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1.
Reprod Fertil Dev ; 32(7): 676-689, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32317092

RESUMO

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between invitro- and invivo-derived embryos.

2.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31409031

RESUMO

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.


Assuntos
Bovinos/fisiologia , Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Capacitação Espermática , Animais , Apoptose , Feminino , Fertilização In Vitro/veterinária , Masculino , Melatonina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo
3.
Adv Mater ; 31(37): e1903277, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31348581

RESUMO

Small interfering RNA (siRNA) holds inherent advantages and great potential for treating refractory diseases. However, lack of suitable siRNA delivery systems that demonstrate excellent circulation stability and effective at-site delivery ability is currently impeding siRNA therapeutic performance. Here, a polymeric siRNA nanomedicine (3I-NM@siRNA) stabilized by triple interactions (electrostatic, hydrogen bond, and hydrophobic) is constructed. Incorporating extra hydrogen and hydrophobic interactions significantly improves the physiological stability compared to an siRNA nanomedicine analog that solely relies on the electrostatic interaction for stability. The developed 3I-NM@siRNA nanomedicine demonstrates effective at-site siRNA release resulting from tumoral reactive oxygen species (ROS)-triggered sequential destabilization. Furthermore, the utility of 3I-NM@siRNA for treating glioblastoma (GBM) by functionalizing 3I-NM@siRNA nanomedicine with angiopep-2 peptide is enhanced. The targeted Ang-3I-NM@siRNA exhibits superb blood-brain barrier penetration and potent tumor accumulation. Moreover, by cotargeting polo-like kinase 1 and vascular endothelial growth factor receptor-2, Ang-3I-NM@siRNA shows effective suppression of tumor growth and significantly improved survival time of nude mice bearing orthotopic GBM brain tumors. New siRNA nanomedicines featuring triple-interaction stabilization together with inbuilt self-destruct delivery ability provide a robust and potent platform for targeted GBM siRNA therapy, which may have utility for RNA interference therapy of other tumors or brain diseases.


Assuntos
Terapia Genética , Glioblastoma/terapia , Nanomedicina , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Terapia Combinada , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos
4.
ACS Chem Neurosci ; 9(7): 1616-1624, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29708326

RESUMO

Ethanol is a principle ingredient of alcoholic beverages with potential neurotoxicity and genotoxicity, and the ethanol-associated oxidative DNA damage in the central nervous system is well documented. Natural source compounds may offer new options to protect the brain against ethanol-induced genotoxicity. Veratrum maackii Regel is a toxic rangeland plant linked to teratogenicity which is also used in traditional Chinese medicine as "Lilu" and is reported to contain a family of compounds called stilbenes that can have positive biological activity. In this study, nine stilbenes were isolated from the aerial parts of V. maackii Regel, and their structures were identified as cis-mulberroside A (1), resveratrol-4,3'- O-ß-d-diglucopyranoside (2), mulberroside A (3), gentifolin K (4), resveratrol-3,5- O-ß-d-diglucopyranoside (5), oxyresveratrol- 4'- O-ß-d-glucopyranoside (6), oxyresveratrol-3- O-ß-d-glucopyranoside (7), oxyresveratrol (8), and resveratrol (9) using ESI-MS and NMR techniques. The total concentration of extracted compounds 2-9 was 2.04 mg/g, suggesting that V. maackii Regel is a novel viable source of these compounds. In an in vivo comet assay, compounds 1-9 were observed to decrease DNA damage in mouse cerebellum and cerebral cortex caused by acute ethanol administration. Histological observation also revealed decreased brain injury in mice administered with compounds 1-9 after acute ethanol administration. The protective effects of compound 6 were associated with increasing T-SOD and GSH-PX activities and a decrease in NO and MDA concentrations. These findings suggest that these compounds are potent inhibitors of ethanol-induced brain injury possibly via the inhibition of oxidative stress and may be valuable leads for future therapeutic development.


Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Etanol/efeitos adversos , Substâncias Protetoras/farmacologia , Estilbenos/farmacologia , Veratrum , Transtornos Relacionados ao Uso de Álcool/tratamento farmacológico , Transtornos Relacionados ao Uso de Álcool/metabolismo , Transtornos Relacionados ao Uso de Álcool/patologia , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Masculino , Camundongos , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fototerapia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Distribuição Aleatória , Estilbenos/química , Estilbenos/isolamento & purificação
5.
Chem Commun (Camb) ; 54(29): 3609-3612, 2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29570195

RESUMO

A superior biocompatible spherical nucleic acid (SNA) conjugate was fabricated by grafting siRNA onto the surface of a core composed of a spherical DNA nanostructure that we have termed a DNA nanoclew (DC). After uptake by cultured cancer cells, SNA nanoparticles release engrafted siRNAs by cleavage of the intracellular Dicer enzyme. Moreover, in vitro experiments reveal that such SNAs demonstrate potent gene knockdown at both mRNA and protein levels, while with negligible cytotoxicity.


Assuntos
DNA/química , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Silenciamento de Genes/métodos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Inativação Gênica , Células HeLa , Humanos , Nanopartículas/toxicidade , Hibridização de Ácido Nucleico , Tamanho da Partícula , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/toxicidade , Ribonuclease III/química
6.
Acta Biomater ; 58: 432-441, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28602854

RESUMO

A chemical template strategy was put forward to synthesize monodisperse rattle-type magnetic carbon (Fe3O4@C) hollow microspheres. During the synthesis procedure, monodisperse Fe2O3 microspheres were used as chemical template, which released Fe3+ ions in acidic solution and initiated the in-situ polymerization of pyrrole into polypyrrole (PPy) shell. With the continual acidic etching of Fe2O3 microspheres, rattle-type Fe2O3@PPy microspheres were generated with the cavity appearing between the PPy shell and left Fe2O3 core, which were then transformed into Fe3O4@C hollow microspheres through calcination in nitrogen atmosphere. Compared with traditional physical template, the shell and cavity of rattle-type hollow microspheres were generated in one step using the chemical template method, which obviously saved the complex procedures including the coating and removal of middle shells. The experimental results exhibited that the rattle-type Fe3O4@C hollow microspheres with different parameters could be regulated through controlled synthesis of the intermediate Fe2O3@PPy product. Moreover, when the rattle-type Fe3O4@C hollow microspheres were investigated as drug carrier, they manifested sustained-release behaviour of doxorubicin, justifying their promising applications as carriers in drug delivery. STATEMENT OF SIGNIFICANCE: The aim of the present study was first to synthesize rattle-type Fe3O4@C hollow microspheres through a simple synthesis method as a drug carrier. Here a chemical template synthesis of rattle-type hollow microspheres was developed, which saved the complex procedures including the coating and removal of middle shells in traditional physical template. Second, all the influence factors in the reaction processes were systematically investigated to obtain rattle-type Fe3O4@C hollow microspheres with controlled parameters. Third, the rattle-type Fe3O4@C hollow microspheres were studied as drug carriers and the influences of their structural parameters on drug loading and releasing performance were investigated.


Assuntos
Doxorrubicina , Portadores de Fármacos , Óxido Ferroso-Férrico , Microesferas , Polímeros , Pirróis , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Óxido Ferroso-Férrico/química , Óxido Ferroso-Férrico/farmacocinética , Óxido Ferroso-Férrico/farmacologia , Células HeLa , Humanos , Polímeros/química , Polímeros/farmacocinética , Polímeros/farmacologia , Pirróis/química , Pirróis/farmacocinética , Pirróis/farmacologia
7.
Mater Sci Eng C Mater Biol Appl ; 75: 829-835, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28415536

RESUMO

Template-etching strategy was put forward to synthesize rattle-type magnetic silica (Fe3O4@SiO2) hollow microspheres in a controlled way. During the experiment, monodisperse Fe2O3 microspheres were fabricated as physical template to generate uniform Fe2O3@SiO2 with controlled shell thicknesses through sol-gel method, and the subsequent Fe2O3 template etching process created variable space between Fe2O3 core and SiO2 shell, and the final calcination process transformed rattle-type Fe2O3@SiO2 hollow microspheres into corresponding Fe3O4@SiO2 product in hydrogen/nitrogen atmosphere. Compared with traditional physical template, here template-etching synthesis of rattle-type hollow microspheres saved the insertion of middle shells and their removal, which simplified the synthesis process with controllable core size and shell thickness. The rattle-type Fe3O4@SiO2 hollow microspheres as drug carrier show efficient doxorubicin (DOX) loading, and the release rate of DOX loaded the rattle-type Fe3O4@SiO2 hollow microspheres exhibit a surprising shell-thickness-dependent and a pH responsive drug release features. Additionally, MTT assays in HeLa cells demonstrated that the Fe3O4@SiO2 nanocarriers were non-toxic even at the concentration of 250µgmL-1 for 48h. Thus, our results revealed that the Fe3O4@SiO2-DOX could play an important role in the development of intracellular delivery nanodevices for cancer therapy.


Assuntos
Portadores de Fármacos/química , Compostos Férricos/química , Microesferas , Dióxido de Silício/química
8.
J Biotechnol ; 218: 66-72, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26656225

RESUMO

Bacterial artificial chromosomes (BACs) are vectors that are capable of carrying gene fragments of up to 300 kb in size, and in theory, harbor cis-regulatory elements that are necessary for the expression of specific genes. Therefore, BACs can effectively alleviate or even eliminate the position effect induced by gene-integration, rendering these as ideal expression vectors of exogenous genes. However, the number of relevant studies involving BACs as vectors of exogenous genes are limited. In the present study, we converted the BAC regulatory region of the Mus musculus Wap gene into a mammary gland-specific expression vector. Using the galK-based positive-negative selection method, we seamlessly replaced the Wap gene in a BAC with Homo sapiens GPX3, MT2, and Luc genes while keeping the original mammary gland-specific regulatory sequence intact, without introducing any extra sequences (Loxp/Frt). To improve the efficiency of creating BAC transgenic mice, we used a Tol2 transposon system optimized for mammalian codons and eliminated 100 kb of sequence from the BAC 5' end (173 kb), which resulted in an 8.5% rate of successful gene transmission via pronuclear injection. The results of the present study indicate that seamlessly constructed BAC expression vectors can be used for the transmission of the GPX3 gene.


Assuntos
Cromossomos Artificiais Bacterianos , Técnicas de Transferência de Genes , Glândulas Mamárias Animais/fisiologia , Animais , Elementos de DNA Transponíveis , Feminino , Deleção de Genes , Genes Bacterianos , Genes Reporter , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Masculino , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos
9.
Int J Clin Exp Med ; 7(8): 1958-66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25232376

RESUMO

AIM: To explore Trichostatin A (TSA) effect on SGC-7901 gastric cancer cells. METHODS: MTT, fluorescence microscopy, and flow cytometry were used to assess TSA effect on cell growth and apoptosis in SGC-7901. Immunocytochemistry was used to evaluate the expression of acetylated histone H4 in SGC-7901 cells.Gene expression profile was determined by microarray assays. Glycoprotein hormones alpha subunit (CGA) gene and protein expressions in SGC-7901 cells were evaluated by Real-time PCR and Western blot, respectively. In addition, CGA protein levels in gastric adenocarcinoma and normal adjacent tissues were assessed by immunohistochemistry. RESULTS: TSA inhibited SGC-7901 cell growth. In addition, cell proliferation was significantly decreased (P = 0.02) in TSA treatment groups (0.93 ± 0.07) compared with controls (1.15 ± 0.07). Apoptosis related morphological changes, including nuclear chromatin condensation and fluorescence strength, were observed by fluorescence microscopy. These findings corroborated the increased expression of acetylated histone H4 observed in TSA treated cells compared to controls, as determined by immunocytochemistry. Interestingly, treatment of SGC-7901 cells with TSA (75 ng/ml) resulted in CGA gene down-regulation (P = 0.0381). Accordingly, CGA protein levels were decreased in TSA treated SGC-7901 cells. Finally, immunohistochemistry analysis showed that CGA expression was significantly higher in gastric adenocarcinoma tissues than normal adjacent tissues (P = 0.001). CONCLUSION: TSA induces cell apoptosis and increases the levels of acetylated histone H4 in SGC-7901 cells. In addition, TSA treatment decreases the expression in gastric cancer cells of the CGA gene, which is upregulated in gastric adenocarcinoma tissues.

10.
Int J Clin Exp Med ; 7(12): 4857-66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25663982

RESUMO

In this study, we investigated the effect of trichostatin A (TSA) on the gastric cancer cell line BGC-823. The effect of TSA on growth inhibition and apoptosis of BGC-823 cells was examined. The gene expression profile was determined by microarray. Western blotting was used to study the levels of acetylated histone H4 and Glycoprotein non-metastatic melanoma protein B (GPNMB) proteins. GPNMB gene expression was measured by real-time PCR. GPNMB protein levels in gastric adenocarcinoma tissues and adjoining normal tissues were detected by immunohistochemistry. The results showed that a significant decrease in cell population following treatment with 75 ng/mL TSA for 48 h (0.87 ± 0.04) as compared to control (1.14 ± 0.06) (P = 0.02). Apoptotic cells were increased in TSA (75 ng/mL for 48 h) treated group as compared to the control group (from 2.02% to 19.74%) by flow cytometry. The expression of acetylated histone H4 was increased in TSA treated (75 ng/mL for 48 h) group (from 1.00 ± 0.26 to 1.87 ± 0.33, F = 5.862, P = 0.0038) as compared to the control group by Western blotting. After 48 h TSA treatment (75 ng/mL), BGC-823 cells showed decrease in GPNMB gene expression (from 1.00 ± 0.21 to 0.59 ± 0.11, F = 6.214, P = 0.0018). Immunohistochemistry showed that GPNMB expression in gastric adenocarcinoma was significantly higher than the adjoining normal tissues (P = 0.000). To conclusion, our results support that TSA can induce apoptosis, and increase acetylated histone H4 in BGC-823 cells. GPNMB expression is decreased in BGC-823 cells after TSA treatment. GPNMB is overexpressed in gastric adenocarcinoma tissue. GPNMB involved in TSA-induced apoptosis might participate in gastric cancer.

11.
PLoS One ; 8(9): e74202, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098638

RESUMO

Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Telômero/genética , Telômero/fisiologia , Análise de Variância , Animais , Diferenciação Celular/fisiologia , Primers do DNA/genética , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica/fisiologia , Técnicas Histológicas , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Telomerase/metabolismo , Telômero/ultraestrutura , Homeostase do Telômero/fisiologia
12.
Cell Reprogram ; 15(1): 92-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23379582

RESUMO

Pluripotent stem cells can be created successfully through the inner cell mass (ICM), nuclear transfer, and defined-factor induction. Unfortunately, the epigenetic characteristics of the cells produced are poorly understood. In this article, we compared expression levels of enzymes involved in epigenetic modifications across six pluripotent stem cell lines. Six of the 11 genes evaluated here (Dnmt3a, Dnmt3b, Tet1, Ezh2, Mll1, and Lsd1) showed abnormally low levels of expression in the two germ-line chimeric induced pluripotent stem cell (iPSC) lines. We also conducted locus-specific analysis of DNA methylation at 9 loci. Although iPSCs did express Oct4, the Oct4 promoter region was shown to have a higher level of DNA methylation. The Xist and Line-1 repeating sequences differed relatively little in methylation level across the cell lines, but Peg3, Peg10, and H19 exhibited high degrees of variation in the pattern of DNA methylation. Meg3 in the Dlk1-Dio3 imprinting cluster was incompletely methylated in embryonic stem cells (ESCs) and nuclear transfer (nt) ESCs. However, in germ-line chimeric iPSCs, Meg3 was almost entirely methylated. ESC and ntESC lines showed twice as much Meg3 expression than in the iPSC lines. The genomic 5mC contents detected by reverse-phase high-performance liquid chromatography (HPLC) indicated that, despite their germ-line chimeric abilities, iPSCs remained incompletely reprogrammed, even though no direct evidence is shown here.


Assuntos
Desdiferenciação Celular , Metilação de DNA , DNA/metabolismo , Loci Gênicos , Impressão Genômica , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Linhagem Celular , DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Família Multigênica/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
13.
Sci China Life Sci ; 55(12): 1029-37, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23233217

RESUMO

Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences (ITSs). Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG (vITSs) are also localized at the centromeric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere sequences, TTTAGGG (bITSs), also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluorescence in situ hybridization (FISH) and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres (TTS). Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved telomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.


Assuntos
Suínos/genética , Telômero , Animais , Hibridização in Situ Fluorescente , Troca de Cromátide Irmã
14.
Sci China Life Sci ; 54(6): 553-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21706416

RESUMO

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells. However, this retrovirus system infects only mouse and rat cells, which limits its use in establishing iPS cells from other mammals. In our study, we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts. We transfected four human reprogramming factors (Oct4, Sox2, Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells. We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF. Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies. Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.


Assuntos
Desdiferenciação Celular/fisiologia , Diferenciação Celular/fisiologia , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Suínos , Animais , Bovinos , Linhagem Celular , Corpos Embrioides/citologia , Corpos Embrioides/fisiologia , Humanos , Cariotipagem , Camundongos , Camundongos SCID , Ratos , Retroviridae/fisiologia , Teratoma/metabolismo , Teratoma/patologia
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