缺氧小鼠脑中神经节苷脂和单胺类神经递质水平

脑缺氧和缺氧后的再灌注常导致一系列复杂的生理生化改变．通过小鼠重复缺氧后,观察其脑组织中的神经节苷脂和单胺类递质水平变化情况,发现重复缺氧时,随着缺氧次数的增加,a．神经节苷脂水平（以唾液酸含量表示）非常明显地持续下降(P<0.01),其中GM1与GD1b相对组分百分比值下降尤为突出(GM1:P<0.05,GD1b:P<0.01); b.单胺类神经递质中NE和DOPA水平下降,DA和HIAA水平升高(P<0.01)．结果提示脑组织经重复缺氧后,中枢神经组织细胞膜受到一定程度的损伤,其结果可能影响到单胺类神经递质的合成、释放、重摄取和贮存的全过程;据此推测脑组织缺氧时神经节苷脂与单胺类神经递质水平改变时存在着相互联系．     

基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测

极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4 [flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox], Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。     

Cre-LoxP系统在炭疽芽胞杆菌基因敲除中的应用及eag基因的敲除

将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因．以B.anthracis基因组为模板扩增得到上下游同源臂,联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体,转化B．anthracis AP422,通过一系列筛选得到带有抗性标记的重组菌．然后,通过转入Cre重组酶表达质粒,去除抗性标记,得到eag基因缺失的重组菌,并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定．最终建立了Cre-LoxP系统在B．anthracis中的应用方法,并成功敲除eag基因.     

cry3A和vhb基因在转基因马铃薯中的表达

分别构建了含cry3A和cry3A+vhb基因的植物表达载体pBCry3A和pBC₃Vhb,并通过根癌农杆菌介导转化了马铃薯. 对转化再生植株进行PCR和DNA印迹分析表明,外源基因已整合到马铃薯基因组中, 且连续三代无性繁殖后转基因仍存在. ELISA分析表明cry3A基因在转基因植株中得到了高效表达, 在单转cry3A植株中最高表达量达0.1%, 转cry3A与vhb双基因株系中为0.065%. 水涝试验显示,转双基因且vhb mRNA的RT-PCR呈阳性的马铃薯植株,对低氧胁迫有较好的耐受性, 表明获得的上述转双基因马铃薯株系可能会具有很好的抗虫和耐涝性能.     

LASS1基因克隆及其在大鼠脑皮层的表达与神经元衰老的相关性初步研究

长寿保障基因LAG1是从酵母中克隆的与酵母寿命相关的基因,随酵母生命衰老而表达发生变化. 对大鼠中同源基因LASS1进行克隆、测序和序列分析,发现其mRNA序列不同于GenBank中的预测序列,开放阅读框包含1 053碱基对,编码蛋白由350个氨基酸组成,内含Lag1蛋白家族保守的Lag1p motif和TLC结构域. 从新生、1月龄、6月龄、12月龄和24月龄大鼠脑顶叶皮质提取总RNA,用半定量RT-PCR及RNA印迹方法对LASS1在大鼠脑皮质中的表达随年龄变化情况进行分析. 结果表明,出生后LASS1表达量随年龄增加而增高,至6月龄达高峰,然后随年龄增加而逐渐下降,至24月老龄鼠达最低. 衰老相关β半乳糖苷酶(SA-β-gal)对鼠脑皮层染色发现,神经元阳性染色随年龄增长明显增加. 大鼠LASS1基因表达在正常衰老过程中发生变化,为进一步研究该基因的作用奠定了基础.     

Sec-10 影响神经肌肉接头突触后敏感性的研究

exocyst复合物在分泌过程中起重要作用．可是分子机理尚未研究透彻．通过TMP/UV的基因敲除方法,得到了exocyst复合物中一种组分sec-10的缺陷型线虫．药学结果显示这种线虫神经信号传递存在缺陷,可是电生理的方法证明在神经肌肉接头处的已报道离子型受体和野生型相比并未发生改变．因此猜测,sec-10并未直接影响神经肌肉接头处的离子型受体,而是通过其他途径来行使功能．     

不同视觉经验中zif268基因的表达模式

zif268基因编码一转录因子ZIF268. 在发育的视皮层中,zif268基因的表达模式受发育的调节. 在具有正常视觉经验的视皮层中,zif268基因有较高水平的表达. 视觉废置后,视皮层内该基因的表达水平显著降低. 通过视觉刺激可显著增强该基因的表达. 有关zif268基因表达模式的研究对于阐明该基因在哺乳动物视觉系统中的生理功能起到借鉴的作用.     

SOCS-3基因在Balb/c小鼠脑组织中的表达与脑不对称性

为探讨细胞因子信号传导抑制因子-3（suppresso of cytokine signaling-3, SOCS-3）在Balb/c小鼠皮层、海马、下丘脑中的表达及其与脑不对称性的关系.通过伸爪取食法将Balb/c小鼠分为左利组和右利组,取左、右侧皮层,左、右侧海马及下丘脑,以RT-PCR法间接测定SOCS-3 mRNA水平.结果表明：a.在左利鼠右侧皮层中,SOCS-3基因表达水平显著高于左侧皮层(P＜0.05);右利鼠左侧皮层SOCS-3表达显著高于左利鼠左侧皮层(P＜0.05).b.海马中SOCS-3表达：左利鼠右侧海马SOCS-3表达显著高于左侧海马(P＜0.05);右利鼠左侧海马SOCS-3表达显著高于左利鼠左侧海马(P＜0.05).c.右利鼠下丘脑中SOCS-3显著高于左利鼠(P＜0.05).上述研究结果提示,Balb/c小鼠脑中SOCS-3表达与脑不对称性有关.     

单增李斯特菌vip基因缺失

单增李斯特菌(Listeria monocytogenes)是广泛存在于自然界及食物中的食源性致病菌,作为胞内寄生菌,它可以引起强烈的细胞免疫,是潜在的优良疫苗载体。vip是单增李斯特菌的毒力基因,与其侵袭能力密切相关。因此构建vip基因敲除株可为单增李斯特菌疫苗载体的研发打下重要基础。从单增李斯特菌EGDe基因组中扩增出vip基因上、下游序列,连接到穿梭载体pKSV7中得到敲除载体pKSV7-Δvip,将其以电穿孔的方式转入单增李斯特菌后,通过同源重组利用氯霉素和温度双重压力筛选得到vip基因的敲除突变株,并对敲除菌株的生长曲线进行分析发现vip敲除对细菌的生长没有显著影响,为进一步研究vip基因功能、单增李斯特的致病机制和疫苗载体的研发提供参考。     

小鼠胚胎Sry基因的RNA干涉研究

为了研究 Sry 基因的调控网络,采用 siRNA 技术使 Sry 基因沉默,探讨了有效沉默 Sry 基因的途径和最佳条件. 设计、合成针对小鼠 Sry 基因的发夹状寡核苷酸链,退火后连入真核表达载体pSilencer 4.1-CMV neo vector,构建以小鼠 Sry 基因为靶点的 siRNA 干涉载体 pSilencer 4.1/Sry217及 pSilencer 4.1/Sry565,通过尾静脉注射法将载体质粒导入妊娠小鼠体内,于小鼠妊娠第 11.5 天,即 11.5 dpc (days post coitum,性交后天数) 取出胚胎,采用双重 PCR 法对胚胎进行性别鉴定,鉴定为雄性的胚胎采用半定量 RT-PCR 法检测Sry 基因的表达量,研究不同干扰序列、不同注射时间及注射剂量对 Sry 基因表达量的影响. 研究结果,确定了质粒的最佳注射时间为 9.5 dpc,注射剂量为 20 μg,注射干扰质粒 pSilencer 4.1/Sry565 对 Sry基因的抑制效率达 85% 左右. 结果表明,siRNA 可以显著抑制雄性胚胎 Sry 基因的表达.     

Presenilin-1/Presenilin-2双基因敲除小鼠的繁育及基因型鉴定

目的:繁殖及鉴定Presenilins双基因敲除小鼠,为进一步研究阿尔茨海默症(AD)奠定基础。方法:将引进的野生型及PS1/PS2双基因敲除小鼠进行饲养并繁殖,繁殖成功的子代小鼠基因型有野生型、杂合子和纯合子3种。提取子代小鼠鼠尾基因组DNA,用PCR法和琼脂糖凝胶电泳鉴定基因类型。结果:PS1/PS2双基因敲除小鼠的饲养和繁殖均获得成功,繁殖结果符合孟德尔遗传规律,同时获得更多基因型小鼠和Presenilins双基因敲除小鼠。结论:正确的饲养繁殖以及鉴定方法是获得PS1/PS2双基因敲除小鼠的有效途径。     

Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration

Mutations in presenilins are the major cause of familial Alzheimer's disease, but the pathogenic mechanism by which presenilin mutations cause memory loss and neurodegeneration remains unclear. Here we demonstrate that conditional double knockout mice lacking both presenilins in the postnatal forebrain exhibit impairments in hippocampal memory and synaptic plasticity. These deficits are associated with specific reductions in NMDA receptor-mediated responses and synaptic levels of NMDA receptors and alphaCaMKII. Furthermore, loss of presenilins causes reduced expression of CBP and CREB/CBP target genes, such as c-fos and BDNF. With increasing age, mutant mice develop striking neurodegeneration of the cerebral cortex and worsening impairments of memory and synaptic function. Neurodegeneration is accompanied by increased levels of the Cdk5 activator p25 and hyperphosphorylated tau. These results define essential roles and molecular targets of presenilins in synaptic plasticity, learning and memory, and neuronal survival in the adult cerebral cortex.     

Characterization of Age-Dependent and Progressive Cortical Neuronal Degeneration in Presenilin Conditional Mutant Mice

Presenilins are the major causative genes of familial Alzheimer''s disease (AD). Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO) mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold) and apoptotic (TUNEL+, 7.4-fold) neurons, which represent ∼0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (∼9%) and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen cortical neurons. Together, our findings show that loss of presenilins in cortical neurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss of cortical neurons in the aging brain. The low occurrence and significant delay of apoptosis among cortical neurons lacking presenilins suggest that loss of presenilins may induce apoptotic neuronal death through disruption of cellular homeostasis rather than direct activation of apoptosis pathways.     

Isomer-specific effects of conjugated linoleic acid on proliferative activity of cultured neural progenitor cells

We used forebrain-specific conditional presenilin 1 (PS1) and presenilin 2 (PS2) double knockout mice (dKO mice), which exhibit neurodegenerative disease-like symptoms, including inflammation of the brain and periphery, to investigate whether periodontal and salivary tissues display alterations. Mandibles were dissected for alveolar bone height analysis. Maxillae were fixed and decalcified for histological observation and osteoclast detection. Submandibular glands were fixed for histological observation. The submandibular gland and the gingiva of the mandibular incisor teeth were used to assay inflammatory mediators. At 9 months, the number of osteoclasts had significantly increased in the periodontal ligament and the periodontal tissues exhibited obvious histomorphological abnormalities in the dKO mice compared to the control mice at the same age. Alveolar bone loss in dKO mice increased with age. The salivary tissues in dKO mice exhibited obvious age-dependent histomorphological abnormalities. The levels of the inflammatory mediators IL-1β, TNF-α, and GM-CSF in the submandibular gland and gingiva also increased in an age-dependent manner. These findings suggest that inflammation in the dKO brain could expand to the periphery, including the oral tissue, which could ultimately induce abnormalities in the periodontal and salivary tissues.     

Identification of a novel family of putative methyltransferases that interact with human and Drosophila presenilins

Mutations in the presenilin genes have been shown to cause the majority of cases of early-onset familial Alzheimer's disease (AD). In addition to their role in AD, presenilins are also known to function during development by interacting with the Notch pathway. To determine if presenilins have additional functions during development and AD we have used a yeast two-hybrid approach to search for proteins that can bind to presenilins. Here, we show the identification and characterization of a novel putative methyltransferase (Metl) that interacts with the loop region of Drosophila presenilin as well as human presenilin-1 and presenilin-2, suggesting that this interaction is evolutionarily conserved and functionally important. Metl appears to be a member of a conserved family of methyltransferases that share homology with, but are distinct from, the UbiE family of methyltransferases involved in ubiquinone and menaquinone biosynthesis. In Drosophila, the metl gene gives rise to two major isoforms by alternative splicing that are broadly expressed throughout development and found in the central nervous system in an overlapping pattern with Drosophila presenilin. Finally, we show that two independent dominant adult phenotypes produced by overexpression of presenilin can be enhanced by overexpression of metl in the same tissue. Taken together, these results suggest that presenilin and Metl functionally and genetically interact during development.     

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Late-stage neuropathological hallmarks of Alzheimer's disease (AD) are β-amyloid (βA) and hyperphosphorylated tau peptides, aggregated into plaques and tangles, respectively. Corresponding phenotypes have been mimicked in existing transgenic mice, however, the translational value of aggressive over-expression has recently been questioned. As controlled gene expression may offer animal models with better predictive validity, we set out to design a transgenic mouse model that circumvents complications arising from pronuclear injection and massive over-expression, by targeted insertion of human mutated amyloid and tau transgenes, under the forebrain- and neurone-specific CaMKIIα promoter, termed PLB1(Double). Crossing with an existing presenilin 1 line resulted in PLB1(Triple) mice. PLB1(Triple) mice presented with stable gene expression and age-related pathology of intra-neuronal amyloid and hyperphosphorylated tau in hippocampus and cortex from 6 months onwards. At this early stage, pre-clinical (18)FDG PET/CT imaging revealed cortical hypometabolism with increased metabolic activity in basal forebrain and ventral midbrain. Quantitative EEG analyses yielded heightened delta power during wakefulness and REM sleep, and time in wakefulness was already reliably enhanced at 6 months of age. These anomalies were paralleled by impairments in long-term and short-term hippocampal plasticity and preceded cognitive deficits in recognition memory, spatial learning, and sleep fragmentation all emerging at ～12 months. These data suggest that prodromal AD phenotypes can be successfully modelled in transgenic mice devoid of fibrillary plaque or tangle development. PLB1(Triple) mice progress from a mild (MCI-like) state to a more comprehensive AD-relevant phenotype, which are accessible using translational tools such as wireless EEG and microPET/CT.     

Tau/APP/PS1三转基因小鼠模型的建立及生物学特征

目的:建立Tau/APP/PS1三转基因小鼠模型,从分子生物学、行为学及病理学角度研究其生物学特征。方法:将自行建立的Tau转基因小鼠与Jackson实验室引种的APP/PS1双转基因小鼠杂交、传代;PCR鉴定小鼠基因型;RT-PCR检测外源基因的转录;Western blot测定外源基因的蛋白表达;Bielschowsky氏染色法和ABC免疫组化法观察大脑神经纤维缠结和老年斑等病理改变;Morris水迷宫观测学习记忆的改变。结果:Tau/APP/PS1三转基因小鼠的大脑可转录和表达Tau、APP和PS1三种外源基因,6~8月龄时大脑皮层和海马可见神经元纤维缠结和老年斑,其学习记忆获得能力在6月龄开始受损。结论:建立的Tau/APP/PS1三转基因小鼠具有Tau和Aβ两种病理改变和学习记忆障碍,为深入探究Tau与Aβ的关系、阐明AD的发病机制以及研发靶点治疗药物提供实验工具。     

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).     

Exogenous induction of cerebral beta-amyloidosis in betaAPP-transgenic mice

A key commonality of most age-related neurodegenerative diseases is the accumulation of aggregation-prone proteins in the brain. Except for the prionoses, the initiation and propagation of these proteopathies in vivo remains poorly understood. In a previous study, we found that the deposition of the amyloidogenic peptide Abeta can be induced by injection of dilute extracts of Alzheimeric neocortex into the brains of Tg2576 transgenic mice overexpressing the human beta-amyloid precursor protein. The present study was undertaken to assess the pathology after long-term (12 months) incubation, and to clarify the distinctive anatomical distribution of seeded Abeta-immunoreactivity. All mice were injected at 3 months of age; 5 months later, as expected, Abeta deposits were concentrated mostly in the injected hemisphere. After 12 months, abundant, transgene-derived Abeta deposits were present bilaterally in the forebrain, but plaque load was still clearly greater in the extract-injected hemisphere. There was also evidence of tau hyperphosphorylation in axons of the corpus callosum that had been injured by the injection, most prominently in transgenic mice, but also, to a lesser degree, in non-transgenic mice. Five months following injection of AD-extract, an isolated cluster of Abeta-immunoreactive microglia was sometimes evident in the ipsilateral entorhinal cortex; the strong innervation of the hippocampus by entorhinal cortical neurons suggests the possible spread of seeded pathology from the injection site via neuronal transport mechanisms. Finally, using India Ink to map the local dispersion of injectate, we found that Abeta induction is especially potent in places where the injectate is sequestered. The AD-seeding model can illuminate the emergence and spread of cerebral beta-amyloidosis and tau hyperphosphorylation, and thus could enhance our understanding of AD and its pathogenic commonalties with other cerebral proteopathies.