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Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.
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Proteínas Adaptadoras del Transporte Vesicular/genética , Blastocisto/metabolismo , Linaje de la Célula/genética , Ectodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Biomarcadores/metabolismo , Blastocisto/citología , Ectodermo/citología , Ectodermo/crecimiento & desarrollo , Desarrollo Embrionario , Femenino , Fertilización In Vitro , Perfilación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Masculino , Oocitos/citología , Oocitos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Porcinos , Transcripción GenéticaRESUMEN
Lipid droplets (LD) provide a source of energy, and their importance during embryogenesis has been increasingly recognized. In particular, pig embryos have larger amounts of intercellular lipid bilayers than other mammalian species, suggesting that porcine embryos are more dependent on lipid metabolic pathways. The objective of the present study was to detect the effect of stearoyl-coenzyme A desaturase 1 (SCD1) on LD formation and to associate these effects with the mRNA abundance of LD formation-related genes (SREBP, ARF1, COPG2, PLD1 and ERK2) in in vitro-produced porcine embryos. To determine the effect of SCD1 on LD formation and related genes, we examined the effects of SCD1 inhibition using CAY10566 (an SCD1 inhibitor, 50 µM) on parthenogenetic embryos. SCD1 inhibition downregulated the mRNA levels of LD formation-related genes and embryo development. Our results revealed that SCD1 functions in the regulation of LD formation via phospholipid formation and embryo development. In addition, we treated parthenogenetic embryos with oleic acid (100 µM), which led to a significant increase in the blastocyst formation rate, LD size and number compared to controls. Remarkably, the adverse effects of the SCD1 inhibitor could be counteracted by oleic acid. These data suggest that porcine embryos can use exogenous oleic acid as a metabolic energy source.
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Embrión de Mamíferos/fisiología , Regulación del Desarrollo de la Expresión Génica , Gotas Lipídicas/fisiología , Lípidos/química , Lipogénesis/genética , Estearoil-CoA Desaturasa/metabolismo , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Femenino , Gotas Lipídicas/enzimología , Estearoil-CoA Desaturasa/genética , PorcinosRESUMEN
This study describes a three-dimensional visualization system with spatial information for the effective control of a tele-operated robot. The environmental visualization system for operating the robot is very important. The tele-operated robot performs tasks in a disaster area that is not accessible to humans. The visualization system should perform in real-time to cope with rapidly changing situations. The visualization system should also provide accurate and high-level information so that the tele-operator can make the right decisions. The proposed system consists of four fisheye cameras and a 360° laser scanner. When the robot moves to the unknown space, a spatial model is created using the spatial information data of the laser scanner, and a single-stitched image is created using four images from cameras and mapped in real-time. The visualized image contains the surrounding spatial information; hence, the tele-operator can not only grasp the surrounding space easily, but also knows the relative position of the robot in space. In addition, it provides various angles of view without moving the robot or sensor, thereby coping with various situations. The experimental results show that the proposed method has a more natural appearance than the conventional methods.
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Hermetic sealing is an important technology for isolating and protecting air-sensitive materials and is key in the development of foldable and stretchable electronic devices. Here we report an ultra gas-proof polymer hybrid thin layer prepared by filling the free volume of the polymer with Al2O3 using gas-phase atomic layer infiltration. The high-density polymer-inorganic hybrid shows extremely low gas transmission rate, below the detection limit of the Ca corrosion test (water vapor transmission rate <10-7 g m-2 day-1). Furthermore, because of the remarkable nanometer-scale thinness of the complete polymer-inorganic hybrid, it is highly flexible, which makes it useful for hermetic sealing of stretchable and foldable devices.
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Holographic data storage (HDS) has attracted much attention as a next-generation storage medium. Because HDS suffers from two-dimensional (2D) inter-symbol interference (ISI), the partial-response maximum-likelihood (PRML) method has been studied to reduce 2D ISI. However, the PRML method has various drawbacks. To solve the problems, we propose a modified decision feedback equalizer (DFE) for HDS. To prevent the error propagation problem, which is a typical problem in DFEs, we also propose a reliability factor for HDS. Various simulations were executed to analyze the performance of the proposed methods. The proposed methods showed fast processing speed after training, superior bit error rate performance, and consistency.
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To fight cancer at its roots by targeting cancer stem cells is a promising approach for therapy. Previously, an indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), was reported effectively inhibiting the growth of lung cancer cells through impairment of cellular mitochondria functions. To address more on drug efficiency, the study further exploited if EMMQ can impede the propagation of tumorspheres stemmed from non-small cell lung cancer cells. EMMQ inhibited proliferation of spheroids in culture. In animal models, administration of the drug attenuated the spheroid tumorigenicity. The activated apoptosis alleviated growth of xenograft tumors in immune-deficient mice as established by the enriched tumorspheres. More evidence suggested that the reduced stemness of the spheroid tumors is attributed to apoptotic death. The findings supported that EMMQ is an eligible approach to eradicate the minor but tumorigenic lung cancer tumorspheres.
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Apoptosis/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Indoles/farmacología , Indoles/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Quinolinas/farmacología , Quinolinas/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Trasplante de Neoplasias , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Ensayo de Tumor de Célula MadreRESUMEN
Non-intrusive electrocardiogram (ECG) monitoring has many advantages: easy to measure and apply in daily life. However, motion noise in the measured signal is the major problem of non-intrusive measurement. This paper proposes a method to reduce the noise and to detect the R peaks of ECG in a stable manner in a sitting arrangement using non-intrusive sensors. The method utilizes two capacitive ECG sensors (cECGs) to measure ECG, and another two cECGs located adjacent to the sensors for ECG are added to obtain the information on motion. Then, active noise cancellation technique and the motion information are used to reduce motion noise. To verify the proposed method, ECG was measured indoors and during driving, and the accuracy of the detected R peaks was compared. After applying the method, the sum of sensitivity and positive predictivity increased 8.39% on average and 26.26% maximally in the data. Based on the results, it was confirmed that the motion noise was reduced and that more reliable R peak positions could be obtained by the proposed method. The robustness of the new ECG measurement method will elicit benefits to various health care systems that require noninvasive heart rate or heart rate variability measurements.
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Técnicas Biosensibles/instrumentación , Electrocardiografía/instrumentación , Frecuencia Cardíaca/fisiología , Procesamiento de Señales Asistido por Computador/instrumentación , Capacidad Eléctrica , Humanos , Movimiento (Física) , Relación Señal-RuidoRESUMEN
Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 (20 µg/mL) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions.
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X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.
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Blastocisto/fisiología , Factores de Transcripción de Octámeros/fisiología , Partenogénesis/fisiología , Inactivación del Cromosoma X/fisiología , Cromosoma X/genética , Animales , Femenino , Técnicas de Transferencia de Gen , Genes Ligados a X , Humanos , Lentivirus , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , PorcinosRESUMEN
BACKGROUND: Class III ß-tubulin (TUBB3) is a prognostic marker in various tumors, but the role of TUBB3 in advanced gastric cancer is not clearly defined. We analyzed the significance of TUBB3 expression, along with that of excision repair cross-complementation group 1 (ERCC1) in recurrent and metastatic gastric cancer patients receiving taxane-based first-line palliative chemotherapy. METHODS: We reviewed the cases of 146 patients with advanced gastric adenocarcinoma who received taxane-based first-line palliative chemotherapy between 2004 and 2010 at Chonnam National University Hwasun Hospital (Gwangju, Korea). Immunohistochemical staining for TUBB3 and ERCC1 was performed using paraffin wax-embedded tumor tissues. We evaluated the patients' response to chemotherapy, progression-free survival (PFS), and overall survival (OS). RESULTS: In total, 146 patients with advanced gastric cancer received docetaxel and cisplatin (n = 15) or paclitaxel and cisplatin (n = 131). The median PFS was significantly shorter for patients with high-level TUBB3 expression than for patients with low-level TUBB3 expression (3.63 vs. 6.67 months, P = 0.001). OS was not associated with TUBB3 expression (13.1 vs. 13.1 months, P = 0.769). By multivariate analysis, only TUBB3 was related to a shorter PFS (HR 2.74, 95% CI 1.91-3.91, P = 0.001). Patients with high-level ERCC1 expression showed a lower response rate than patients with low-level ERCC1 expression (24 vs. 63.2%, P = 0.001); however, ERCC1 had no clinical effect on PFS or OS. CONCLUSIONS: TUBB3 was a strong predictive marker in recurrent and metastatic gastric cancer patients receiving taxane-based first-line palliative chemotherapy. No clinical impact of ERCC1 was evident in this setting.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Tubulina (Proteína)/metabolismo , Adulto , Anciano , Hidrocarburos Aromáticos con Puentes/administración & dosificación , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Metástasis de la Neoplasia , Pronóstico , Recurrencia , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Taxoides/administración & dosificación , Resultado del TratamientoRESUMEN
Fertilized embryos develop and move freely in the reproductive tract until implantation. Subsequently, the embryos continue to develop after attachment to the uterus. Because of the absence of the uterus, in vitro culturing of embryos is limited to a period of approximately a week. Hatched blastocysts were seeded on feeder cells to extend the culture period. We cultured the colonies formed from the blastocysts for an additional 14 days. From the colonies, four types of cells were established, and each type was isolated to extract RNA. RNA sequencing was conducted using NovaSeq6000. Sequencing reads were aligned to genes and transcripts. Raw data from our previous study were used to compare these samples with the cultured cell lines. We analyzed differentially expressed genes and Gene Ontology terms between new samples and cultured cell lines. Our data can provide essential information for extending the period of embryo culture in vitro.
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OBJECTIVE: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. METHODS: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. RESULTS: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. CONCLUSION: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.
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OBJECTIVE: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. METHODS: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. RESULTS: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. CONCLUSION: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.
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Fatty acid has a various role in preimplantation embryo development. Especially, Linoleic acid, polyunsaturated fatty acid, has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B. But to date, the function of NF-κB has not been demonstrated in porcine preimplantation embryos. We demonstrated that linoleic acid had a positive effect on embryo development at a certain concentration(25 µM), but developmental failure was observed at higher concentration. Furthermore, the expression level of NF-κB increased, unlike that of IL-6, as the concentration of linoleic acid increased. Interestingly, the concentration of NF-κB was found to increase even at the concentration of linoleic acid at which embryo development decreased. We found that pro-apoptotic gene expression was downregulated in the linoleic acid-treated group. It was also found that MCL-1, an anti-apoptotic gene known to be unaffected by IL-6, was found to be increased at the mRNA level in the linoleic acid-treated group. As the concentration of NF-kB increased, the nuclear translocation of C-JUN gradually increased dependent on the linoleic acid concentration. It was confirmed that NF-κB is an important factor in porcine embryos by treated ammonium pyrrolidinedithiocarbamate (APDC 0.1 µM, an inhibitor of NF-κB) affected NF-κB protein expression, IL-6 expression, and blastocyst production. These data supported porcine embryos can use exogenous linoleic acid as a metabolic energy source via NF-κB.
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Ácido Linoleico , FN-kappa B , Animales , Apoptosis , Femenino , Interleucina-6 , Ácido Linoleico/farmacología , FN-kappa B/metabolismo , Embarazo , ARN Mensajero/metabolismo , PorcinosRESUMEN
OBJECTIVES: Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a species-specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions. MATERIALS AND METHODS: To determine the functions of OCT4 in lineage specification and embryo proliferation, loss- and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes. RESULTS: In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased. CONCLUSION: Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation.
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Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros , Embarazo , Femenino , Porcinos , Animales , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Blastocisto/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.
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Many types of embryonic stem cells have been induced from pre-implantation blastocysts to study the specification of early lineages. Various cell lines have been established using chemicals, including excessive inhibitory molecules. Previous studies have also aimed to purify cell populations representing a single embryonic lineage from a protocol. In this study, we used a novel culture condition to induce cells from blastocyst seeding and analyzed their characteristics. Next, signaling inhibitors were introduced during the cell culture period. Furthermore, we investigated the cell types using RNA sequencing. Each type of cell population showed a distinct morphology and reactivity with alkaline phosphatase. Marker proteins enabled each cell type to be distinguished by immunocytochemistry, and genes such as Sox17, Gata4, Gata6, T, and Cdx2 showed applicability for the discrimination of cell types. Signaling inhibitors suppressed the production of some cell types, and gene expression and marker protein patterns were collapsed. RNA-sequencing suggested cell-type-specific marker genes and the correlation among samples. In conclusion, four types of cells could be induced from porcine embryos using a single protocol, and they could be isolated manually. Our data will help promote the study of lineage segregation based on embryonic cells.
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Skin aging is a major risk factor for the dermal diseases, and interventions to attenuate cellular senescence are expected to reduce the risk for age-related diseases involving skin atrophy. However, blocking cell death or extending proliferation causally results in side effects and an increased cancer risk. For identification of a safer approach, we focused on PDK1 inhibition, which could revert cellular senescence and reduce senescence factors in skin in vitro, in a human skin equivalent model and in an exploratory, placebo-controlled, interventional trial. Natural phytochemical kaempferol tetrasaccharides resulted in a significant reduction in cellular senescence, and an increase in collagen fiber was observed in the skin cell and human skin equivalent. Clinical enhancement in skin appearance was noted in multiple participants, and an immunohistochemical study revealed improvement in the histological appearance of skin tissue and extracellular matrix. This change was associated with relative improvement in histological markers of senescence and clinical appearance of the aged skin and an increase in collagen fiber, an essential factor for preventing skin atrophy and consistency of the basement membrane. These results indicate that PDK1 inhibition is a potentially effective antiaging intervention, suggesting a diagnostic role and preventive actions of PDK1 in senescence-associated skin atrophy.
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Fibroblastos , Quempferoles , Humanos , Anciano , Quempferoles/farmacología , Quempferoles/uso terapéutico , Piel , Senescencia Celular , Colágeno/metabolismo , Atrofia/tratamiento farmacológico , Atrofia/metabolismoRESUMEN
This study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in fibroblast growth factor (FGF)- or leukemia inhibitory factor (LIF)-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. Porcine embryonic fibroblasts were coinfected with the pOCT4-ΔPE-eGFP (distal enhancer [DE]-green fluorescent protein [GFP]) and pOCT4-ΔDE-DsRed2 (proximal enhancer [PE]-red fluorescent protein [RFP]) vectors, and GFP and RFP expression were verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 DE and PE were activated in different expression patterns simultaneously as changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems confirmed that porcine iPSCs are in primed state after reprogramming in FGF2- or LIF-containing media. This work demonstrates the applicability of porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.
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Diferenciación Celular , Reprogramación Celular , Células Madre Embrionarias/citología , Fibroblastos/citología , Proteínas Fluorescentes Verdes/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Animales , Células Cultivadas , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Células Madre Pluripotentes/metabolismo , PorcinosRESUMEN
Aim: Therapy to overcome drug resistance by modulating epidermal growth factor receptor (EGFR) is a viable approach to suppress the proliferation of human non-small cell lung cancer (NSCLC) cells. A previous study demonstrated that the seeds of an aqueous Brucea javanica (BJ) (L.) Merr (Simaroubaceae) extract containing quassinoid mixtures effectively inhibited the growth and alleviated tumorigenesis in H1975 cells of NSCLC by targeting T790M/L858R EGFR. This study aimed to further determine whether the aqueous BJ extract affects the enriched H1975 spheroids in suspension culture and mouse xenograft tumor models. Methods: The spheroids of NSCLC adenocarcinoma H1975 cells were enriched in a serum-free media. The growth rate of sphere propagation by aqueous BJ extract was determined in suspended culture and in colony-formation assay. BJ extract was fed orally to nude mice bearing xenograft tumors. The resected tumors were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and proliferating cell nuclear antigen assessment. Various markers were used to determine the pluripotency of tumors from mice treated with different concentrations of BJ extract. Results: BJ extract was demonstrated to be effective against the propagation of the enriched spheroids. In animal models, oral administration of the aqueous BJ extract reduced spheroid tumorigenicity. The alleviated growth of the established xenograft tumors can be attributed to the reduced drug resistance and induced apoptosis without distinct adverse effects. More evidence supports activated apoptotic death attenuated spheroid stemness of tumors. Conclusion: As an effective treatment regime to assuage lung cancer, the indigenous BJ extract promises to obliterate drug resistance and the growth of cancer stem cell tumors from NSCLC cells harboring T790M/L858R EGFR.