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1.
J Vis Exp ; (139)2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30247461

RESUMO

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

2.
J Neurosci ; 38(19): 4598-4609, 2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29661967

RESUMO

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.

3.
Stem Cells Dev ; 27(5): 347-354, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29336219

RESUMO

Various somatic stem cells divide asymmetrically; however, it is not known whether embryonic stem cells (ESCs) divide symmetrically or asymmetrically, not only while maintaining an undifferentiated state but also at the onset of differentiation. In this study, we observed single ESCs using time-lapse imaging and compared sister cell pairs derived from the same mother cell in either the maintenance or differentiation medium. Mouse ESCs were cultured on E-cadherin-coated glass-based dishes, which allowed us to trace single cells. The undifferentiated cell state was detected by green fluorescent protein (GFP) expression driven by the Nanog promoter, which is active only in undifferentiated cells. Cell population analysis using flow cytometry showed that the peak width indicating distribution of GFP expression broadened when cells were transferred to the differentiation medium compared to when they were in the maintenance medium. This finding suggested that the population of ESCs became more heterogeneous at the onset of differentiation. Using single-cell analysis by time-lapse imaging, we found that although the total survival ratio decreased by changing to differentiation medium, the one-live-one-dead ratio of sister cell pairs was smaller compared with randomly chosen non-sister cell pairs, defined as an unsynchronized cell pair control, in both media. This result suggested that sister cell pairs were more positively synchronized with each other compared to non-sister cell pairs. The differences in interdivision time (the time interval between mother cell division and the subsequent cell division) between sister cells was smaller than that between non-sister cell pairs in both media, suggesting that sister cells divided synchronously. Although the difference in Nanog-GFP intensity between sister cells was smaller than that between non-sister cells in the maintenance medium, it was the same in differentiation medium, suggesting asymmetrical Nanog-GFP intensity. These data suggested that ESCs may divide asymmetrically at the onset of differentiation resulting in heterogeneity.

4.
Cell Stem Cell ; 22(1): 128-137.e9, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29276142

RESUMO

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

5.
Small ; 13(18)2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28272774

RESUMO

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.

6.
J Biomed Mater Res A ; 105(6): 1583-1592, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27643636

RESUMO

The development of new three-dimensional (3D) cell culture system that maintains the physiologically relevant signals of hepatocytes is essential in drug discovery and tissue engineering research. Conventional two-dimensional (2D) culture yields cell growth, proliferation, and differentiation. However, gene expression and signaling profiles can be different from in vivo environment. Here, we report the fabrication of a 3D culture system using an artificial scaffold and our custom-made inkjet 3D bioprinter as a new strategy for studying liver-specific functions of hepatocytes. We built a 3D culture platform for hepatocytes-attachment and formation of cell monolayer by interacting the galactose chain of galactosylated alginate gel (GA-gel) with asialoglycoprotein receptor (ASGPR) of hepatocytes. The 3D geometrical arrangement of cells was controlled by using 3D bioprinter, and cell polarity was controlled with the galactosylated hydrogels. The fabricated GA-gel was able to successfully promote adhesion of hepatocytes. To observe liver-specific functions and to mimic hepatic cord, an additional parallel layer of hepatocytes was generated using two gel sheets. These results indicated that GA-gel biomimetic matrices can be used as a 3D culture system that could be effective for the engineering of liver tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1583-1592, 2017.


Assuntos
Alginatos/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Materiais Biocompatíveis/metabolismo , Galactose/metabolismo , Hepatócitos/citologia , Impressão Tridimensional , Engenharia Tecidual/instrumentação , Alginatos/química , Animais , Materiais Biocompatíveis/química , Adesão Celular , Células Cultivadas , Desenho de Equipamento , Galactose/análogos & derivados , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Hepatócitos/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Camundongos Endogâmicos ICR
7.
Adv Healthc Mater ; 5(15): 1992, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27511954

RESUMO

E-cadherin-modified poly(lactic-co-glycolic acid) (hE-cad-PLGA) microparticles were fabricated and then mediated the 3D cell aggregates of human mesenchymal stem cells (MSCs) on page 1949 by Jun Yang and co-workers. The hE-cad-Fc matrix and the PLGA microparticles synergistically regulate the proliferation and bioactive factors secretions of MSCs by activating EGFR, AKT and ERK1/2 signaling pathways. The hE-cad-PLGA microparticles offer a novel route to expand multipotent stem cell-based clinical applications.

8.
Adv Healthc Mater ; 5(15): 1949-59, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27245478

RESUMO

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/ß-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications.


Assuntos
Caderinas/química , Ácido Láctico/química , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/metabolismo , Ácido Poliglicólico/química , Agregação Celular , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo
9.
Bioconjug Chem ; 27(7): 1599-605, 2016 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-27269811

RESUMO

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.

10.
Anticancer Res ; 36(2): 673-6, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26851023

RESUMO

Antigen-presenting cells (APCs) play a pivotal role in cancer immunotherapy. APCs in conventionally used flasks are harvested by enzymatic digestion or cell scraping for application to cancer immunotherapy. However, these methods may impair functional molecules expressed on the APC surface and reduce their effects in cancer immunotherapy. Recently, we found that APCs could be harvested by shaking at 4°C in flasks coated with poly[N-p-vinylbenzyl-O-2-acetoamide-2-deoxy-ß-D-glucopyranosyl-(1→4)-2-acetoamide-2-deoxy-ß-D-gluconamide] (PVGlcNAc) or a copolymer consisting of sulfonylurea (SU) linked to poly[N-p-vinyl-benzyl-4-O-ß-D-galactopyranosyl-D-gluconamide] [P(VLA-co-SU)]. In the present study, we compared the functions of cytotoxic T-lymphocytes (CTLs) induced by APCs generated in PVGlcNAc- or P(VLA-co-SU)-coated flasks and conventional flasks. APCs from PVGlcNAc- or P(VLA-co-SU)-coated flasks showed higher expression of cluster of differentiation (CD)80/86, CD11c, and major histocompatibility complex class II alloantigen I-A(d), and higher cytotoxicity than APCs from conventional flasks. These results suggest that the use of PVGlcNAc- or P(VLA-co-SU)-coated flasks is optimal for harvesting APCs. The generated APCs also have a higher antigen-presenting ability compared to those generated in conventional flasks. Our results may contribute to the development of effective cancer immunotherapies.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Separação Celular/métodos , Dissacarídeos/metabolismo , Lactose/análogos & derivados , Teste de Cultura Mista de Linfócitos , Poliestirenos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Biomarcadores/metabolismo , Antígeno CD11c/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe II/metabolismo , Lactose/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Polivinil/metabolismo , Linfócitos T Citotóxicos/imunologia
11.
Biomacromolecules ; 17(3): 756-66, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26859785

RESUMO

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.


Assuntos
Caderinas/farmacologia , Diferenciação Celular , Proliferação de Células , Matriz Extracelular/química , Células Endoteliais da Veia Umbilical Humana/citologia , Caderinas/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Tecidos Suporte/efeitos adversos , Tecidos Suporte/química
12.
PLoS One ; 10(8): e0135170, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26244942

RESUMO

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and ß-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.


Assuntos
Caderinas/genética , Diferenciação Celular/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Western Blotting , Caderinas/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sobrevivência Celular/genética , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Microscopia Confocal , Neuritos/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Imagem com Lapso de Tempo , beta Catenina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
13.
Int J Nanomedicine ; 10: 2313-23, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25848252

RESUMO

Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-ß-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose-asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Dissacarídeos , Hepatócitos/citologia , Hepatócitos/fisiologia , Compostos de Vinila , Animais , Receptor de Asialoglicoproteína/metabolismo , Caderinas/química , Caderinas/genética , Adesão Celular/fisiologia , Separação Celular/métodos , Sobrevivência Celular , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Dissacarídeos/química , Hepatócitos/efeitos dos fármacos , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Soro , Esferoides Celulares , Compostos de Vinila/química
14.
PLoS One ; 10(3): e0116022, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25738937

RESUMO

RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.


Assuntos
Apatitas/efeitos adversos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Apatitas/química , Apatitas/farmacocinética , Terapia Genética/métodos , Células HT29 , Humanos , Proteínas Inibidoras de Apoptose/genética , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/efeitos adversos , Neoplasias/terapia , RNA Interferente Pequeno/uso terapêutico
15.
Anal Bioanal Chem ; 407(9): 2631-5, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25633218

RESUMO

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.


Assuntos
Córnea/citologia , Microscopia de Força Atômica/métodos , Animais , Córnea/química , Células Endoteliais/química , Células Endoteliais/citologia , Células Epiteliais/química , Células Epiteliais/citologia , Glutaral/química , Suínos , Fixação de Tecidos
16.
Gen Comp Endocrinol ; 212: 156-62, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24815888

RESUMO

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.


Assuntos
Proteína Semelhante a Receptor de Calcitonina/metabolismo , Membrana Celular/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Receptores de Adrenomedulina/metabolismo , Adrenomedulina/metabolismo , Animais , Western Blotting , Peptídeo Relacionado com Gene de Calcitonina , Peixes , Citometria de Fluxo , Glicosilação , Técnicas Imunoenzimáticas , Ligantes , Transporte Proteico
17.
Biosci Biotechnol Biochem ; 79(4): 558-65, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25485793

RESUMO

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.


Assuntos
Fatores Biológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Pinctada/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores Biológicos/genética , Fatores Biológicos/isolamento & purificação , Fatores Biológicos/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Linhagem Celular , Proliferação de Células , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Complemento C1q/genética , Complemento C1q/metabolismo , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Pinctada/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais
18.
Vaccine ; 32(47): 6199-205, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25261380

RESUMO

To demonstrate the potential of pH-sensitive carbonate apatite (CO3Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO3Ap complexes, or pCI-neo/CO3Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO3Ap (10 µg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100 µg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO3Ap (100 µg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10 µg of pCI-neo-sOVA delivered by CO3Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO3Ap nanoparticles for DNA vaccine delivery.


Assuntos
Apatitas/química , Imunidade Celular , Imunidade Humoral , Nanopartículas/química , Vacinas de DNA/imunologia , Animais , Epitopos de Linfócito T/imunologia , Feminino , Hipersensibilidade Tardia/imunologia , Interferon gama/imunologia , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Plasmídeos/imunologia , Baço/imunologia , Vacinas de DNA/química
19.
J Nanosci Nanotechnol ; 14(6): 4007-13, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24738344

RESUMO

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.


Assuntos
Caderinas/farmacocinética , Moléculas de Adesão Celular/farmacocinética , Proteínas da Matriz Extracelular/farmacocinética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Poliésteres/química , Tecidos Suporte , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacocinética , Caderinas/química , Caderinas/genética , Adesão Celular/fisiologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacocinética , Desenho de Equipamento , Análise de Falha de Equipamento , Proteínas da Matriz Extracelular/química , Humanos , Teste de Materiais , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Engenharia Tecidual/instrumentação
20.
J Nanosci Nanotechnol ; 14(1): 564-76, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24730283

RESUMO

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.


Assuntos
Membrana Celular/química , Quitosana/química , DNA/genética , Nanocápsulas/química , RNA Interferente Pequeno/genética , Transfecção/métodos , DNA/administração & dosagem , DNA/química , Difusão , Inativação Gênica , Concentração de Íons de Hidrogênio , Nanocápsulas/ultraestrutura , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química
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