and observed its efficacy on AD-related neuropathology and cognitive functions. Afterwards, we directly isolated microglia from middle-aged and aging APPswe/PS1dE9 mice and determined effects of TREM2 overexpression on microglial Aβ phagocytosis and Aβ-binding receptors expression in vitro. In aging APPswe/PS1dE9 mice, TREM2 overexpression has no beneficial effect on AD-related neuropathology and spatial cognitive functions. Of note, in vitro experiments showed a significant reduction of Aβ phagocytosis in microglia from aging APPswe/PS1dE9 mice, possibly attributing to the declined expression of Aβ-binding receptors. Meanwhile, this phagocytic deficit in microglia from aging APPswe/PS1dE9 mice cannot be rescued by TREM2 overexpression. Taken together, our study shows that TREM2 overexpression fails to provide neuroprotection in aging APPswe/PS1dE9 mice, possibly attributing to deficits in microglial Aβ phagocytosis at the late-stage of disease progression. These findings indicate that TREM2-mediated protection in AD is at least partially dependent on the reservation of microglial phagocytic functions, emphasizing the importance of early therapeutic interventions for this devastating disease.     

Early memory deficits precede plaque deposition in APPswe/PS1dE9 mice: involvement of oxidative stress and cholinergic dysfunction

A large body of evidence has shown that cognitive deficits occur early, before amyloid plaque deposition, suggesting that soluble amyloid-β protein (Aβ) contributes to the development of early cognitive dysfunction in Alzheimer disease (AD). However, the underlying mechanism(s) through which soluble Aβ exerts its neurotoxicity responsible for cognitive dysfunction in the early stage of AD remains unclear so far. In this study, we used preplaque APPswe/PS1dE9 mice ages 2.5 and 3.5 months to examine alterations in cognitive function, oxidative stress, and cholinergic function. We found that only soluble Aβ, not insoluble Aβ, was detected in these preplaque APPswe/PS1dE9 mice. APPswe/PS1dE9 mice 2.5 months of age did not show any significant changes in the measures of cognitive function, oxidative stress, and cholinergic function, whereas 3.5-month-old APPswe/PS1dE9 mice exhibited spatial memory impairment in the Morris water maze, accompanied by significantly decreased acetylcholine (ACh), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) as well as increased malondialdehyde (MDA) and protein carbonyls. In 3.5-month-old preplaque APPswe/PS1dE9 mice, correlational analyses revealed that the performance of impaired spatial memory was inversely correlated with soluble Aβ, MDA, and protein carbonyls, as well as being positively correlated with ACh, ChAT, SOD, and GSH-px; soluble Aβ level was inversely correlated with ACh, ChAT, SOD, and GSH-px, as well as being positively correlated with MDA and protein carbonyls; ACh level showed a significant positive correlation with ChAT, SOD, and GSH-px, as well as a significant inverse correlation with MDA and protein carbonyls. Collectively, this study provides direct evidence that increased oxidative damage and cholinergic dysfunction may be early pathological responses to soluble Aβ and involved in early memory deficits in the preplaque stage of AD. These findings suggest that early antioxidant therapy and improving cholinergic function may be a promising strategy to prevent or delay the onset and progression of AD.