RESUMEN
Exosomes are secreted from a variety of cells and transmit parental cell-derived biomolecules, such as nucleic acids and proteins, to recipient cells in distant organs. In addition to their important roles in both physiological and pathological conditions, exosomes are expected to serve as natural drug carriers without any cytotoxicity, immunogenicity, or tumorigenicity. However, the use of exosomes as drug delivery tools is limited due to the low uptake efficiency of the target cells, insufficient release of the contents from the endosome to the cytosol, and possible adverse effects caused by the delivery to non-target cells. In the present study, we examined the effects of the modification of exosomes with carbonate apatite or a lactose-carrying polymer. Using newly generated monitoring exosomes that contain either firefly luciferase or fused mCherry/enhanced green fluorescent protein, we demonstrated that the modification of exosomes with carbonate apatite improved their release from the endosome into the cytosol in recipient cells. Meanwhile, the modification of exosomes with a lactose-carrying polymer enhanced the selective delivery to parenchymal hepatocytes. These modified exosomes may provide an efficient strategy for macromolecule therapy for incurable diseases that cannot be treated with conventional small-molecule compounds.
RESUMEN
In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.
Asunto(s)
Encéfalo/fisiología , Células-Madre Neurales/fisiología , Proteínas Proto-Oncogénicas c-fyn/fisiología , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Cadherinas/genética , Cateninas/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/genética , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Bulbo Olfatorio/citología , Bulbo Olfatorio/crecimiento & desarrollo , Bulbo Olfatorio/fisiologíaRESUMEN
Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.
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Células Madre Embrionarias/citología , Microfluídica/métodos , Nanofibras/química , Animales , Microambiente Celular , HumanosRESUMEN
The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3ß (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro.
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Biomimética/métodos , Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Adsorción , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Integrinas/metabolismo , Ratones , Ingeniería de ProteínasRESUMEN
In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/ß-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering.
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Cadherinas/farmacología , Diferenciación Celular , Proliferación Celular , Matriz Extracelular/química , Células Endoteliales de la Vena Umbilical Humana/citología , Cadherinas/genética , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Andamios del Tejido/efectos adversos , Andamios del Tejido/químicaRESUMEN
We present a characterization of chemically treated cells using atomic force microscopy (AFM) which can observe changes in morphology and elasticity of cells. Since AFM has the significant advantage that it does not require fixation of samples, the method is simple and can capture various properties of living cells. In this study, corneal epithelial and endothelial cells were examined. The topography images of the corneal cells without glutaraldehyde (GA) fixation were successfully obtained. The images showed a natural three-dimensional shape of these cells, which scanning electron microscope (SEM) images could not provide. The AFM images of GA-fixed cells were taken and compared with a SEM image reported in the literature. Our results show that longer time for GA fixation makes the surface of the corneal endothelial tissue stiffer. Also, longer treatment results in relatively large structural variation in samples. Combined with conventional histochemical methods, this approach helps us gain an overall understanding of the influence of such chemical treatment.
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Córnea/citología , Microscopía de Fuerza Atómica/métodos , Animales , Córnea/química , Células Endoteliales/química , Células Endoteliales/citología , Células Epiteliales/química , Células Epiteliales/citología , Glutaral/química , Porcinos , Fijación del TejidoRESUMEN
Adrenomedullins (AM) is a multifaceted distinct subfamily of peptides that belongs to the calcitonin gene-related peptide (CGRP) superfamily. These peptides exert their functional activities via associations of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs) RAMP2 and RAMP3. Recent studies established that RAMPs and CLRs can modify biochemical properties such as trafficking and glycosylation of each other. However there is very little or no understanding regarding how RAMP or CLR influence ligand-induced events of AM-receptor complex. In this study, using pufferfish homologs of CLR (mfCLR1-3) and RAMP (mfRAMP2 and mfRAMP3), we revealed that all combinations of CLR and RAMP quickly underwent ligand-induced internalization; however, their recycling rates were different as follows: mfCLR1-mfRAMP3>mfCLR2-mfRAMP3>mfCLR3-mfRAMP3. Functional receptor assay confirmed that the recycled receptors were resensitized on the plasma membrane. In contrast, a negligible amount of mfCLR1-mfRAMP2 was recycled and reconstituted. Immunocytochemistry results indicated that the lower recovery rate of mfCLR3-mfRAMP3 and mfCLR1-mfRAMP2 was correlated with higher proportion of lysosomal localization of these receptor complexes compared to the other combinations. Collectively our results indicate, for the first time, that the ligand-induced internalization, recycling, and reconstitution properties of RAMP-CLR receptor complexes depend on the receptor-complex as a whole, and not on individual CLR or RAMP alone.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/metabolismo , Membrana Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Proteína 2 Modificadora de la Actividad de Receptores/metabolismo , Proteína 3 Modificadora de la Actividad de Receptores/metabolismo , Receptores de Adrenomedulina/metabolismo , Adrenomedulina/metabolismo , Animales , Western Blotting , Péptido Relacionado con Gen de Calcitonina , Peces , Citometría de Flujo , Glicosilación , Técnicas para Inmunoenzimas , Ligandos , Transporte de ProteínasRESUMEN
The organic matrix of nacre has been reported for its effect on osteogenesis. It was found that PFMG4 (Pinctada fucata mantle gene 4) with an N-terminal signal peptide could be secreted into nacre of Pinctada fucata (P. fucata). Here, we report that PFMG4 is highly expressed in mantle tissue and has high homology with C1q protein in different species. In MC3T3-E1 osteoblast cells, we found that highly expressed PFMG4 could suppress cell proliferation and type I collagen expression, but it could increase alkaline phosphatase activity and mineralized deposition. These results show that PFMG4 has potential ability in enhancing osteoblast differentiation, suggesting a new idea in developing medicine for the therapy of osteoporosis.
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Factores Biológicos/farmacología , Diferenciación Celular/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Pinctada/química , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores Biológicos/genética , Factores Biológicos/aislamiento & purificación , Factores Biológicos/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Calcificación Fisiológica/genética , Línea Celular , Proliferación Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Complemento C1q/genética , Complemento C1q/metabolismo , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Pinctada/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: Bile reflux contributes to development of Barrett's esophagus (BE) and could be involved in its progression to esophageal adenocarcinoma (EAC). We investigated whether bile acids affect levels or functions of microRNAs (MIRs) 221 and 222, which bind to the 3'-UTR of p27Kip1 messenger RNA to inhibit its translation. Reduced p27Kip1 increases degradation of the transcription factor CDX2; levels of CDX2 have been reported to decrease during progression of BE to EAC. METHODS: We used quantitative reverse transcriptase polymerase chain reaction to compare levels of MIRs 221 and 222 and immunohistochemistry to compare levels of p27Kip1 and CDX2 proteins in areas of BE and EAC from each of 11 patients. We examined the effects of bile acid exposure on levels of MIRs 221 and 222 and CDX2 in EAC cells. We investigated the effects of inhibitors of MIRs 221 and 222 on growth of human EAC xenograft tumors in NOD/SCID/IL-2Rγ(null) mice. RESULTS: Levels of MIRs 221 and 222 increased and levels of p27Kip1 and CDX2 decreased in areas of EAC vs BE. Levels of MIRs 221 and 222 increased, along with activity of nuclear bile acid receptor/farnesoid X receptor (FXR), when cultured cells were exposed to bile acids. Incubation of cells with bile acids increased degradation of CDX2; this process was reduced when cells were also incubated with proteasome inhibitors. Overexpression of MIRs 221 and 222 reduced levels of p27Kip1 and CDX2, and knockdown of these MIRs increased levels of these proteins in cultured cells. Inhibitors of MIRs 221 and 222 increased levels of p27Kip1 and CDX2 in EAC cells and reduced growth of xenograft tumors in NOD/SCID/IL-2Rγ(null) mice. CONCLUSIONS: We observed increased levels of MIRs 221 and 222 in human EAC tissues, compared with areas of BE from the same patient. We found that exposure of esophageal cells to bile acids activates FXR and increases levels of MIRs 221 and 222, reducing levels of p27Kip1 and promoting degradation of CDX2 by the proteasome. Our work opened the perspective of therapeutically targeting this pathway either via FXR antagonists or inhibitors of MIRs as a treatment option for BE and EAC.
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Adenocarcinoma/metabolismo , Ácidos y Sales Biliares/farmacología , Carcinogénesis/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Adenocarcinoma/patología , Anciano , Animales , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Factor de Transcripción CDX2 , Línea Celular Tumoral , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Progresión de la Enfermedad , Neoplasias Esofágicas/patología , Femenino , Proteínas de Homeodominio/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , MicroARNs/efectos de los fármacos , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Successful gene therapy depends on the development of efficient and cell-specific gene delivery systems. Currently, animal viral vectors have been mostly used for in vivo and in clinical trials owing to their high transduction efficiency. However, they suffer from numerous limitations such as biosafety, immunogenicity, gene packaging capacity, complicated production and cell specificity. Therefore non-viral vectors are attractive alternatives to viral gene delivery systems due to their low toxicity, relatively easy production and greater diversity. Among non-viral vectors, chitosan and chitosan derivatives have been extensively utilized as gene carriers owing to their low immunogenicity, biocompatibility, biodegradability, low toxicity and ease of chemical modifications. However, low transfection efficiency of DNA (or low gene silencing of siRNA) and low cell specificity of chitosan should be overcome before clinical trials. The objective of this review is to summarize several parameters affecting the transfection efficiency of DNA (or gene silencing of siRNA) for the promising use of chitosan as gene carriers. Besides, chemical modifications of chitosan with pH-sensitive molecules and specific ligands so as to enhance the transfection efficiency of DNA (or gene silencing of siRNA) and cell specificity will be covered.
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Membrana Celular/química , Quitosano/química , ADN/genética , Nanocápsulas/química , ARN Interferente Pequeño/genética , Transfección/métodos , ADN/administración & dosificación , ADN/química , Difusión , Silenciador del Gen , Concentración de Iones de Hidrógeno , Nanocápsulas/ultraestructura , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/químicaRESUMEN
Extracellular matrix (ECM) plays a fundamental role in regulating cell attachment, proliferation, migration and differentiation. Both synthetic and biologically derived materials have been explored as an ECM in regenerative medicine and tissue engineering. To biomimick the extracellular matrix, we combined the advantages of the biological properties of nanofibrous scaffolds and the fusion protein to apply for the culture of human mesenchymal stem cells in vitro. In this study, we fabricated well random-oriented/aligned nanofibrous scaffolds with PCL, modified with hE-cadherin-Fc fusion protein and studied the synergistic effect of the scaffolds. The random-oriented/aligned architecture was observed in the nanofibrous scaffolds by SEM. XPS and WCA measurements evidenced that hE-cadherin-Fc was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was well improved after fusion protein coating. The hE-cadherin-Fc modified markedly promoted the adhesion and proliferation of hMSCs and guided hMSCs to a spindlier morphology compared with unmodified nanofibrous scaffolds. Furthermore, hMSCs on the hE-cadherin-Fc-coated nanofibrous scaffolds also had differentiation potential. These results suggested that the combination of PCL nanofibrous scaffolds and hE-cadherin-Fc fusion protein may be a promising artificial ECM for the behavior of hMSCs in vitro.
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Cadherinas/farmacocinética , Moléculas de Adhesión Celular/farmacocinética , Proteínas de la Matriz Extracelular/farmacocinética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Poliésteres/química , Andamios del Tejido , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacocinética , Cadherinas/química , Cadherinas/genética , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacocinética , Diseño de Equipo , Análisis de Falla de Equipo , Proteínas de la Matriz Extracelular/química , Humanos , Ensayo de Materiales , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Ingeniería de Tejidos/instrumentaciónRESUMEN
The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked ß-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.
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Acetilglucosamina/metabolismo , Apoptosis , Lectinas/metabolismo , Preñez , Vimentina/metabolismo , Animales , Sitios de Unión , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos ICR , Embarazo , Propiedades de Superficie , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
The cytoskeleton protein vimentin is dramatically altered following pathological events such as fibrosis and tumorigenesis. Vimentin binds to multivalent N-acetylglucosamine (GlcNAc) molecules at the cell surface and interacts with O-linked ß-GlcNAc proteins. Moreover, dying cells can be engulfed by neighboring cells through surface interactions between vimentin and many O-GlcNAc proteins in cell debris. Here, we show that vimentin was altered by its interaction with GlcNAc-bearing molecules such as GlcNAc-bearing polymers. The interaction with GlcNAc-bearing polymers promoted the cell surface recruitment of vimentin followed by the phosphorylation of vimentin serine 71 and the increase in tetrameric vimentin disassembled from vimentin filaments in HeLa cells. Moreover, it was found that GlcNAc-bearing polymers and O-GlcNAc proteins from dying cells promoted vimentin expression and cell migration in the Madin-Darby canine kidney and Michigan Cancer Foundation-7 cells. These results suggest that interactions between surface vimentin and GlcNAc molecules, including the O-GlcNAc proteins from dying cells, may play a pivotal role in vimentin expression and the migration of cancer cells. We propose new mechanisms of vimentin expression in cancer cells.
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Acetilglucosamina/metabolismo , Glicoproteínas de Membrana/metabolismo , Vimentina/metabolismo , Animales , Muerte Celular , Movimiento Celular , Perros , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Mutación Missense , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Serina/genética , Vimentina/química , Vimentina/genéticaRESUMEN
BACKGROUND: RNA interference (RNAi) is a powerful approach in functional genomics to selectively silence messenger mRNA (mRNA) expression and can be employed to rapidly develop potential novel drugs against a complex disease like cancer. However, naked siRNA being anionic is unable to cross the anionic cell membrane through passive diffusion and therefore, delivery of siRNA remains a major hurdle to overcome before the potential of siRNA technology can fully be exploited in cancer. pH-sensitive carbonate apatite has recently been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis. Moreover, internalized siRNA was found to escape from the endosomes in a time-dependent manner and efficiently silence gene expression. RESULTS: Here we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes has led to the sensitization of a human cervical cancer cell line to doxorubicin- and paclitaxel depending on the dosage of the individual drug whereas no such enhancement in cell death was observed with cisplatin irrespective of the dosage following intracellular delivery of the siRNAs. CONCLUSION: Thus, PLCG2 and CALM1 genes are two potential targets for gene knockdown in doxorubicin and paclitaxel-based chemotherapy of cervical cancer.
RESUMEN
It was believed for a long time that mRNA is very unstable, and can not be used for therapeutic purposes. In the last decade, however, many research groups proved its transfection feasibility along with advantages and applications. Our investigation is aimed at establishing a potent and efficient mRNA delivery system. We previously reported that an inorganic-organic hybrid carrier by exploiting the advantages of inorganic nano apatite particles onto organic carrier DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} and showed potential effect of carbonate apatite particles on each of the mRNA delivery steps in dividing and non-dividing cell. Here, we report on the development of a more efficient mRNA carrier by complexing ECM protein, fibronectin with the DOTAP-apatite carrier. The carrier showed enhanced uptake of luciferase mRNA both qualitatively and quantitatively. Accelerated cellular endocytosis rate was evaluated using labeled endosome. Finally expression of lucifearse mRNA was higher for fibronectin complexed carrier in compared to the uncoated one.
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Portadores de Fármacos , Endocitosis , Ácidos Grasos Monoinsaturados , Fibronectinas , Terapia Genética , Compuestos de Amonio Cuaternario , ARN Mensajero/administración & dosificación , Transfección/métodos , Animales , Apatitas , Liposomas , Luciferasas/genética , Mamíferos , ARN Mensajero/uso terapéuticoRESUMEN
To enhance vascularization of hydrophobic implants in vivo, a VEGF-Fc fusion protein consisting of vascular endothelial growth factor (VEGF) fused to the immunoglobulin G Fc domain was prepared as an artificial extracellular matrix (ECM). VEGF-Fc was stably immobilized on a polystyrene plate due to the hydrophobicity of the Fc domain, and significantly enhanced the adhesion of human umbilical vein endothelial cells (HUVECs). Additionally, the use of VEGF-Fc as an ECM markedly promoted the proliferation of HUVECs longer than 72 h and induced the reorganization of actin filaments into larger stress fibers within these cells. The VEGF-Fc fusion protein may be a promising artificial ECM for enhancing endothelial cell growth.
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Proliferación Celular , Células Endoteliales/fisiología , Técnicas de Cultivo de Célula/métodos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Poliestirenos/química , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets. Poly (N-isopropyl acrylamide) (PNIPAAm) hydrogel is a temperature-responsive polymer gel whose volume changes isotropically in response to temperature changes below 37 °C. We designed a combined hydrogel substrate consisting of collagen-immobilized PNIPAAm as the local stimulation side and polyacrylamide (PAAm) as the non-stimulation side to assess propagation of mechanical transduction. Mardin-Darby canine kidney (MDCK) cells adhered to the collagen-immobilized PNIPAAm gel increased it area and were flattened as the gel swelled with temperature decrease. E-cadherin in these cells became undetectable in some domains, and actin stress fibers were more clearly observed at the cell base. In contrast, E-cadherin in cells adhered to the collagen-immobilized PAAm side was equally stained as that in cells adhered to the collagen-immobilized PAAm side even after temperature decrease. ERK1/2 MAPK activation of cells on the non-stimulated substrate occurred after partial stretching of the cell sheet suggesting the propagation of signaling. These results indicate that a change in the balance of mechanical tension induced by partial stretching of cell sheets leads to activation and propagation of the cell signaling.
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Hidrogel de Polietilenoglicol-Dimetacrilato/química , Estrés Mecánico , Resinas Acrílicas/química , Animales , Cadherinas/metabolismo , Adhesión Celular , Línea Celular , Movimiento Celular , Forma de la Célula , Colágeno/química , Perros , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transducción de Señal , TemperaturaRESUMEN
Hepatic tissue engineering may be an effective approach for the treatment of liver disease; however, its practical application requires hepatic cell separation technologies that do not involve cell surface modification and maintain cell activity. In this study, we developed hepatocyte cell separation materials using a thermoresponsive polymer and a polymer with high affinity to hepatocytes. A block copolymer of poly(N-p-vinylbenzyl-O-ß-D-galactopyranosyl-(1â4)-D-gluconamide) (PVLA) and poly(N-isopropylacrylamide) (PNIPAAm) [PVLA-b-PNIPAAm] was prepared through two steps of atom transfer radical polymerization. On the prepared PVLA-b-PNIPAAm brush, HepG2 cells (model hepatocytes) adhered at 37 °C and detached at 20 °C, attributed to the temperature-modulated affinity between PVLA and HepG2. Cells from the immortalized human hepatic stellate cell line (TWNT-1) did not adhere to the copolymer brush, and RAW264.7 cells (mouse macrophage; model Kupffer cells) adhered to the copolymer brush, regardless of temperature. Using the difference in cell adhesion properties on the copolymer brush, temperature-modulated cell separation was successfully demonstrated. A mixture of HepG2, RAW264.7, and TWNT-1 cells was seeded on the copolymer brush at 37 °C for adherence. By reducing the temperature to 20 °C, adhered HepG2 cells were selectively recovered with a purity of approximately 85% and normal activity. In addition, induced pluripotent stem (iPS) cell-derived hepatocytes adhered on the PVLA-b-PNIPAAm brush at 37 °C and detached from the copolymer brush at 20 °C, whereas the undifferentiated iPS cells did not adhere, indicating that the prepared PVLA-b-PNIPAAm brush could be utilized to separate hepatocyte differentiated and undifferentiated cells. These results indicated that the newly developed PVLA-b-PNIPAAm brush can separate hepatic cells from contaminant cells by temperature modulation, without affecting cell activity or modifying the cell surface. Thus, the copolymer brush is expected to be a useful separation tool for cell therapy and tissue engineering using hepatocytes.
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Hepatocitos , Poliestirenos , Ratones , Animales , Humanos , Temperatura , Poliestirenos/farmacología , Polímeros/farmacologíaRESUMEN
We studied effect of artificial extracellular matrices (ECMs), such as collagen I, poly (N-p-vinylbenzyl-4-O-ß-D-galactopyranosyl-D-gluconamide)(PVLA) and E-cadherin-IgG Fc (E-cad-Fc) on hepatic metabolism to identify the mechanism of in vivo hepatocellular functional and metabolic integrity. mRNA expression of liver function marker, cytochrome P450 (CYP) and transporter genes in hepatocytes were compared among used ECMs using real-time RT-PCR. mRNA expressions of Cyp2c29 and Cyp2d22 among CYP genes in hepatocytes on PVLA were recovered after 3days due to enhanced liver-specific function by the spheroid formation of hepatocytes whereas mRNA expressions of CYP genes in hepatocytes on collagen and E-cad-Fc drastically decreased with time. mRNA expressions of the Cyp2c29 and Cyp2d22 in hepatocytes on PVLA were more recovered in the presence of epidermal growth factor (EGF) due to the more and bigger spheroid formation of hepatocytes. Multidrug resistance-associated protein 2 (Mrp2) protein was accumulated at intracellular lumen as similar to bile duct in hepatocyte spheroid formed on PVLA, indicating that spheroid formation of hepatocytes is very important for maintaining liver functions.
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Sistema Enzimático del Citocromo P-450/genética , Matriz Extracelular/metabolismo , Expresión Génica , Hepatocitos/enzimología , Animales , Cadherinas/química , Cadherinas/metabolismo , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Disacáridos/química , Disacáridos/metabolismo , Matriz Extracelular/química , Galactosa/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Mensajero/genética , Esferoides Celulares/enzimología , Compuestos de Vinilo/química , Compuestos de Vinilo/metabolismoRESUMEN
The ability of carbonate apatite (CO(3)Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO(3)Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO(3)Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO(3)Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO(3)Ap and OVA-containing alumina salt (Alum), suggesting that CO(3)Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO(3)Ap.