Since silver-nanoparticles (NPs) possess an antibacterial activity, they were commonly used in medical products and devices, food storage materials, cosmetics, various health care products, and industrial products. Various silver-NP based medical devices are available for clinical uses, such as silver-NP based dressing and silver-NP based hydrogel (silver-NP-hydrogel) for medical applications. Although the previous data have suggested silver-NPs induced toxicity in vivo and in vitro, there is lack information about the mechanisms of biological response and potential toxicity of silver-NP-hydrogel.
Methods
In this study, the genotoxicity of silver-NP-hydrogel was assayed using cytokinesis-block micronucleus (CBMN). The molecular response was studied using DNA microarray and GO pathway analysis.
Results and discussion
The results of global gene expression analysis in HeLa cells showed that thousands of genes were up- or down-regulated at 48?h of silver-NP-hydrogel exposure. Further GO pathway analysis suggested that fourteen theoretical activating signaling pathways were attributed to up-regulated genes; and three signal pathways were attributed to down-regulated genes. It was discussed that the cells protect themselves against silver NP-mediated toxicity through up-regulating metallothionein genes and anti-oxidative stress genes. The changes in DNA damage, apoptosis and mitosis pathway were closely related to silver-NP-induced cytotoxicity and chromosome damage. The down-regulation of CDC14A via mitosis pathway might play a role in potential genotoxicity induced by silver-NPs.
Conclusions
The silver-NP-hydrogel induced micronuclei formation in cellular level and broad spectrum molecular responses in gene expression level. The results of signal pathway analysis suggested that the balances between anti-ROS response and DNA damage, chromosome instability, mitosis inhibition might play important roles in silver-NP induced toxicity. The inflammatory factors were likely involved in silver-NP-hydrogel complex-induced toxic effects via JAK-STAT signal transduction pathway and immune response pathway. These biological responses eventually decide the future of the cells, survival or apoptosis. 相似文献
Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC. 相似文献
The existence of two sophisticated parallel splicing machineries in multicellular organisms has raised intriguing questions--ranging from their impact on proteome expansion to the evolution of splicing and of metazoan genomes. Exploring roles for the distinct splicing systems in vivo has, however, been restricted by the lack of techniques to selectively inhibit their function in cells. In this study, we show that morpholino oligomers complementary to the branch-site recognition elements of U2 or U12 small nuclear RNA specifically suppress the function of the two splicing systems in mammalian cells. The data provide the first evidence for a role of distinct spliceosomes in pre-mRNA splicing from endogenous mammalian genes and establish a tool to define roles for the different splicing machineries in vivo. 相似文献
Hydrogel nanoparticles of cross-linked polyvinylpyrrolidone (PVP-NP) (35-50 nm in diameter) containing fluoresceinated dextran (FITC-Dx) were encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes(1)). Incubation of these loaded F-virosomes with human hepatoblastoma cells (HepG2) in culture resulted in membrane-fusion-mediated delivery of NPs to the cell cytoplasm, as inferred from the ability of cells to internalize FITC-Dx loaded PVP-NP (PVP(f)-NP) in the presence of azide (an inhibitor of the endocytotic process). Introduction of PVP(f)-NP into the HepG2 cells was assured by selective accumulation of FITC fluorescence in the cytosolic compartment. The structural integrity of the internalized PVP(f)-NP was also confirmed by fluorescence microscopy and ultracentrifugation analysis. The potential usefulness of PVP-NP-mediated cytosolic release of water soluble drugs both in vitro and in vivo has been established for the first time. 相似文献
This paper examines the recent emergence of miniaturized optical fiber based sensing and actuating devices that have been successfully integrated into fluidic microchannels that are part of microfluidic and lab‐on‐chip systems. Fluidic microsystems possess the advantages of reduced sample volumes, faster and more sensitive biological assays, multi‐sample and parallel analysis, and are seen as the de facto bioanalytical platform of the future. This paper considers the cases where the optical fiber is not merely used as a simple light guide delivering light across a microchannel, but where the fiber itself is engineered to create a new sensor or tool for use within the environment of the fluidic microchannel.
We report the use of plasmonic responses of single gold nanoparticles (AuNPs) with various sizes for the analysis of biomolecular recognition. We also describe the relationship between particle size and plasmonic response induced by the binding of receptors and target analytes. To investigate the plasmonic response of AuNPs, Rayleigh light scattering spectra were collected from individual AuNPs using a dark-field microspectroscopy system. Using prostate-specific antigen (PSA) as a model, the linear dynamic range was obtained in the concentration range of 10(-4) to 10 ng/ml, with the smallest detectable concentration at 0.1 pg/ml corresponding to localized surface plasmon resonance (LSPR) λ(max) shifts of approximately 2.95 nm. This result indicates that individual AuNPs can be used for development of a very sensitive, robust, simple, and label-free biosensor to detect protein biomarkers. Furthermore, the method possesses great potential for monitoring other biological interactions. 相似文献
Synthetic DNA linkers containing a single mismatched nucleotide (C:A) are repaired without bias at high efficiency when introduced into mammalian cells on a SV40 shuttle vector. From the pattern of repair in vectors containing multiple linkers, it appears that DNA synthesis following mismatch excision can replace a length of DNA as short as 40 nucleotides. Furthermore, results from the introduction of linker molecules containing combinations of single-strand nicks suggest that transient unsealed nicks do not drive the direction of mismatch repair in mammalian cells, as has previously been proposed. 相似文献
The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.
Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes. 相似文献
We demonstrate a new method for the simultaneous measurement of the activation of key regulatory enzymes within single cells. To illustrate the capabilities of the technique, the activation of protein kinase C (PKC), protein kinase A (PKA), calcium-calmodulin activated kinase II (CamKII), and cdc2 protein kinase (cdc2K) was measured in response to both pharmacological or physiological stimuli. This assay strategy should be applicable to a broad range of intracellular enzymes, including phosphatases, proteases, nucleases, and other kinases. 相似文献
An improved dual-laser flow cytometric system for quantitative analysis and sorting of mammalian cells has been developed using a low-power argon and high-power krypton laser as illumination sources, thus permitting the excitation of fluorescent dyes having absorption regions ranging from the ultraviolet to infrared. Cells stained in liquid suspension with fluorescent dyes enter a flow chamber where they intersect two spatially separated laser beams. Separate pairs of quartz beam-shaping optics focus each beam onto the cell stream. Electro-optical sensors measure fluorescence and light scatter signals from cells that are processed electronically and displayed as frequency distribution histograms. Cells also can be electronically separated and microscopically identified. The ease and versatility of operation designed into this system represent a marked technological improvement for dual-laser excited flow systems. Details of this instrument are described along with illustrative examples of cells stained with mithramycin and rhodamine and analyzed for DNA content, total protein, and nuclear and cytoplasmic diameter. 相似文献
Normal cells depend on autophagy to maintain cellular homeostasis by recycling damaged organelles and misfolded proteins and degrading toxic agents. Similar to apoptosis, targeting autophagy has been under attention in cancer therapy. However, autophagy has both pro-survival and pro-death functions in tumors, and its targeting requires further elucidation. The current review focuses on using nanoparticles for targeting autophagy in cancer treatment. Nanocarriers can deliver autophagy regulators along with chemotherapeutic agents leading to intracellular accumulation in cancer cells and synergistic cancer therapy. Furthermore, genetic tools such as siRNA and shRNA can be used for targeting molecular components that regulate autophagy, such as the ATG12-ATG5-ATG16L1 complex. A number of nanostructures, such as gold and zinc oxide nanoparticles, can be used to enhance oxidative stress-mediated apoptosis and autophagy, reducing cancer progression. Further, using nanoparticles to modulate autophagy potentiates the anti-tumor effects of cisplatin and gefitinib during chemotherapy. Polymeric nanoparticles, lipid-based nanostructures and carbon-based nanomaterials are among other nanoparticles capable of regulating autophagy in cancer cells. Of note, various regulatory components of autophagy such as ATGs, Beclin-1 and LC3-II can be affected by nanomaterials. Based on the role of nanomaterial-induced autophagy as pro-survival or pro-death, further targeting can potentiate the fight against cancer cells. 相似文献
Transparency is widespread in nature, ranging from transparent insect wings to ocular tissues that enable you to read this text, and transparent marine vertebrates. And yet, cells and tissue models in biology are usually strongly light scattering and optically opaque, precluding deep optical microscopy. Here we describe the directed evolution of cultured mammalian cells toward increased transparency. We find that mutations greatly diversify the optical phenotype of Chinese Hamster Ovary cells, a cultured mammalian cell line. Furthermore, only three rounds of high-throughput optical selection and competitive growth are required to yield fit cells with greatly improved transparency. Based on 15 monoclonal cell lines derived from this directed evolution experiment, we find that the evolved transparency frequently goes along with a reduction of nuclear granularity and physiological shifts in gene expression profiles. In the future this optical plasticity of mammalian cells may facilitate genetic clearance of living tissues for in vivo microscopy. 相似文献
The study investigated the feasibility of lyophilization for long-term preservation of somatic cells and embryonic development after whole cell intracytoplasmic injection (WCICI) into enucleated pig oocyte. Confluent cultured porcine fetal fibroblast (pFF) cells were lyophilized and stored at 4 °C for at least 6 months. Results showed that compared to non-lyophilized control cells, lyophilized cells had drastically reduced cellular viability (P < 0.01). WCICI of reconstituted lyophilized cells could support complete embryonic development. However, the rates of cleavage (64.7 ± 2.7 vs. 43.5 ± 4.7%) and blastocyst formation (18.2 ± 0.6 vs. 10.2 ± 1.6%) were lower than that of control (P < 0.05). Total nuclei number per blastocyst (30.4 ± 4.5 vs. 25.2 ± 4.7) and intensity of acetylation at histone H3 (AcH3) protein (55.9 ± 3.5 vs. 53.3 ± 3.8) did not differ (P > 0.05). The development ability of embryos, produced from lyophilized somatic cells, was further increased (19.5 ± 2.4 vs. 10.2 ± 1.6%; P < 0.05) by treatment with trichostatin A (TSA) for 24 h post-activation. These TSA-treated embryos also had AcH3 level comparable with in vitro fertilized embryos (63.1 ± 3.2 vs. 69.9 ± 1.3). In conclusion, our results suggest that lyophilized somatic cells can direct embryonic development up to blastocyst stage after WCICI into pig oocytes. Treatment of embryos, produced from lyophilized somatic cells, with TSA can further increase their in vitro developmental potential. 相似文献