快速老化模型小鼠海马正反向抑制消减cDNA文库的构建

目的:构建快速老化模型小鼠(SAM)海马正反向抑制消减cDNA文库,以揭示SAMP8学习记忆脑老化的机制,同时为研究阿尔茨海默病(AD)的发病机制提供线索。方法:以快速老化模型小鼠SAMP8和SAMR1海马的总RNA为材料,采用抑制消减杂交方法和蓝白斑筛选克隆构建文库,并用PCR鉴定了文库的质量。结果:成功构建了12月龄雄性SAMP8和SAMR1海马的正反向抑制消减cDNA文库,其中正向文库包含864个克隆,反向文库包含960个克隆,阳性克隆率为96.16%,插入片段范围为250～2000bp。结论:SAMP8和SAMR1海马的正反向抑制消减cDNA文库的构建,为进一步筛选鉴定SAMR1和SAMP8海马差异表达基因提供了丰富的实验材料。     

黄连解毒汤对快速老化小鼠海马基因表达的影响

目的：研究黄连解毒汤（HL）对快速老化小鼠亚系SAMP8海马差异表达基因的影响,揭示HL改善认知功能障碍的分子机制。方法：应用快速老化小鼠亚系SAMP8和SAMR1海马差异表达cDNA芯片,比较了SAMP8和SAMR1、石杉碱甲处理的SAMP8和SAMP8对照,以及HL处理的SAMP8和SAMP8对照的基因表达谱,并对HL的药物反应基因进行了比较;采用实时荧光定量PCR技术对芯片结果进行了验证。结果：给予SAMP8HL后,对SAMP8海马基因表达具有明显的调节作用。差异表达基因包括信号转导基因Dusp12、Rps6ka1、Penk1、Nope,Leng8,蛋白质代谢相关基因Ttc3、Amfr、Prr6、Ube2以,核酸代谢基因Fhit、Itm2c、Cstf2t、Ddx3x、Ercc5、Pcgfr6,能量代谢基因Stubl,免疫反应相关基因Clqb,转录调节基因Dlertdl6le、Gcn512、Ssu72,细胞生长相关基因Ngrn、Anln,递质转运相关基因Slc17a7,以及功能未知基因12个。结论：提示HL对于认知功能的改善可能是通过调节信号转导、突触传递、蛋白质和能量代谢、细胞增殖分化等途径发挥作用的;研究中发现的HL作用基因可能是改善学习记忆的潜在靶标。     

拟南芥cDNA芯片和油菜-拟南芥比较作图相结合筛选甘蓝型油菜抗菌核病先验基因

以拟南芥cDNA芯片筛选核盘菌侵染时甘蓝型油菜的局部抗(耐)病相关基因, 共获得在病斑周围局部组织中受核盘菌胁迫后表达变化达2倍以上的基因61个. 这些基因中, 36个基因为上升表达, 25个基因为下降表达. RT-PCR和Northern杂交验证了部分芯片杂交筛选的结果, 表明拟南芥cDNA芯片可用于甘蓝型油菜基因转录谱的研究. 结合油菜-拟南芥基因组比较作图, 将一些cDNA芯片筛选得到的油菜菌核病抗性相关基因定位于甘蓝型油菜抗菌核病QTL区间内. 取定位于QTL峰值区域的少数基因为先验基因, 值得优先对这些基因进行深入研究.     

小鼠小脑区域性特异表达基因的分离和筛选——乳化酚递减杂交

哺乳类脑组织有着极其复杂和众多的基因表达,许多遗传性神经退行性疾病起源于不同的脑功能区域的基因表达缺陷。分离和筛选小脑特异表达基因是认识小脑疾病的重要途径。本文采用递减杂交法,大脑前皮层和肝组织单链cDNA为驱动物与小脑双链cDNA进行乳化酚杂交,经过重组噬菌体λgt11 DNA连接选择,特异性地克隆那些仅在小脑中优势表达的cDNA。杂交后的小脑eDNA文库容量仅是起始文库容量的0.049%(2.5×10~3/5.15×10~6)。用小脑和肝cDNA探针与PERT cDNA进行克隆杂交和斑点印迹杂交,结果显示绝大多数克隆仅与小脑cDHA杂交阳性。选择10个克隆cDNA与小脑等5种组织mRNA做Northern杂交,有2个小脑特异表达克隆,6个小脑优势表达克隆。对其中PC7和PD8克隆cDNA做部分DNA序列分析,它们是以前未报道过的新基因。     

应用寡核苷酸芯片筛选维甲酸诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因

目的:应用寡核苷酸芯片筛选维甲酸(RA)诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因。方法:从人胎脑及不同类型神经系统肿瘤组织中获取目的基因,查询相应基因mRNA序列,设计并合成探针,制备了含218种基因的神经功能相关的寡核苷酸芯片。应用RA诱导SH-SY5Y8d分化成成熟神经元,提取对照组和实验组每天的总RNA,通过逆转录荧光标记cDNA探针并与芯片杂交,洗片后扫描获取图像,数据分析获得差异表达基因,并通过RT-PCR进行验证。结果:发现13种基因表达上调,没有得到下调基因。RT-PCR验证结果基本与芯片结果一致。结论:SH-SY5Y经RA诱导分化成神经元存在一些差异表达的基因,寡核苷酸芯片技术可为研究SH-SY5Y诱导分化成神经元的分子作用机理提供技术平台。     

cDNA微阵列技术研究NaHCO3胁迫下星星草基因表达谱

为研究NaHCO3胁迫下星星草基因的表达,分别将荧光染料Cy5-dCTP和Cy3-dCTP用反转录方法标记在处理和对照星星草cDNA上制成探针,并与载有星星草基因的cDNA芯片进行杂交。通过对芯片的杂交信号强度分析来研究基因的表达情况。分析结果显示,共有25个基因在NaHCO3胁迫处理前后差异表达,其中17个基因在NaHCO3胁迫下表达下调,8个基因在NaHCO3胁迫下表达上调。生物信息学分析表明这些基因的功能涉及了信号传导与转录调控、细胞防御、细胞代谢等多个方面。从而获得了NaHCO3胁迫下星星草的基因表达谱,定量地阐述了NaHCO3胁迫和非胁迫条件下星星草基因的差异表达情况。     

慢性髓细胞性白血病病人骨髓单个核细胞中差异表达基因的筛选

目的:筛选慢性髓细胞性白血病(CML)病人骨髓单个核细胞与正常人的差异表达基因,探讨CML的发病机制.方法:提取正常人和CML病人单个核细胞的RNA,逆转录成cDNA并用地高辛标记,应用全基因组表达谱基因芯片对差异表达基因进行研究,采用Jubilant病理/疾病分类法对CML相关差异表达基因进行分析.结果:共筛选出CM...     

猪瘟病毒感染猪白细胞凋亡基因表达谱变化的研究

为了了解CSFV感染引起猪细胞凋亡的分子机制,利用基因芯片分析了猪感染CSFV前后基因组转录水平的变化。分离攻毒前和攻毒后猪外周血白细胞(Peripheral blood leucocyte,PBL),提取总RNA,经反转录和体外转录得到cRNA,与Affymetrix猪全基因组cDNA芯片进行杂交分析,筛选差异表达的凋亡基因。结果显示,感染猪的PBLs中共筛选到24个涉及细胞凋亡有关基因发生2倍以上变化,并对差异表达的12个宿主细胞凋亡相关基因,用荧光定量RT-PCR进行了验证。功能分析显示,这些凋亡基因的表达变化与CSFV诱导宿主细胞凋亡的机制有关。本文首次用猪全基因组芯片研究了CSFV感染诱导宿主基因的表达变化,建立了CSFV感染猪细胞凋亡相关基因的表达谱,初步阐释了CSFV感染诱导宿主细胞凋亡的分子机制。     

应用cDNA微阵列技术筛选大鼠脊髓损伤修复相关基因

脊髓损伤是一类常见的、高致残率的中枢神经系统疾病,由于多种复杂因素影响其损伤后的修复过程,损伤脊髓的再生能力非常有限。本研究采用cDNA微阵列技术筛选大鼠脊髓损伤后出现的差异表达基因。实验组动物在T8-T9进行脊髓全横断手术,对照组动物只打开椎板;4．5d后取脊髓进行RNA提取并在反转录过程中进行Cy3／Cy5标记,然后与预制的、带有4041条特异性探针的芯片进行杂交。Cy5／Cy3信号比值≥2．0视为脊髓损伤后出现差异表达的基因。通过筛选,我们得到了65个上调表达基因（21个已知基因,30个已知EST和14个未知基因）和79个下调基因（20个已知基因,42个已知EST和17个未知基因）。进一步通过半定量RT-PCR对其中的5个上调已知基因（Timpl,Tagln,Vim,Fc gamma receptor,Ctss）和三个下调已知基因（stearyl-CoA desaturase,F2,Ensa）的表达情况进行了验证,结果显示与芯片结果一致。这些基因可能在脊髓损伤后的修复过程中起一定的作用,对其深入研究将有助于揭示脊髓损伤修复的分子机制。     

用基因芯片结合荧光差异显示—PCR筛选和识别胃腺癌转移相关的基因

使用来源于同一个胃腺癌病人的原发灶RF－1（ATCC编号：CRL－1864）和转移灶RF－48细胞系（ATCC编号,CRL－1863）作为研究肿瘤转移分子机制的模型,RF－1（实验组）和RF－48（对照组）的mRNA通过逆转录方法,将Cy3和Cy5两种荧光染料分别标记到两种细胞的cDNA上,制备成cDNA探针,并与表达谱芯片（双点4096条基因）进行杂交与扫描,重复2次实验,利用计算机数据处理判断基因是否在上述两种细胞中有表达差异,共筛选出差异表达的基因共138条,其中81条在RF－48细胞中表达明显上调,57条在RF－48细胞中表达显著下调,同时也通过荧光差异显示－PCR（FDD－PCR）技术,克服了45个涉及胃腺癌转移相关基因,包括未被发现的基因3个,在两种筛选方法中都存在差异表达的基因共有7条,对部分可能与肿瘤志移机制有关的差异表达基因的作用进行了分析和讨论,基因芯片技术可高通量,大规模地研究基因表达水平,FDD－PCR技术可克隆出未发现的新基因,二者结合,初步筛选出与转移相关的基因,有助于揭示胃腺癌转移的分子机制。     

应用基因芯片技术检测肝组织的基因表达

为检测正常肝组织中的基因表达状况,采用cDNA microarray技术对正常肝组织中表达的1500个基因进行定量,结果为97个基因较高表达,1010个基因中度表达,380个基因低表达,结果是cDNA microarray技术是大规模检测基因表达的有效方法。     

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.     

Combination of PCR subtraction and cDNA microarray for differential gene expression profiling.

PCR subtraction hybridization has been used effectively to enrich and single out differentially expressed genes. However identification of these genes by means of cloning and sequencing individual cDNAs is a tedious and lengthy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent detection method. mRNA from human prolactin (hPRL) or human prolactin antagonist (hPRL-G129R) treated and non-treated breast cancer cells was isolated, and cDNAs were synthesized and used for the PCR subtraction to enrich the differentially expressed genes in the treated cells. The PCR-amplified and subtracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membrane and identified by chemiluminescence. The results suggest that the strategy of combining all three methods will allow for a more efficient, nonradioactive way of identifying differentially expressed genes in target cells.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.     

Frontiers of Model Animals for Neuroscience:Two Prosperous Aging Model Animals forPromoting Neuroscience Research

A model animal showing spontaneous onset is a useful tool for investigating the mechanism of disease. Here, I would like to introduce two aging model animals expected to be useful for neuroscience research: the senescence-accelerated mouse (SAM) and the klotho mouse. The SAM was developed as a mouse showing a senescence-related phenotype such as a short lifespan or rapid advancement of senescence. In particular, SAMP8 and SAMP10 show age-related impairment of learning and memory. SAMP8 has spontaneous spongy degeneration in the brain stem and spinal cord with aging, and immunohistochemical studies reveal excess protein expression of amyloid precursor protein and amyloid β in the brain, indicating that SAMP8 is a model for Alzheimer’s disease. SAMP10 also shows age-related impairment of learning and memory, but it does not seem to correspond to Alzheimer’s disease because senile plaques primarily composed of amyloid β or neurofibrillary tangles primarily composed of phosphorylated tau were not observed. However, severe atrophy in the frontal cortex, entorhinal cortex, amygdala, and nucleus accumbens can be seen in this strain in an age-dependent manner, indicating that SAMP10 is a model for normal aging. The klotho mouse shows a phenotype, regulated by only one gene named α-klotho, similar to human progeria. The α-klotho gene is mainly expressed in the kidney and brain, and oxidative stress is involved in the deterioration of cognitive function of the klotho mouse. These animal models are potentially useful for neuroscience research now and in the near future.     

Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.