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In the fumarylacetoacetate hydrolase deficient (Fah-/-) mouse, massive liver repopulation can be easily obtained after transplanted hepatocytes. Understanding the mechanisms of complete liver repopulation in Fah-/- mice will be useful for future clinical application. Here, we found that the endogenous hepatocytes in liver of Fah-/- mice undertook senescence during the time of tyrosinemia symptoms. Increase of senescent hepatocytes in Fah-/- mice provided proliferative advantage to the transplanted hepatocytes. Importantly, senescent hepatocytes upregulated the expression of extracellular matrix enzyme, contributing to degradation of extracellular matrix components and weakness of cell adhesion and connection. The liver exhibiting a loose architecture provided the space for the engraftment and expansion of transplanted hepatocytes. These findings underscore the underlying mechanisms of completed liver repopulation in Fah-/- mice. Senescence followed by loose hepatic parenchyma is a preconditioning for liver repopulation, which would be a promising strategy to achieve therapeutic liver repopulation in clinical settings.
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Senescencia Celular , Hepatocitos/citología , Hígado/citología , Animales , Puntos de Control del Ciclo Celular , Proliferación Celular , Ciclohexanonas , Hepatocitos/trasplante , Hidrolasas/deficiencia , Hidrolasas/metabolismo , Ratones , NitrobenzoatosRESUMEN
Exercise plays an important role in the prevention and treatment of chronic liver disease and associated metabolic disorders. A single bout of exercise induces tissue blood flow redistribution, which decreases splanchnic circulation and leads to physiologic hypoxia in the gastrointestinal system and liver. The transcription factor, hypoxia inducible factor-1α (HIF-1α), and its regulator, prolylhydroxylase 2 (PHD2), play pivotal roles in the response to oxygen flux by regulating downstream gene expression levels in the liver. We hypothesized that exercise increases the HIF-1α levels in the liver, and that the hepatic PHD2/HIF-1α axis is involved in postexercise restoration of systemic energy homeostasis. Through constant O2 consumption, CO2 production, food and water intake, and physical activity detection with metabolic chambers, we observed that one 30-min session of swimming exercise enhances systemic energy metabolism in mice. By using the noninvasive bioluminescence imaging ROSA26 oxygen-dependent domain Luc mouse model, we reveal that exercise increases in vivo HIFα levels in the liver. Intraperitoneal injections of the PHD inhibitor, dimethyloxalylglycine, mimicked exercise-induced HIFα increase, whereas the HIF-1α inhibitor, PX-478, blocked this effect. We next constructed liver-specific knockout (LKO) mouse models with albumin- Cre-mediated, hepatocyte-specific Hif1a and Phd2 deletion. Compared with their controls, Hif1a-LKO and Phd2-LKO mice exhibited distinct patterns of hepatic metabolism-related gene expression profiles. Moreover, Hif1a-LKO mice failed to restore systemic energy homeostasis after exercise. In conclusion, the current study demonstrates that a single bout of exercise disrupts systemic energy homeostasis, increasing the HIF-1α levels in the liver. These findings also provide evidence that the hepatic PHD2/HIF-1α axis is involved in postexercise systemic metabolic homeostasis.-Luo, B., Xiang, D., Wu, D., Liu, C., Fang, Y., Chen, P., Hu, Y.-P. Hepatic PHD2/HIF-1α axis is involved in postexercise systemic energy homeostasis.
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Homeostasis/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Hígado/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Animales , Línea Celular Tumoral , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Ratones Transgénicos , Oxígeno/metabolismo , Prolil Hidroxilasas/genética , ARN Mensajero/genéticaRESUMEN
Hepatocellular carcinoma (HCC) is among the most common lethal cancers of the digestive system with poor prognosis rates and ineffective therapeutic options. Matrine, a traditional Chinese medicine found in the roots of sophora species, has been used in the clinical treatment of liver fibrosis, chronic hepatitis B and other diseases. We have synthesized a matrine derivatives named WM622 (C26H35ON3S2) with a significant inhibitory effect on transplanted tumors in vivo. The half inhibitory concentration (IC50) of WM622 is 34 µM, which is much lower than matrine. WM622 inhibited the proliferation and promoted apoptosis of hepatocellular carcinoma cells significantly, and the cell cycle was blocked in G0/G1 phase. The protein phosphorylation levels of EGFR, AKT, PI3K and GSK3ß (p-EGFR, p-AKT, p-PI3K, and p-GSK3ß) were also decreased by WM622 treatment dose dependently. In tumor-bearing mice, WM622 could reduce the tumor volumes. In conclusion, the study demonstrated that WM622 could inhibit the proliferation of the hepatocellular carcinoma both in vivo and in vitro by inducing apoptosis, blocking cell cycle in G0/G1 phase and inhibiting the PI3K/AKT signal pathways.
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Alcaloides , Carcinoma Hepatocelular/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolizinas , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Alcaloides/química , Alcaloides/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Quinolizinas/química , Quinolizinas/farmacología , Fase de Descanso del Ciclo Celular/genética , Transducción de Señal/genética , MatrinasRESUMEN
UNLABELLED: A better understanding of hepatocyte senescence could be used to treat age-dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53-p21 and p16(ink4a)-pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. CONCLUSION: These findings suggest that the hepatocyte "ploidy conveyer" is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy.
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Proliferación Celular , Senescencia Celular/fisiología , Hepatocitos/citología , Hepatocitos/trasplante , Regeneración Hepática/fisiología , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citometría de Flujo , Hepatocitos/fisiología , Hidrolasas/genética , Operón Lac , Hígado/citología , Hígado/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Poliploidía , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: To observe the sensitivity of stroke volume variation (SVV) for assessing volume change during induction period of general anesthesia. METHODS: Patients who underwent orthopaedic surgery under general anesthesia and mechanical ventilation were divided into two groups randomly. Patients in the group â were subjected to progressive central hypovolemia and correction of hypovolemia sequentially; patients in the Group â ¡ were exposed to hypervolemia alone. Each step was implemented after 5 minutes when the hemodynamics was stable. SVV and cardiac index (CI) were recorded, and Pearson's product-moment correlation was used to analyze correlation between SVV and CI. RESULTS: Forty patients were included in this study, 20 cases in each group. For group â patients, SVV was increased significantly along with blood volume reduction, and changes in CI were negatively correlated with changes in SVV (r=-0.605, P<0.01); SVV decreased significantly along with correction of blood volume; changes in CI were negatively correlated with changes in SVV (r=-0.651, P<0.01). For group â ¡ patients, along with blood volume increase, SVV did not change significantly; changes in CI revealed no significant correlation with changes in SVV (r=0.067, P>0.05). CONCLUSION: SVV is a useful indicator for hypovolemia, but not for hypervolemia.
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Presión Arterial , Gasto Cardíaco , Volumen Sistólico , Adulto , Anciano , Volumen Sanguíneo , Presión Venosa Central , Femenino , Humanos , Masculino , Persona de Mediana Edad , Periodo PerioperatorioRESUMEN
Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.
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OBJECTIVE: To observe the effects of short-term exposure to opioid analgesics on human sperm motility. METHODS: Twenty normal semen samples were collected, each divided into 19 groups, one as the control and the others treated in vitro with six opioid analgesics at three different concentrations, respectively, and sperm motility was assessed by computer-assisted sperm analysis at 15 min, 2 h and 4 h. RESULTS: Compared with the control group, fentanyl, alfentanil and sufentanil at 1 x 10(-5), 2 x 10(-3) and 0.05 mg/ml significantly decreased the percentage of grade a + b sperm at 15 min, 2 h and 4 h (P<0.05), and so did butorphanol at 2 x 10(-3) and 0.05 mg/ml (P<0.05) and dezocine at 0.05 and 0.5 mg/ml (P<0.05), but neither showed any remarkable effect at 1 x 10(-5) mg/ml at the three time points (P>0.05). Pentazocine effected no significant difference at 3 x 10(-5) and 0. 05 mg/ml (P>0.05) but a gradual increase in the percentage of grade a + b sperm at 0.5 mg/ml at the three time points (P<0.05). Butorphanol totally inhibited sperm motility at 0.05 mg/ml at 15 min and at 2 x 10(-3) mg/ml at 2 h, and so did dezocine at 0.05 and 0.5 mg/ml, but such inhibitory effect was not observed with fentanil, alfentanil and sulfentanil at 0.05 mg/ml. As for the sperm motility decreasing effect at 0.05 mg/ml at 15 min, sufentanil, butorphanol and dezocine exhibited significant differences (P<0.05) while fentanyl displayed none from alfentanil (P>0.05). CONCLUSION: Given the same length of time of treatment, butorphanol and dezocine totally inhibit sperm motility at a high concentration, but make no significant change at a low concentration. While fentanyl, alfentanil and sufentanil can significantly decrease sperm motility at the same low concentration, and partially inhibit it at all concentrations. On the contrary, a high concentration of pentazocine can promote human sperm motility.
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Analgésicos Opioides/farmacología , Motilidad Espermática/efectos de los fármacos , Humanos , Técnicas In Vitro , MasculinoRESUMEN
Stable epigenetic silencing of p16(INK4a) is a common event in hepatocellular carcinoma (HCC) cells, which is associated with abnormal cell proliferation and liberation from cell cycle arrest. Understanding the early epigenetic events in silencing p16(INK4a) expression may illuminate a prognostic strategy to block HCC development. Toward this end, we created a reprogram cell model by the fusion mouse HCC cells with mouse embryonic stem cells, in which the ES-Hepa hybrids forfeited HCC cell characteristics along with reactivation of the silenced p16(INK4a). HCC characteristics, in terms of gene expression pattern and tumorigenic potential, was restored upon induced differentiation of these reprogrammed ES-Hepa hybrids. The histone methylation pattern relative to p16(INK4a) silencing during differentiation of the ES-Hepa hybrids was analyzed. H3K27 trimethylation at the p16(INK4a) promoter region, occurring in the early onset of p16(INK4a) silencing, was followed by H3K9 dimethylation at later stages. During the induced differentiation of the ES-Hepa hybrids, H3K4 di- and trimethylations were maintained at high levels during the silencing of p16(INK4a), strongly suggesting that H3K4 methylation events did not cause the silencing of p16(INK4a). Our results suggested that the enrichment of H3K27 trimethylation, independent of H3K9 dimethylation, trimethylation, and DNA methylation, was an early event in the silencing of p16(INK4a) during the tumor development. This unique chromatin pattern may be a heritable marker of epigenetic regulation for p16(INK4a) silencing during the developmental process of hepatocellular carcinogenesis.
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Carcinoma Hepatocelular/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Epigénesis Genética , Silenciador del Gen , Histonas/química , Neoplasias Hepáticas/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Cromatina/metabolismo , Células Madre Embrionarias/citología , Citometría de Flujo , Histonas/metabolismo , RatonesRESUMEN
OBJECTIVE: To observe the effects of remifentanil combined with naloxone on human sperm motility in vitro and to investigate its possible mechanism. METHODS: Twenty normal semen samples were collected, each divided into 13 aliquots, one as the control and the others treated in vitro with different concentrations of remifentanil or remifentanil + naloxone for 35 min. The percentage of progressive mobile sperm was assessed by computer-assisted sperm analysis at 5, 10, 15, 20 and 35 min. RESULTS: Compared with the control group, remifentanil at 0.1, 1, 10 and 100 microg/L significantly decreased sperm motility at 5 and 10 min in a dose-dependent manner, with no significant difference at 15 and 30 min; sperm motility showed no significant difference on 5 -35 min exposure to naloxone at 0.004 -0.04 mg/L, nor on 5, 10, 15 and 20 min exposure at 0.4 -4 mg/L, but was significantly increased at 35 min. Compared with the corresponding concentrations of remifentanil alone, remifentanil + naloxone remarkably increased sperm motility at 0.1 + 0.004, 1 + 0.04, 10 + 0.4, and 100 + 4 mg/L at 5 and 10 min, with no obvious difference at 15 and 30 min. CONCLUSION: The onset and offset of the effect of remifentanil on sperm motility are rapid and its inhibitory effect can be antagonized by naloxone, which may be related with the micro-opioid receptor.
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Naloxona/farmacología , Piperidinas/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adulto , Humanos , Masculino , Naloxona/administración & dosificación , Piperidinas/administración & dosificación , Remifentanilo , Adulto JovenRESUMEN
Previous study showed that senescent hepatocytes from aged liver could be rejuvenated after repopulated in the young recipient liver. The proliferative capacity of hepatocytes was restored with the senescence reversal. However, it is unknown whether metabolic and homeostatic function of aged liver, as well as age-dependent liver steatosis could be rejuvenated or alleviated. Here, we found that senescent hepatocytes from aged liver were rejuvenated after exposing to young blood. An autonomous proliferation of senescent hepatocytes which resulting in ploidy reversal might be the underlying mechanism of senescent reversal. After performing 2/3 partial hepatectomy (2/3PHx) in young blood exposed old liver, delayed DNA synthesis of senescent hepatocytes was rescued and the number of BrdU positive hepatocytes was restored from 4.39±2.30% to 17.85±3.21%, similarly to that in the young mice at 36 hours post 2/3PHx. Moreover, Cyclin A2 and Cyclin E1 overexpression of hepatocytes in aged liver facilitating the G1/S phase transition was contributed to enhance liver regeneration. Furthermore, lipid droplet spread widely in the elderly human liver and old mouse liver, but this aged-associated liver steatosis was alleviated as senescence reversal. Collectively, our study provides new thoughts for effectively preventing age-related liver diseases.
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PURPOSE: Cancer stem cells (CSCs) have been considered involving in tumorigenesis, local recurrence, and therapeutic drug resistance of hepatocellular carcinoma (HCC). To investigate novel and effective methods for targeting hepatic CSCs is crucial for a permanent cure of liver cancer. METHODS: The expression level of SIRT1 was detected in CSCs of HCC tissues and cancer cell lines. Expression of CSC markers, the self-renewal and tumorigenic ability of liver CSCs were analyzed with SIRT1 inhibition. Cellular senescence-related markers were used to detect CSCs senescence after inhibition of SIRT1. RESULTS: SIRT1 was highly expressed in CSCs of HCC cell lines and human HCC tissues. In vitro study revealed that decreasing of SIRT1 level significantly downregulated the stemness-associated genes of liver CSCs and reduced the CSC stemness properties. Also, downregulated SIRT1 suppressed liver CSCs proliferation by decreasing their self-renewal abilities. Furthermore, CSCs with decreased SIRT1 expression showed limited tumorigenicity and formed smaller HCC tumor in vivo. And SIRT1 decreased CSCs became more susceptible to chemotherapeutic drugs. Mechanistically, SIRT1 decreased CSCs became senescence through the activation of p53-p21 and p16 pathway. The data further indicated that the tumor formed from SIRT1-knockdown CSCs exhibited higher senescence-associated ß-galactosidase (SA-ß-Gal) activity but lower proliferative capacity. CONCLUSION: Taken together, these findings pointed that induction of senescence in liver CSCs is an effective tumor suppression method for HCC, and SIRT1 may be served as a promising target for HCC treatment.
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OBJECTIVE: To investigate the effects of the total saponin of Psidium guajava leaf (TSGL) on HIV-1 envelop proteins (env) mediated virus entry into target cells. METHODS: The TSGL was purified and concentrated using SA-1 macropore resin. The effect of TSGL on HIV-1 entry into target cells was tested using a cell-cell fusion assay by mixing CHO-WT and MT-2 cells. The cytotoxicity of TSGL was measured by MTT assay. The activity of TSGL on blocking the HIV-1 gp41 six helical bundle (6-HB) formation was analyzed by ELISA and Native-PAGE (N-PAGE). RESULTS: The TSGL could inhibit HIV env mediated cell-cell fusion with an IC50 of (7.33 +/- 0.40) microg/mL, and displayed little cytotoxicity at that concentration. ELISA assay showed that the TSGL could prevent gp41 6-HB formation with inhibitory activity of 95.93% at 25 microg/mL. N-PAGE study confirmed the inhibitory effect of TSGL on gp41 6-HB formation. CONCLUSIONS: The TSGL can inhibit HIV entry target cells by interfering the envelop subunit gp41 form the critical 6-HB structure.
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Proteína gp41 de Envoltorio del VIH/efectos de los fármacos , Inhibidores de Fusión de VIH/farmacología , VIH-1/efectos de los fármacos , Psidium/química , Saponinas/farmacología , Fusión Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/administración & dosificación , VIH-1/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Hojas de la Planta/química , Saponinas/administración & dosificación , Internalización del Virus/efectos de los fármacosRESUMEN
Background: Exercise induces blood flow redistribution among tissues, leading to splanchnic hypoperfusion. Intestinal epithelial cells are positioned between the anaerobic lumen and the highly metabolic lamina propria with an oxygen gradient. Hypoxia-inducible factor (HIF)-1α is pivotal in the transcriptional response to the oxygen flux. Methods: In this study, the pimonidazole hydrochloride staining was applied to observe the tissue hypoxia in different organs, which might be affected by the blood flow redistribution. The HIF-1α luciferase reporter ROSA26 oxygen-dependent degradation domain (ODD)-Luc/+ mouse model (ODD domain-Luc; female, nâ¯=â¯3-6/group) was used to detect the HIF-1α expression in the intestine. We used 3 swimming models: moderate exercise for 30 min, heavy-intensity exercise bearing 5% bodyweight for 1.5 h, and long-time exercise for 3 h. Results: We found that 1 session of swimming at different intensities could induce tissue hypoxia redistribution in the small intestine, colon, liver and kidney, but not in the spleen, heart, and skeletal muscle. Our data showed that exercise exacerbated the extent of physiological hypoxia in the small intestine. Next, using ODD-Luc mice, we found that moderate exercise increased the in vivo HIF-1α level in the small intestine. The post-exercise HIF-1α level was gradually decreased in a time-dependent manner. Interestingly, the redistribution of tissue hypoxia and the increase of HIF-1α expression were not related to the exercise intensity and duration. Conclusion: This study provided evidence that the small intestine is the primary target organ for exercise-induced tissue hypoxia and HIF-1α redistribution, suggesting that HIF-1α may be a potential target for the regulation of gastrointestinal functions after exercise.
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Ejercicio Físico/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Intestino Delgado/irrigación sanguínea , Intestino Delgado/metabolismo , Animales , Femenino , Humanos , Ratones , Modelos Animales , Acondicionamiento Físico Humano/fisiología , Flujo Sanguíneo RegionalRESUMEN
BACKGROUND: Angiogenesis plays an important role in the development of rheumatoid arthritis (RA), which increases the supply of nutrients, cytokines, and inflammatory cells to the synovial membrane. Genistein (GEN), a soy-derived isoflavone, has been validated that can effectively inhibit the angiogenesis of several tumours. We thus carried out a study in vitro to investigate the effect of GEN in vascular endothelial growth factor (VEGF) expression and angiogenesis induced by the inflammatory environment of RA. METHODS: MH7A cells were used to verify whether GEN can inhibit the expression of VEGF in MH7A cells under inflammatory conditions and demonstrate the mechanism. EA.hy926 âcells were used to verify whether GEN can inhibit the migration and tube formation of vascular endothelial cells in inflammatory environment. RESULTS: GEN dose-dependently inhibited the expression and secretion of interleukin (IL)-6 and VEGF, as well as the nucleus translocation of Signal transducer and activator of transcription 3 (STAT3) in MH7A. Furthermore, GEN inhibited IL-6-induced vascular endothelial cell migration and tube formation in vitro. CONCLUSION: GEN inhibits IL-6-induced VEGF expression and angiogenesis partially through the Janus kinase 2 (JAK2)/STAT3 pathway in RA, which has provided a novel insight into the antiangiogenic activity of GEN in RA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our study provides scientific guidance for the clinical translational research of GEN in the RA treatment.
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There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte-like phenotypes. When transplanted intrasplentically into carbon tetrachloride-injured livers of SCID mice, EGFP-tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three-dimensional architecture, and differentiate into hepatocyte-like cells expressing human albumin and alpha-1-anti-trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury.
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Células de la Médula Ósea/citología , Diferenciación Celular , Hepatocitos/citología , Hígado/patología , Células Madre Mesenquimatosas/citología , Animales , Biomarcadores/metabolismo , Tetracloruro de Carbono , Fusión Celular , Linaje de la Célula , Proliferación Celular , Separación Celular , Células Cultivadas , Células Clonales , ADN/análisis , Hepatocitos/metabolismo , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones SCID , PloidiasRESUMEN
BMI-1 (B-cell-specific Moloney murine leukemia virus integration site 1), a novel oncogene, has attracted much attention in recent years for its involvement in the initiation of a variety of tumors. Recent evidence showed that BMI-1 was highly expressed in neoplastic skin lesions. However, whether dysregulated BMI-1 expression is causal for the transformation of skin cells remains unknown. In this study, we stably expressed BMI-1 in a human keratinocyte cell line, HaCaT. The expression of wild-type BMI-1 induced the malignant transformation of HaCaT cells in vitro. More importantly, we found that expression of BMI-1 promoted formation of squamous cell carcinomas in vivo. Furthermore, we showed that BMI-1 expression led to the downregulation of tumor suppressors, such as p16INK4a and p14ARF, cell adhesion molecules, such as E-Cadherin, and differentiation related factor, such as KRT6. Therefore, our findings demonstrated that dysregulated BMI-1 could indeed lead to keratinocytes transformation and tumorigenesis, potentially through promoting cell cycle progression and increasing cell mobility.
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Transformación Celular Neoplásica/metabolismo , Queratinocitos/patología , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Ciclo Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones SCID , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
OBJECTIVE: To develop a novel gene delivery vector TAT-PEI-beta-CyD. METHODS: beta-cyclodextrin (beta-CyD) was linked by low molecular weight (PEI 600) via 1, 1-carbonyldiimidazole (CDI), and TAT peptide (RRRQRRKKRC) was coupled to PEI 600 by [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. The copolymer was characterized by (1)H-NMR and FT-IR. Physiochemical characteristics of TAT-PEI-beta-CyD/DNA complexes were tested by agarose gel electrophoresis and particle size measurements. Cell viability and transfection efficiency were evaluated in A293 and B16 cells using PEI 25 kDa as a control. RESULT: TAT peptide was successfully coupled to PEI-beta-CyD. The result of gel electrophoresis showed that the TAT-PEI-beta-CyD was able to condense DNA efficiently at N/P ratio of 4. The particle size of TAT-PEI-beta-CyD/DNA complexes was around 100 nm. The cytotoxicity of TAT-PEI-beta-CyD was lower than that of PEI 25 kDa. The transfection efficiency of TAT-PEI-beta-CyD was higher than that of PEI 25 kDa in A293 and B16 cells at N/P ratio of 30. CONCLUSION: The novel vector TAT-PEI-beta-CyD has been developed successfully with low cytotoxicity and high transfection efficiency.
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Técnicas de Transferencia de Gen , Fragmentos de Péptidos/química , Polietileneimina/química , beta-Ciclodextrinas/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Línea Celular , Terapia Genética/métodos , HumanosRESUMEN
OBJECTIVE: To develop a novel non-viral gene delivery vector based on polyethylenimine and beta-cyclodextrin targeting to Her-2 receptor (MC10-PEI-beta-CyD). METHODS: The PEI-beta-CyD was synthesized by low molecular weight polyethylenimine (PEI, Mw 600) cross-linked beta-cyclodextrin (beta-CyD) via N, N-carbonyldiimidazole (CDI). The chemical linker[N-succinimidy-3-(2-pyridyldithio) propionate, SPDP] was used to bind peptide MC10 (MARAKEGGGC) to PEI-beta-CyD to form the vector MC10-PEI-beta-CyD. The (1)H-NMR was used to confirm the structure of vector. The DNA condensing ability,and the particle size of MC10-PEI-beta-CyD/DNA complexes were demonstrated by gel retardation assay and electron microscope observation (TEM). Cell viability was tested by MTT assay. The transfection efficiency was determined on cultured SKOV-3, A549 and MCF-7 cells. RESULT: MC10 was linked onto PEI-beta-CyD successfully. The vector was able to condense DNA at N/P ratio of 5 and particle size was about (170 +/-35)nm. The vector showed low cytotoxicity and high transfection efficiency in cultured SKOV-3, A549 and MCF-7 cells. CONCLUSION: A novel non-viral vector MC10-PEI-beta-CyD with low cytotoxicity and high transfection efficiency has been successfully synthesized.
Asunto(s)
Técnicas de Transferencia de Gen , Péptidos/química , Polietileneimina/química , Receptor ErbB-2/genética , beta-Ciclodextrinas/química , Línea Celular , Marcación de Gen , Vectores Genéticos , Humanos , Polietileneimina/farmacologíaRESUMEN
OBJECTIVE: To construct a drug carrier and gene vector PEG-PEI-Pt. METHODS: Polyethyleneglycol (PEG) was coupled to polyethylenimine (PEI 600) and platinum tetrachloride; PEG-PEI-Pt complex was formed in ethanol. The complex was characterized by XRD, UV-VIS and FT-IR and the DNA condensation was tested by electrophoretic mobility shift assay. The cell viability was evaluated by MTT assay in Hela, B16, A293 and COS-7 cells and in vitro transfection efficiency was measured in A293 and B16 cells. RESULT: The structure of PEG-PEI-Pt was characterized by XRD, UV-VIS and FT-IR. PEG-PEI-Pt complex was able to bind DNA at N/P weight ratio of 0.4:1; the complex showed cytotoxicity on Hela and B16 cells. The complex had higher transfection efficiency in A293 and B16 cells than PEI 600. CONCLUSION: A novel drug carrier and gene vector PEG-PEI-Pt was constructed successfully.
Asunto(s)
Portadores de Fármacos/síntesis química , Técnicas de Transferencia de Gen , Compuestos de Platino/química , Polietilenglicoles/química , Polietileneimina/química , Línea Celular , Terapia Genética/métodos , Humanos , TransfecciónRESUMEN
OBJECTIVE: To develop a novel non-viral gene delivery vector CY11-PEI-beta-CyD and to test its gene transfection efficiency. METHODS: CY11 (CGMQLPLATWY) was conjugated to polyethylenimine-beta-cyclodextrin to form CY11-PEI-beta-CyD with a cross-linker [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. (1)H-NMR and TGA were used to confirm the structure of vector. The DNA condensing ability of CY11-PEI-beta-CyD was investigated by gel retardation assay. Cytotoxicity of CY11-PEI-beta-CyD was determined by MTT assay and transfection efficiency was investigated in COS-7, Hela and B16 cells. RESULT: CY11 was conjugated onto PEI-beta-CyD successfully, confirmed by(1)H NMR and TGA. The novel vector effectively condensed DNA at N/P ratio of 4îIt showed low cytotoxicity up to the concentration was 160 Mgr;g/ml. The transfection efficiency was 17-fold higher than that of PEI 25 kDa at N/P ratio of 20. CONCLUSION: The novel vector CY11 -PEI-beta-CyD with low cytotoxic and high transfection efficiency may be used as a potential carrier for gene delivery.