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1.
Whitney A. Greene Alberto. Mu?iz Mark L. Plamper Ramesh R. Kaini Heuy-Ching Wang 《Journal of visualized experiments : JoVE》2014,(88)
The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. 相似文献
2.
Linda Ravenna Lorenzo Principessa Alessandra Verdina Luisa Salvatori Matteo Antonio Russo Elisa Petrangeli 《PloS one》2014,9(5)
Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1α and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic pro-inflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1α activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression. 相似文献
3.
4.
目的:采用基因表达谱分析方法,探讨小麦耐铝的分子机理。方法:利用抑制消减杂交(SSH)技术,以小麦的铝敏感品种Chisholm及其耐铝近等基因系Chisholm-T(其耐铝性来自小麦品种Atlas66)的根尖为材料,构建了2个铝胁迫后的SSHcDNA文库,共含有1628个表达序列标签(EST),利用这些EST制作了小麦根系的cDNA基因芯片。以cDNA基因芯片为平台,在铝胁迫后6h、1d、3d和7d,分别比较Chisholm和Chisholm-T之间的基因表达谱差异。结果:在各个时间点,耐铝和不耐铝小麦材料之间约有5%的EST表现出差异表达。对所有差异表达的EST进行测序分析,序列数据经Pipe-Online2.0进行毗连序列群(contig)拼接,发现只有8.3%的重复序列。结论:SSH是一种非常有效的差减和均一化的建库方法。对有功能注释的差异表达基因进行功能分类分析,表明这些基因参与了植物体内的电子传递、信号传导、植物保护和次生物质的代谢活动。 相似文献
5.
滋养层细胞侵袭相关基因表达谱分析 总被引:1,自引:0,他引:1
分离收集正常妊娠第8~12周的细胞滋养层细胞和绒毛外滋养层细胞,提取细胞总RNA,制备cRNA探针并与AffymetrixU133plus2.0基因芯片进行杂交,获得正常细胞滋养层细胞和绒毛外滋养层细胞基因表达谱芯片。经计算机分析共筛选到1318个差异表达基因,其中上调基因813个,下调505个。所有差异表达基因按GeneOntoloty功能分类标准进行了功能检索。为胚胎发育早期绒毛外滋养层细胞侵袭的基因调控机制的研究提供了实验基础。 相似文献
6.
NOR1基因是一在正常组织中广泛表达且在肿瘤组织中表达下调的新基因.为进一步研究NOR1基因的功能和寻找其下游基因,利用脂质体技术将NOR1基因转染进HepG2细胞,采用cDNA微阵列技术分析其基因表达谱的改变.试验表明NOR1基因的转染能使Grb2,HBP17,TNFRSF11B等59个基因上调,同时也下调Bik,MAp2K6,ZFP95等103个基因.随后用实时荧光定量PCR对cDNA 微阵列结果中上述3个上调表达基因进行验证,结果表明,基因表达差异具有统计学意义(P<0.05),荧光定量PCR结果与微阵列结果相符.这些结果提示,NOR1基因对肝癌HepG2细胞的生物学行为的影响可能与它对细胞信号转导,细胞周期调控,转录、翻译调控相关基因的表达影响有关. 相似文献
7.
《Chronobiology international》2013,30(3):585-590
We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS‐2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (~07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time‐PCR analysis. DEX stimulation of human BEAS‐2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes. 相似文献
8.
Ornella Spadoni Alessio Crestini Paola Piscopo Lorenzo Malvezzi-Campeggi Irene Carunchio Massimo Pieri Cristina Zona Annamaria Confaloni 《Cellular and molecular neurobiology》2009,29(5):635-641
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse
model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between
AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons,
and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s
disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between
these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice
over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical
cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and
APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining
and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting
the existence of post-translational mechanisms that modify protein levels. 相似文献
9.
Gene Expression Profiles of Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease 总被引:9,自引:0,他引:9
Cholinergic neurons of the nucleus basalis (NB) are selectively vulnerable in Alzheimer's disease (AD), yet the molecular mechanisms associated with their dysfunction remain unknown. We used single cell RNA amplification and custom array technology to examine the expression of functional classes of mRNAs found in anterior NB neurons from normal aged and AD subjects. mRNAs encoding neurotrophin receptors, synaptic proteins, protein phosphatases, and amyloid-related proteins were evaluated. We found that trkB and trkC mRNAs were selectively down-regulated in NB neurons, whereas p75NTR mRNA levels remained stable in end stage AD. TrkA mRNA was reduced by approximately 28%, but did not reach statistical significance. There was a down-regulation of synaptophysin, synaptotagmin, and protein phosphatases PP1 and PP1 mRNAs in AD. In contrast, we found a selective up-regulation of cathepsin D mRNA in NB neurons in AD brain. Thus, anterior NB neurons undergo selective alterations in gene expression in AD. These results may provide clues to the molecular pathogenesis of NB neuronal degeneration during AD. 相似文献
10.
从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RTPCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZaB,电穿孔法将重组体整合人酵母菌P.pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut^-菌用甲醇诱导表达,SDS-PAGE检测其表达产物。Mut^-酵母转化菌经甲醇诱导可分泌表达约65kDa和42kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性。 相似文献
11.
Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous
study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation.
This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment
also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or
BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions
after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors
expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease
in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells. 相似文献
12.
从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RT-PCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZαB,电穿孔法将重组体整合入酵母菌P.pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌用甲醇诱导表达,SDS-PAGE检测其表达产物.Mut+酵母转化菌经甲醇诱导可分泌表达约65kDa和42 kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性. 相似文献
13.
干旱是最重要的环境胁迫,香蕉MaASR1基因在植物响应逆境胁迫时发挥着重要作用,为了深入研究MaASR1基因的过表达使拟南芥抗旱的分子机制,利用全基因组表达芯片来广谱的筛选MaASR1基因转入后自然条件下及干旱处理条件下差异基因的表达情况。对基因芯片的结果进行了详细的生物信息学分析及相关基因的RT-PCR验证,结果表明MaASR1基因异源表达的拟南芥株系在自然生长条件下共有747个差异基因,其中上调基因559个,下调基因188个;在干旱胁迫条件下共得到653个差异基因,其中上调基因256个,下调基因397个;MaASR1基因的转入可以通过影响激素、光合作用、锌指蛋白及不依赖ABA途径的DREB2A等相关基因的表达来提高拟南芥的抗旱性。为解析MaASR1基因作为转录因子提高植物抗旱能力的分子机制奠定基础。 相似文献
14.
Charles Oliver Morton Mirjam Fliesser Marcus Dittrich Tobias Mueller Ruth Bauer Susanne Kneitz William Hope Thomas Richard Rogers Hermann Einsele Juergen Loeffler 《PloS one》2014,9(5)
The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA. 相似文献
15.
Maya Amit Michal Nadler-Holly Ester Feldmesser Keith R. Willison 《Journal of molecular biology》2010,401(3):532-3090
The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed. 相似文献
16.
Tu H Rady PL Juelich T Tyring SK Koldzic-Zivanovic N Smith EM Hughes TK 《Cellular and molecular neurobiology》2007,27(2):161-170
1. Aim: The hypothalamic-pituitary-adrenal (HPA) axis is a mediator for interactions between the immune and neuroendocrine systems.
Pro-inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) have
been shown to activate the HPA axis. Recently, interleukin-10, an important anti-inflammatory cytokine in the immune system,
has been shown to be expressed in the central nervous system and neuroendocrine system. Little is known, however, about IL-10’s
functions in the HPA axis.
2. Methods: The Affymetrix DNA microarray (mouse genome U74Av2 Probe Array) was conducted to determine the gene expression profile regulated
by IL-10 in cells of HPA axis origin.
3. Results: In this study, we analyzed gene expression regulated by IL-10 in cells derived from the HPA axis. The results showed that
quorums of genes are modulated by IL-10 in these neuroendocrine cells.
4. Conclusions: These findings will provide a valuable repository to aid in understanding IL-10’s functions in the HPA axis at the molecular
level. 相似文献
17.
肠上皮干细胞研究进展 总被引:4,自引:0,他引:4
肠上皮干细胞是位于肠黏膜陷窝内的具有自我更新和增殖分化为成熟肠上皮细胞功能的细胞。肠黏膜上皮细胞不断更新及肠黏膜损伤的修复均通过肠上皮干细胞增殖、分化完成。根据抗损伤能力的不同肠上皮干细胞可分为三级,一定程度内可适应不同生理、病理变化。陷窝内干细胞之间存在竞争,占优势的干细胞迅速分裂、增殖,使陷窝表现为单克隆性。肠上皮干细胞所处微环境内的各种细胞因子及端粒酶共同影响肠上皮干细胞的分裂、增殖与分化。目前尚缺乏确切的肠上皮干细胞标记,Msi-1、Hes-1和D整合素可能是肠上皮干细胞的自然标记物,但有待进一步研究证实。 相似文献
18.
Wang B Li F Luan W Xie Y Zhang C Luo Z Gui L Yan H Xiang J 《Marine biotechnology (New York, N.Y.)》2008,10(6):664-675
Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated
Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged
samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements
in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most
upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated
in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were
analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help
for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio.
Wang and Li contributed equally to this work. 相似文献
19.
1. The cellular prion protein (PrPC) is expressed widely in neural and nonneural tissues at the highest level in neurons in the central nervous system (CNS).2. Recent studies indicated that transgenic mice with the cytoplasmic accumulation of PrPC exhibited extensive neurodegeneration in the cerebellum, although the underlying mechanism remains unknown. To identify the genes whose expression is controlled by overexpression of PrPC in human cells, we have established a stable PrPC-expressing HEK293 cell line designated P1 by the site-specific recombination technique.3. Microarray analysis identified 33 genes expressed differentially between P1 and the parent PrPC-non-expressing cell line among 12,814 genes examined. They included 18 genes involved in neuronal and glial functions, 5 related to production of extracellular matrix proteins, and 2 located in the complement cascade.4. Northern blot analysis verified marked upregulation in P1 of the brain-specific protein phosphatase 2A beta subunit (PPP2R2B), a causative gene of spinocerebellar ataxia 12, and the cerebellar degeneration-related autoantigen (CDR34) gene associated with development of paraneoplastic cerebellar degeneration.5. These results indicate that accumulation of PrPC in the cell caused aberrant regulation of a battery of the genes important for specific neuronal function. This represents a possible mechanism underlying PrPC-mediated selective neurodegeneration. 相似文献
20.
Anna Bennis Theo G. M. F. Gorgels Jacoline B. ten Brink Peter J. van der Spek Koen Bossers Vivi M. Heine Arthur A. Bergen 《PloS one》2015,10(10)