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1.
Cell Physiol Biochem ; 54(1): 27-39, 2020 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-31935048

RESUMEN

BACKGROUND/AIMS: To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. METHODS: Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. RESULTS: Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CONCLUSION: CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Microdominios de Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Canales de Potasio Shal/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/análisis , Caveolas/metabolismo , Células Cultivadas , Humanos , Transporte Iónico , Potasio/metabolismo , Mapas de Interacción de Proteínas , Ratas Sprague-Dawley , Canales de Potasio Shal/análisis
2.
Cell Physiol Biochem ; 54(1): 15-26, 2020 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-31916734

RESUMEN

BACKGROUND/AIMS: The primary cilium is a nanoscale membrane protrusion believed to act as a mechano-chemical sensor in a range of different cell types. Disruptions in its structure and signalling have been linked to a number of medical conditions, referred to as ciliopathies, but remain poorly understood due to lack of techniques capable of investigating signal transduction in cilia at nanoscale. Here we set out to use latest advances in nanopipette technology to address the question of ion channel distribution along the structure of primary cilium. METHODS: We used glass nanopipettes and Scanning Ion Conductance Microscopy (SICM) to image 3D topography of intact primary cilia in inner medullary collecting duct (IMCD) cells with nanoscale resolution. The high-resolution topographical images were then used to navigate the nanopipette along the structure of each cilium and perform spatially resolved single-channel recordings under precisely controlled mechanical and chemical stimulation. RESULTS: We have successfully obtained first single-channel recordings at specific locations of intact primary cilia. Our experiments revealed significant differences between the populations of channels present at the ciliary base, tip and within extra-ciliary regions in terms of mean conductance and sensitivity to membrane displacement as small as 100 nm. Ion channels at the base of cilium, where mechanical strain is expected to be the highest, appeared particularly sensitive to the mechanical displacement. CONCLUSION: Our results suggest the distribution of ion channels in the membrane of primary cilia is non-homogeneous. The relationship between the location and function of ciliary ion channels could be key to understanding signal transduction in primary cilia.


Asunto(s)
Membrana Celular/metabolismo , Cilios/metabolismo , Canales Iónicos/metabolismo , Nanotecnología/métodos , Potenciales de Acción/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Mecanotransducción Celular , Ratones
3.
Toxicol Lett ; 322: 87-97, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-31935479

RESUMEN

1,2-Dichloroethane (1,2-DCE) is a widely used chlorinated organic toxicant, but little is known about the cerebellar dysfunction induced by excessive exposure to it. To uncover 1,2-DCE-induced neurotoxicity in cerebellar granular cells (CGCs), and to investigate the underlying mechanisms, we explored this, both in vitro and in vivo. Our findings showed significant cell viability inhibition in human CGCs (HCGCs) treated with 1,2-DCE. Flow cytometry and mitochondrial membrane potential analyses discovered an increase in apoptotic-mediated cell death in HCGCs after 1,2-DCE treatment. This HCGC apoptosis was involved in the increases of protein expression in Cytochrome c, Caspase-3, Bad, Bim, transformation related protein 53, Caspase-8, tumor necrosis factor-α, and Survivin. Quantitative real-time PCR (qPCR) and western blot confirmed the increases in Cytochrome c, Caspase-3, cleaved Caspase-3, and Bad in HCGCs after 1,2-DCE treatment. Bax inhibitor peptide V5 rescued 1,2-DCE-induced HCGC apoptosis. Furthermore, 80 CD-1 male mice were exposed to 1,2-DCE by inhalation at 0, 100, 350, and 700 mg/m3 for 6 h/day for 4 weeks. An open field test found abnormal neurobehavioral changes in the mice exposed to 1,2-DCE. Histopathological examination showed significantly shrunken and hypereosinophilic cytoplasm with nuclear pyknosis in mouse CGCs from the 700 mg/m3 1,2-DCE group. TdT-mediated dUTP nick-end labeling assay verified significant increases in apoptotic positive cells in the mouse CGCs after 1,2-DCE exposure. We confirmed the increases in the expressions of Cytochrome c, Caspase-3, cleaved Caspase-3 and Bad in the mice exposed to 1,2-DCE. These findings suggest that 1,2-DCE exposure can induce CGC apoptosis and cerebellar dysfunction, at least in part, through mitochondrial pathway.


Asunto(s)
Apoptosis/efectos de los fármacos , Cerebelo/efectos de los fármacos , Dicloruros de Etileno/toxicidad , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Conducta Animal/efectos de los fármacos , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/patología , Cerebelo/fisiopatología , Humanos , Locomoción/efectos de los fármacos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/metabolismo , Neuronas/patología , Medición de Riesgo , Transducción de Señal
4.
Braz Oral Res ; 33: e117, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31939498

RESUMEN

The aim of this study was to evaluate the effect of mineral trioxide aggregate (MTA) and Brazilian propolis on the cell viability, mineralization, anti-inflammatory ability, and migration of human dental pulp cells (hDPCs). The cell viability was evaluated with CCK-8 kit after 1, 5, 7, and 9 days. The deposition of calcified matrix and the expression of osteogenesis-related genes were evaluated by Alizarin Red staining and real-time PCR after incubation in osteogenic medium for 21 days. The expression of inflammation-related genes in cells was determined after exposure to 1 µg/mL LPS for 3 h. Finally, the numbers of cells that migrated through the permeable membranes were compared during 15 h. Propolis and MTA significantly increased the viability of hDPCscompared to the control group on days 7 and 9. In the propolis group, significant enhancement of osteogenic potential and suppressed expression of IL-1ß and IL-6 was observed after LPS exposure compared to the MTA and control groups. The number of migration cells in the propolis group was similar to that of the control group, while MTA significantly promoted cell migration. Propolis showed comparable cell viability to that of MTA and exhibited significantly higher anti-inflammatory and mineralization promotion effects on hDPCs.


Asunto(s)
Compuestos de Aluminio/farmacología , Antiinflamatorios/farmacología , Compuestos de Calcio/farmacología , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Óxidos/farmacología , Própolis/farmacología , Silicatos/farmacología , Antraquinonas , Brasil , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Combinación de Medicamentos , Humanos , Interleucina-1beta/análisis , Interleucina-6/análisis , Odontoblastos/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Estadísticas no Paramétricas , Factor de Necrosis Tumoral alfa/análisis
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 48-51, 2020 Jan 10.
Artículo en Chino | MEDLINE | ID: mdl-31922596

RESUMEN

OBJECTIVE: To carry out multipath cytogenetic analysis of a rare case of acute myeloid leukemia (AML) with 11q23 aberration and D13S319 deletion. METHODS: G+R banding technique was used to analyze the chromosomal karyotype of the patient after 24 h of cell culture. Combined interphase and metaphase fluorescence in situ hybridization (FISH) was used to detect specific chromosomal sites for complex translocations and minor missing fragments. RESULTS: The patient was found to harbor MLL-AF10 fusion gene due to rearrangement of the mixed lineage leukemia (MLL) gene in conjunct with deletion of the D13S319 locus on chromosome 13. CONCLUSION: Whether MLL gene rearrangement and absence of D13S319 locus has a double impact on AML should attract more attention. For AML patient with clonal abnormalities such as 13q-, del(13)(q14), -13 or der(13), FISH assay should be proof and considered to determine the size of missing fragment so as targeted therapy may be implemented.


Asunto(s)
Hibridación Fluorescente in Situ , Leucemia Mieloide Aguda , Células Cultivadas , Cromosomas Humanos Par 11/genética , Humanos , Interfase , Cariotipificación , Leucemia Mieloide Aguda/genética , Metafase , Translocación Genética
6.
Plast Reconstr Surg ; 145(2): 409-418, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31985633

RESUMEN

BACKGROUND: Irradiated allogeneic costal cartilage is an alternative option of cartilage graft in patients with insufficient autologous cartilage. However, complications can occur during long-term follow-up. This study investigated whether Tutoplast-processed cartilage, one of the irradiated allogeneic costal cartilages, acts as a scaffold for adipose-derived stem cells and chondrogenesis. METHODS: In vitro setting, human adipose-derived stem cells seeded onto Tutoplast-processed cartilage were cultured in chondrogenic medium and observed using a scanning electron microscope. Next, 3 types of irradiated cartilage-including Tutoplast-processed cartilage, undifferentiated stem cells on Tutoplast-processed cartilage (undifferentiated group), and chondrogenic differentiated stem cells on Tutoplast-processed cartilage (chondrogenic group)-were implanted subcutaneously into nude mice. Gross, histologic, and gene expression analyses of Tutoplast-processed cartilages were performed at postoperative weeks 2 and 4. RESULTS: Human adipose-derived stem cells subjected to in vitro three-dimensional culture differentiated into chondrocytes and expressed cartilage-specificgenes. Adipose-derived stem cells seeded onto Tutoplast-processed cartilage were differentiated into chondrocytes in chondrogenic medium. In the chondrogenic group, the chondrogenic-differentiated cells attached to the surface of the Tutoplast-processed cartilage were maintained during the follow-up and were distinct from the existing Tutoplast-processed cartilage. Moreover, the chondrogenic group had higher expression of cartilage-specific genes compared with the undifferentiated group. CONCLUSIONS: Adipose-derived stem cells seeded onto Tutoplast-processed cartilage underwent chondrogenic differentiation, generating new cartilage, which was maintained after implantation without critical complications. The findings are clinically valuable in terms of overcoming the limitations of irradiated allogeneic costal cartilage, and broaden the surgical options for treatments requiring cartilage.


Asunto(s)
Cartílago/fisiología , Condrogénesis/fisiología , Células Madre Mesenquimatosas/fisiología , Agrecanos/metabolismo , Animales , Biomarcadores/metabolismo , Cartílago/efectos de la radiación , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno Tipo X/metabolismo , Femenino , Humanos , Técnicas In Vitro , Inyecciones Subcutáneas , Músculos Intercostales , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones Desnudos , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Modelos Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Trasplante Heterólogo , Trasplante Homólogo
7.
Plast Reconstr Surg ; 145(2): 420-431, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31985635

RESUMEN

BACKGROUND: Secondary lymphedema is a refractory disease, for which adipose-derived stem cells have shown some therapeutic potential. However, the mechanism of this action remains poorly understood. METHODS: The authors identified podoplanin-expressing adipose-derived stem cells, which allowed them to divide adipose-derived stem cells into podoplanin-positive and podoplanin-negative groups that they characterized in vitro. The authors then used a mouse hindlimb model for lymphedema to trace the fate of podoplanin-positive, podoplanin-negative, and unsorted adipose-derived stem cells in vivo. RESULTS: When induced in culture, podoplanin-positive cells were noted to up-regulate the expression of lymphatic endothelial cell markers, including LYVE-1, and assumed a cobblestone morphology. In addition, a substantial increase in lymphangiogenic cytokines was detected in the podoplanin-positive supernatant. The above findings were largely absent from the podoplanin-negative and unsorted groups. In the mouse model, the implanted cells relieved the limb lymphedema by promoting lymphangiogenesis, with the podoplanin-positive group showing the most significant effect. Immunocolocalization further revealed that the podoplanin-positive cells incorporated into lymphatic vessels were positive for LYVE-1. CONCLUSIONS: These data demonstrated that actions by means of both paracrine and differentiation pathways were involved in the adipose-derived stem cell-mediated therapeutic effects. The podoplanin-positive cells possessed lymphatic paracrine and differentiation abilities and may represent lymphatic endothelial cell precursor cells. The podoplanin-negative cells, which constitute a considerable proportion of the adipose-derived stem cells, may play an important paracrine role by secreting mesenchymal stem cell-related factors.


Asunto(s)
Linfangiogénesis/fisiología , Vasos Linfáticos/fisiología , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/fisiología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Femenino , Proteínas Fluorescentes Verdes , Linfedema/fisiopatología , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Fisiológica/fisiología , Fenotipo
8.
Life Sci ; 242: 117211, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31891720

RESUMEN

Ventricular hypertrophy is a risk factors for arrhythmias, ischemia and sudden death. It involves cellular modifications leading to a pathological remodeling and is associated with heart failure. The activation of the G protein-coupled estrogen receptor (GPER) mediates beneficial actions in the cardiovascular system. Our goal was to prevent and regress the hypertrophy by the activation of GPER in neonatal cardiac myocytes (NRCM) and SHR male rats. Aldosterone increased the neonatal cardiomyocytes cell surface area after 48 h of incubation. The aldo-induced hypertrophy was blocked by the mineralocorticoid receptor (MR) inhibitor Eplererone or the reduction of MR expression by siRNA. The activation of GPER by the agonist G-1 totally prevented the increase surface area by Ald. The transfection of neonatal rat cardiac myocytes with a siRNA against GPER or the incubation with GPER blockers G-15 and G-36 inhibited the protection of G-1. The significant increase of cell surface area after 48 h of incubation with Ald was totally regressed in 24 h by the presence of G-1, indicating that the activation of GPER not only prevent the hypertrophy but also regress the hypertrophy when it is already established. In the in vivo model, G-1 or Vehicle was constantly infused via the minipump to SHR. The reduction of the hypertrophy by G-1 was evident by the cross-sectional area, BNP and ANP markers and by echocardiography. In this studied we demonstrated that the activation of GPER prevented and regressed the hypertrophy induced by Ald in NRCM and regressed hypertrophy in SHR rats.


Asunto(s)
Cardiomegalia/prevención & control , Receptores Acoplados a Proteínas G/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Cardiomegalia/diagnóstico por imagen , Células Cultivadas , Ciclopentanos/farmacología , Ecocardiografía , Eplerenona/farmacología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Quinolinas/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/fisiología
9.
Invest Ophthalmol Vis Sci ; 61(1): 1, 2020 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-31995153

RESUMEN

Purpose: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. Methods: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. Results: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. Conclusions: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.


Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Fosfotransferasas/metabolismo , Células Ganglionares de la Retina/metabolismo , Proteínas de Transporte Vesicular/deficiencia , Proteínas tau/metabolismo , Animales , Western Blotting , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente Indirecta , Ácido Glutámico/toxicidad , Glicoproteínas de la Membrana Asociadas a los Lisosomas/metabolismo , Masculino , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/metabolismo , Roscovitina/farmacología , Transfección , Enzimas Activadoras de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
10.
Hum Genet ; 139(2): 227-245, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31919630

RESUMEN

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.


Asunto(s)
Ataxia/patología , Metilación de ADN , Proteína del Retraso Mental del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Mutación , Proteoma/análisis , Temblor/patología , Ataxia/genética , Ataxia/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína del Retraso Mental del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , ARN Interferente Pequeño/genética , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Temblor/genética , Temblor/metabolismo
11.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(1): 109-115, 2020 Jan 15.
Artículo en Chino | MEDLINE | ID: mdl-31939245

RESUMEN

Objective: To separate peripheral blood mesenchymal stem cells (PBMSC) and peripheral blood endothelial progenitor cells (PBEPC) from peripheral blood, and investigate the biological characteristics of composite cell sheets of PBMSC and PBEPC. Methods: The peripheral blood of healthy adult New Zealand white rabbits was extracted and PBMSC and PBEPC were separated by density gradient centrifugation. Morphological observation and identification of PBMSC and PBEPC were performed. The 3rd generation of PBMSC and PBEPC were used to construct a composite cell sheet at a ratio of 1∶1, and the 3rd generation of PBMSC was used to construct a single cell sheet as control. The distributions of cells in two kinds of cell sheets were observed by HE staining. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) in the supernatants of cell sheets were observed by ELISA at 1, 5, and 10 days after osteogenic induction. Results: The morphology of PBMSC was spindle-shaped or polygonal, and PBMSC had good abilities of osteogenic and adipogenic differentiation. The morphology of PBEPC was paved stone-like, and the tube-forming test of PBEPC was positive. Two kinds of cell sheets were white translucent. The results of HE staining showed that the composite cell sheet had more cell layers and higher cell density than the single cell sheet. The expressions of ALP, OCN, and VEGF in the supernatant of the two groups of cell sheets increased with the time of induction. The expression of OCN in the group of composite cell sheet was significantly higher than that in the group of single cell sheet on the 5th and 10th day, ALP on the 10th day was significantly higher than that in the group of single cell sheet, VEGF expression on the 1st, 5th, and 10th day was significantly higher than that in the group of single cell sheet, all showing significant differences ( P<0.05), and there was no significant difference between the two groups at other time points ( P>0.05). Conclusion: PBMSC have stable differentiation ability, and they have good application prospects because of their minimally invasive access. Composite cell membranes constructed by co-culture of two kinds of cells and induction of membrane formation provides a new idea and exploration for tissue defect repair.


Asunto(s)
Células Progenitoras Endoteliales , Células Madre Mesenquimatosas , Animales , Diferenciación Celular , Células Cultivadas , Osteogénesis , Conejos , Ingeniería de Tejidos , Factor A de Crecimiento Endotelial Vascular
12.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(1): 116-123, 2020 Jan 15.
Artículo en Chino | MEDLINE | ID: mdl-31939246

RESUMEN

Objective: To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology. Methods: Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression. Results: HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a "honeycomb" network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant ( P>0.05). Conclusion: Three-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.


Asunto(s)
Quiste Dermoide , Células Madre Mesenquimatosas , Animales , Células de la Médula Ósea , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Humanos , Conejos , Ingeniería de Tejidos
13.
Genes Dev ; 34(3-4): 149-165, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31919189

RESUMEN

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.


Asunto(s)
Núcleo Celular/inmunología , Cromatina/inmunología , Infecciones por Escherichia coli/inmunología , Neutrófilos/inmunología , Activación Transcripcional/genética , Activación Transcripcional/inmunología , Células Cultivadas , Escherichia coli , Histonas/metabolismo , Humanos
14.
J Photochem Photobiol B ; 203: 111731, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31935633

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic inflammatory disease with a poor prognosis and very few available treatment options. Low-level laser therapy (LLLT) has been gaining prominence as a new and effective anti-inflammatory and immunomodulatory agent. Can lung inflammation and the airway remodeling be regulated by LLLT in an experimental model of IPF in C57Bl/6 mice? The present study investigated if laser attenuates cellular migration to the lungs, the airway remodeling as well as pro-fibrotic cytokines secretion from type II pneumocytes and fibroblasts. Mice were irradiated (780 nm and 30 mW) and then euthanized fifteen days after bleomycin-induced lung fibrosis. Lung inflammation and airway remodeling were evaluated through leukocyte counting in bronchoalveolar lavage fluid (BALF) and analysis of collagen in lung, respectively. Inflammatory cells in blood were also measured. For in vitro assays, bleomycin-activated fibroblasts and type II pneumocytes were irradiated with laser. The pro- and anti-inflammatory cytokines level in BALF as well as cells supernatant were measured by ELISA, and the TGFß in lung was evaluated by flow cytometry. Lung histology was used to analyze collagen fibers around the airways. LLLT reduced both migration of inflammatory cells and deposition of collagen fibers in the lungs. In addition, LLLT downregulated pro-inflammatory cytokines and upregulated the IL-10 secretion from fibroblasts and pneumocytes. Laser therapy greatly reduced total lung TGFß. Systemically, LLLT also reduced the inflammatory cells counted in blood. There is no statistical difference in inflammatory parameters studied between mice of the basal group and the laser-treated mice. Data obtained indicate that laser effectively attenuates the lung inflammation, and the airway remodeling in experimental pulmonary fibrosis is driven to restore the balance between the pro- and anti-inflammatory cytokines in lung and inhibit the pro-fibrotic cytokines secretion from fibroblasts.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Citocinas/metabolismo , Fibrosis Pulmonar Idiopática/radioterapia , Rayos Láser , Animales , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Citocinas/análisis , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de la radiación , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Fibrosis Pulmonar Idiopática/patología , Terapia por Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Regulación hacia Arriba/efectos de la radiación
15.
J Photochem Photobiol B ; 203: 111738, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31954290

RESUMEN

This study aimed to compare the synthesis and secretion of VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, and FGF-2 between pulp fibroblasts from human primary teeth (HPF) and stem cell from human deciduous teeth (SHED) before and after photobiomodulation. HPF were obtained from explant technique and characterized by immunohistochemistry, while SHED were obtained from digestion technique and characterized by flow cytometry. HPF (control group) and SHED were plated, let to adhere, and put on serum starvation to synchronize the cell cycles prior to photobiomodulation. Then, both cell lineages were irradiated with 660-nm laser according to the following groups: 2.5 and 3.7 J/cm2. MTT and crystal violet assays respectively verified viability and proliferation. ELISA Multiplex Assay assessed the following proteins: VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, FGF-2, at 6, 12, and 24 h after photobiomodulation, in supernatant and lysate. Two-way ANOVA/Tukey test evaluated cell viability and proliferation, while angiogenic production and secretion values were analyzed by one-way ANOVA (P < .05). Statistically similar HPF and SHED viability and proliferation patterns occurred before and after photobiomodulation (P > .05). HPF exhibited statistically greater values of all angiogenic proteins than did SHED, at all study periods, except for FGF-2 (supernatant; 12 h); VEGFR1 (lysate; non-irradiated; 12 h); and VEGFR1 (lysate; non-irradiated; 24 h). Photobiomodulation changed the synthesis and secretion of angiogenic proteins by HPF. HPF produced and secreted greater values of all tested angiogenic proteins than did SHED before and after irradiation with both energy densities of 2.5 and 3.7 J/cm2.


Asunto(s)
Fibroblastos/efectos de la radiación , Rayos Láser , Células Madre/efectos de la radiación , Linaje de la Célula/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre/citología , Células Madre/metabolismo , Diente Primario/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
16.
Clin Sci (Lond) ; 134(2): 261-271, 2020 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-31922199

RESUMEN

Acute myeloid leukemia (AML) is a malignant disorder of hemopoietic stem cells. AML can escape immunosurveillance of natural killer (NK) by gene mutation, fusions and epigenetic modification. The mechanism of AML immune evasion is not clearly understood. Here we show that CD48 high expression is a favorable prognosis factor that is down-regulated in AML patients, which can help AML evade from NK cell recognition and killing. Furthermore, we demonstrate that CD48 expression is regulated by methylation and that a hypomethylating agent can increase the CD48 expression, which increases the NK cells killing in vitro. Finally, we show that CD48 high expression can reverse the AML immune evasion and activate NK cells function in vivo. The present study suggests that a combination the hypomethylating agent and NK cell infusion could be a new strategy to cure AML.


Asunto(s)
Antígeno CD48/inmunología , Epigénesis Genética/inmunología , Silenciador del Gen/inmunología , Leucemia Mieloide/inmunología , Escape del Tumor/inmunología , Enfermedad Aguda , Animales , Antimetabolitos Antineoplásicos/farmacología , Antígeno CD48/genética , Línea Celular Tumoral , Células Cultivadas , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Metilación de ADN/inmunología , Decitabina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Estimación de Kaplan-Meier , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Masculino , Ratones Endogámicos BALB C , Escape del Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto
17.
Toxicol Lett ; 319: 197-203, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31785464

RESUMEN

The chemical warfare agent sulfur mustard (SM) affects all cells in the epidermis including melanocytes which are responsible for melanin synthesis. After exposure to SM, pigment abnormalities like hypo- and hyperpigmentation can occur. The underlying molecular pathomechanisms of SM exposure on human melanogenesis have not been elucidated so far. In our study, we investigated the effect of SM on human melanocytes and melanogenesis. Normal human epidermal melanocytes (NHEM) were used as in vitro model and they were exposed to different concentrations of SM (4.5 µM-100 µM). Melanin production was analyzed by absorption measurements at 405 nm. In addition, quantitative real-time PCR (qPCR) and Western blot experiments were performed to determine the expression of essential melanogenesis-related proteins including tyrosinase (TYR), tyrosinase-related protein (TRP) 1 and 2 and microphthalmia transcription factor (MITF). Our findings demonstrated that exposure to low SM concentrations increased melanin synthesis accompanied with an increase in protein expression. In contrast, high SM concentrations led to decreased melanin content and a downregulation in expression of all investigated melanogenesis-associated proteins. We concluded that low SM concentrations may cause hyperpigmentation while high SM concentrations decreased melanin content which may explain hypopigmented skin areas in SM exposed patients.


Asunto(s)
Sustancias para la Guerra Química/toxicidad , Melaninas/biosíntesis , Gas Mostaza/toxicidad , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hiperpigmentación/inducido químicamente , Hipopigmentación/inducido químicamente , Oxidorreductasas Intramoleculares/efectos de los fármacos , Melaninas/genética , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/biosíntesis , Factor de Transcripción Asociado a Microftalmía/genética , Monofenol Monooxigenasa/biosíntesis , Monofenol Monooxigenasa/genética , Tripsina/biosíntesis , Tripsina/genética
18.
Fitoterapia ; 140: 104444, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31790768

RESUMEN

Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. (Zingiberaceae) is a perennial plant native to the East Indies and is widely distributed in South America, Oceania, and Asia. The mature fruits of the plant have been used in traditional medicine in China. In this study, we compared the chemical constituents in the methanol extracts of the leaves, the placenta, the pericarps, and the seeds obtained from the same plant using LC-MS, and we examined the NO inhibitory activities of the respective extracts and the isolated compounds. As a result of LC-MS analyses, kavalactone derivatives (1-6) were detected in the methanol extracts of the leaves, placenta, and pericarps. Of these, compound 6 was identified as a new asymmetrical cyclobutane dimer of 5,6-dehydrokawain. Quantitative analysis showed that the total amounts of kavalactone derivatives were highest in the methanol extract of the pericarps. Moreover, the results of measurements of the anti-inflammatory activity revealed that the pericarps extract showed the strongest activity. The compounds responsible for the anti-inflammatory activity of the extracts from A. zerumbet were identified. Of these, five were known kavalactone derivatives and one was a new kavalactone derivative (aniba dimer C). The results showed that the pericarps of A. zerumbet are a rich source of kavalactone derivatives, and that the pericarps of A. zerumbet can be utilized as an important medicinal resource.


Asunto(s)
Alpinia/química , Antiinflamatorios/farmacología , Lactonas/farmacología , Animales , Antiinflamatorios/aislamiento & purificación , Células Cultivadas , Hepatocitos/efectos de los fármacos , Japón , Lactonas/aislamiento & purificación , Estructura Molecular , Óxido Nítrico/metabolismo , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Extractos Vegetales/química , Hojas de la Planta/química , Ratas
19.
Toxicol Lett ; 320: 9-18, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-31765691

RESUMEN

Pranoprofen (PPF) is a wildly used anti-inflammatory ophthalmic drug. It was reported that PPF could decrease early epithelialization of scrape wounds in rabbit cornea and could reduce cell activities of cultured human corneal endothelial cells. However, effects of PPF on corneal stromal cells playing important roles in corneal wound healing remain unknown. In this study,in vitro model of cultured human corneal stomal (HCS) cells and in vivo model of rabbit corneas were used to investigate the effects and underlying mechanisms of PPF. Our findings showed that high concentrations of PPF treatment (0.1 % to 0.0125 %) caused limited chromatin condensation and quickly decreased cell viability that was proved to initiate necroptosis in HCS cells through activating receptor interacting protein kinase (RIPK) and mixed lineage kinase domain-like (MLKL). While low concentrations of PPF treatment (0.00625 %) induced DNA fragmentation, apoptotic body formation, ROS generation, activation of caspases and increase in cytoplasmic content of Bad, Bax and cytoplasmic cytochrome c that suggested apoptosis happened through ROS-mediated caspase-dependent and caspase-independent pathways. Studies of rabbit corneas treated with 0.1 % PPF (the clinical concentration) showed that PPF could induce apoptosis of rabbit corneal stromal cells. This work would be helpful for better understanding cytotoxic effects PPF on human corneal cells.


Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Apoptosis/efectos de los fármacos , Benzopiranos/toxicidad , Sustancia Propia/efectos de los fármacos , Propionatos/toxicidad , Células del Estroma/efectos de los fármacos , Animales , Caspasas/metabolismo , Células Cultivadas , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Sustancia Propia/metabolismo , Sustancia Propia/patología , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Proteínas Quinasas/metabolismo , Conejos , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Células del Estroma/metabolismo , Células del Estroma/patología , Factores de Tiempo
20.
Toxicol Lett ; 319: 225-236, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31760063

RESUMEN

N-Butylbenzenesulfonamide (NBBS) is a plasticizer detected in the environment suggesting potential human exposure. These studies investigated the in vitro hepatic clearance and disposition of [14C]NBBS in rodents following a single gavage (2, 20 or 200 mg/kg) or intravenous (IV) administration (20 mg/kg). NBBS was cleared slower in hepatocytes from humans compared to rodents. [14C]NBBS was well-absorbed in male rats following gavage administration and excreted extensively in urine (70-76 %) and feces (11-15 %) 72 h following administration. Following a 20 mg/kg gavage dose in male rats, 25 % of the dose was excreted in bile by 24 h suggesting that observed fecal excretion was due to biliary excretion. The radioactivity was distributed to tissues with 14 % and 8 % of the administered dose remaining in tissues at 24 and 72 h, respectively. There was no apparent dose-dependent effect in disposition in male rats. Disposition patterns were similar in female rats (urine, 83 %; feces, 14 %) and male (urine, 69 %; feces, 11 %) and female (urine, 72 %; feces, 9 %) mice following gavage administration of 20 mg/kg. The disposition following IV administration was similar to that of gavage. Urinary radiochemical profiles were similar between doses, routes, species, and sexes. Among numerous metabolites identified, oxidative metabolites of NBBS predominated.


Asunto(s)
Hepatocitos/metabolismo , Plastificantes/farmacocinética , Sulfonamidas/farmacocinética , Administración Intravenosa , Animales , Bilis/metabolismo , Células Cultivadas , Heces/química , Femenino , Humanos , Intubación Gastrointestinal , Masculino , Ratones , Ratones Endogámicos , Plastificantes/metabolismo , Plastificantes/toxicidad , Ratas , Ratas Sprague-Dawley , Sulfonamidas/administración & dosificación , Sulfonamidas/metabolismo , Distribución Tisular
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