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1.
Int J Nanomedicine ; 15: 7185-7198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33061375

RESUMO

Background: Next generation of coating materials on the surface of implants is designed with a paradigm shift from an inert material to an osteoimmunomodulatory material. Regulating immune response to biomedical implants through influencing the polarization of macrophage has been proven to be an effective strategy. Methods: Through anodization and hydrothermal treatment, magnesium ion incorporated TiO2 nanotube array (MgN) coating was fabricated on the surface of titanium and it is hypothesized that it has osteoimmunomodulatory properties. To verify this assumption, systematic studies were carried out by in vitro and in vivo experiments. Results: Mg ion release behavior results showed that MgN coating was successfully fabricated on the surface of titanium using anodization and hydrothermal technology. Scanning electron microscopy (SEM) images showed the morphology of the MgN coating on the titanium. The expression of inflammation-related genes (IL-6, IL-1ß, TNF-α) was downregulated in MgN group compared with TiO2 nanotube (NT) and blank Ti groups, but anti-inflammatory genes (IL-10 and IL-1ra) were remarkably upregulated in the MgN group. The in vitro and in vivo results demonstrated that MgN coating influenced macrophage polarization toward the M2 phenotype compared with NT and blank-Ti groups, which enhanced osteogenic differentiation of rat bone mesenchymal stem cells rBMSCs in conditioned media (CM) generated by macrophages. Conclusion: MgN coating on the titanium endowed the surface with immune-regulatory features and exerted an advantageous effect on osteogenesis, thereby providing excellent strategies for the surface modification of biomedical implants.


Assuntos
Inflamação/patologia , Macrófagos/patologia , Magnésio/farmacologia , Nanoestruturas/química , Osseointegração/efeitos dos fármacos , Titânio/farmacologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Osteogênese/efeitos dos fármacos , Células RAW 264.7 , Ratos Wistar , Propriedades de Superfície
2.
Int J Nanomedicine ; 15: 8045-8057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116517

RESUMO

Purpose: To study the cytotoxic evaluation, antimicrobial and confocal analysis of zinc oxide nanoparticles (ZnO NPs) obtained from a novel plant product fennel (Foeniculum vulgare Mill.) seed extract (FSE). Methods: ZnO NPs were analyzed using UV-Vis spectroscopy, XRD, FTIR, TEM and EDX techniques. The MTT cell cytotoxicity assay measured the proliferation and survival of MCF-7 cells treated at different concentrations of FSE-derived ZnO NPs. The antimicrobial activity towards pathogenic bacteria and yeast strains was investigated. Results: The UV-Vis spectra showed two peaks at 438 nm and 446 nm, confirming nanoparticle formation. The SEM morphology results showed porous ranging from 23-51 nm. The antitumor activity value (IC50) was at 50 µg/mL and 100 µg/mL. Besides, morphological changes of MCF-7, cells treated at different concentrations of FSE of ZnO NPs were observed in cell cultures transfected with a transient pCMV6-XL4-GFP-expressing vector containing C-terminal domain GFP-tagged proteins, which resulted in an apoptotic effect. Antimicrobial IZ ranged up No Inhibition to 18.00 ± 0.4. The IZ revealed at the highest concentration was E. faecium VRE and yeast Cryptococcus sp. (18.00 ± 0.4. mm), followed by S. aureus (17.00 ± 0.2 mm) and P. aeruginosa and the yeast C. parapsilosis (16 ± 0.4 mm). The IZ was equal to that caused by the nystatin to Cryptococcus sp., which was significantly highest than ampicillin treatments of S. aureus, P. aeruginosa, C. albicans, and C. parapsilosis. The MIC value of the FSE-derived ZnO NPs tested against E.faecium and C.albicans was 6.00 µg/mL (E. faecium and C. albicans). It was 32.00 µg/mL (S. aureus, S. typhimurium and Cryptococcus sp.), 64.00 µg/mL (P. aeruginosa), and 128 µg/mL (C. parapsilosis). Conclusion: As far as it is to our knowledge, this study established, for the first time, the biological activities of biosynthesized ZnO NPs from FSE and their synergistic therapeutic potential.


Assuntos
Foeniculum/química , Química Verde/métodos , Nanopartículas Metálicas/química , Extratos Vegetais/química , Sementes/química , Óxido de Zinco/farmacologia , Antibacterianos/farmacologia , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Difração de Raios X
3.
Anticancer Res ; 40(11): 6295-6303, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109567

RESUMO

BACKGROUND/AIM: The global prevalence of head and neck squamous cell carcinoma (HNSCC) remains high, and its prognosis poor. We investigated the anticancer effects of melatonin in human tongue squamous cell carcinoma cells (SCC-25) and its mechanisms of action. MATERIALS AND METHODS: MTT assay was used to determine cell viability. To assess the effects of melatonin on SCC-25 cell metastasis, we conducted cell formation, wound healing, transwell migration and invasion assay. Western blot analysis was performed to measure the levels of autophage marker proteins. RESULTS: We found that melatonin treatment significantly reduced the viability and colony formation ability of SCC-25 cells, impairing cell migration and invasion. Western blotting assay revealed that melatonin increased the levels of autophagy markers, such as LC-3B and Beclin-1. Consequently, melatonin induces autophage in SCC-25 cells. CONCLUSION: Melatonin may be a promising anticancer agent for the treatment of human tongue squamous cell carcinoma.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Melatonina/farmacologia , Neoplasias da Língua/patologia , Apoptose/efeitos dos fármacos , Extratos Celulares , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Ensaio Tumoral de Célula-Tronco
4.
Int J Nanomedicine ; 15: 6433-6449, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922010

RESUMO

Background: Electrospun nanofibers based on Colocasia esculenta tuber (CET) protein are considered as a promising material for wound dressing applications. However, the use of these nanofibers in aqueous conditions has poor stability. The present study was performed to obtain insights into the crosslinked electrospun CET's protein-chitosan (CS)-poly(ethylene oxide) (PEO) nanofibers and to evaluate their potential for wound dressing applications. Methods: The electrospun nanofibers were crosslinked with glutaraldehyde (GA) vapor and heat treatment (HT) to enhance their physicochemical stability. The crosslinked nanofibers were characterized by protein profiles, morphology structures, thermal behavior, mechanical properties, and degradation behavior. Furthermore, the antibacterial properties and cytocompatibility were analyzed by antibacterial assessment and cell proliferation. Results: The protein profiles of the electrospun CET's protein-CS-PEO nanofibers before and after HT crosslinking contained one major bioactive protein with a molecular weight of 14.4 kDa. Scanning electron microscopy images of the crosslinked nanofibers indicated preservation of the structure after immersion in phosphate buffered saline. The crosslinked nanofibers resulted in higher ultimate tensile strength and lower ultimate strain compared to the non-crosslinked nanofibers. GA vapor crosslinking showed higher water stability compared to HT crosslinking. The in vitro antibacterial activity of the crosslinked nanofibers showed a stronger bacteriostatic effect on Staphylococcus aureus than on Escherichia coli. Human skin fibroblast cell proliferation on crosslinked GA vapor and HT nanofibers with 1% (w/v) CS and 2% (w/v) CET's protein demonstrated the highest among all the other crosslinked nanofibers after seven days of cell culture. Cell proliferation and cell morphology results revealed that introducing higher CET's protein concentration on crosslinked nanofibers could increase cell proliferation of the crosslinked nanofibers. Conclusion: These results are promising for the potential use of the crosslinked electrospun CET's protein-CS-PEO nanofibers as bioactive wound dressing materials.


Assuntos
Antibacterianos/farmacologia , Quitosana/química , Colocasia/química , Reagentes para Ligações Cruzadas/química , Nanofibras/química , Proteínas de Plantas/química , Tubérculos/química , Polietilenoglicóis/química , Animais , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Nanofibras/ultraestrutura , Proteínas de Plantas/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Estresse Mecânico , Temperatura
5.
Int J Nanomedicine ; 15: 3903-3920, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606657

RESUMO

Background: Researchers are trying to study the mechanism of neural stem cells (NSCs) differentiation to oligodendrocyte-like cells (OLCs) as well as to enhance the selective differentiation of NSCs to oligodendrocytes. However, the limitation in nerve tissue accessibility to isolate the NSCs as well as their differentiation toward oligodendrocytes is still challenging. Purpose: In the present study, a hybrid polycaprolactone (PCL)-gelatin nanofiber scaffold mimicking the native extracellular matrix and axon morphology to direct the differentiation of bone marrow-derived NSCs to OLCs was introduced. Materials and Methods: In order to achieve a sustained release of T3, this factor was encapsulated within chitosan nanoparticles and chitosan-loaded T3 was incorporated within PCL nanofibers. Polyaniline graphene (PAG) nanocomposite was incorporated within gelatin nanofibers to endow the scaffold with conductive properties, which resemble the conductive behavior of axons. Biodegradation, water contact angle measurements, and scanning electron microscopy (SEM) observations as well as conductivity tests were used to evaluate the properties of the prepared scaffold. The concentration of PAG and T3-loaded chitosan NPs in nanofibers were optimized by examining the proliferation of cultured bone marrow-derived mesenchymal stem cells (BMSCs) on the scaffolds. The differentiation of BMSCs-derived NSCs cultured on the fabricated scaffolds into OLCs was analyzed by evaluating the expression of oligodendrocyte markers using immunofluorescence (ICC), RT-PCR and flowcytometric assays. Results: Incorporating 2% PAG proved to have superior cell support and proliferation while guaranteeing electrical conductivity of 10.8 × 10-5 S/cm. Moreover, the scaffold containing 2% of T3-loaded chitosan NPs was considered to be the most biocompatible samples. Result of ICC, RT-PCR and flow cytometry showed high expression of O4, Olig2, platelet-derived growth factor receptor-alpha (PDGFR-α), O1, myelin/oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) high expressed but low expression of glial fibrillary acidic protein (GFAP). Conclusion: Considering surface topography, biocompatibility, electrical conductivity and gene expression, the hybrid PCL/gelatin scaffold with the controlled release of T3 may be considered as a promising candidate to be used as an in vitro model to study patient-derived oligodendrocytes by isolating patient's BMSCs in pathological conditions such as diseases or injuries. Moreover, the resulted oligodendrocytes can be used as a desirable source for transplanting in patients.


Assuntos
Materiais Biomiméticos/farmacologia , Células da Medula Óssea/citologia , Diferenciação Celular , Nanofibras/química , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Tecidos Suporte/química , Compostos de Anilina/química , Animais , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica , Gelatina/química , Grafite/química , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/ultraestrutura , Células-Tronco Neurais/metabolismo , Oligodendroglia/efeitos dos fármacos , Poliésteres/química , Ratos , Suínos , Tri-Iodotironina/farmacologia
6.
Int J Nanomedicine ; 15: 4351-4362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606682

RESUMO

Purpose: The present study synthesized silver nanoparticles (AgNPs) using the aqueous extract of a traditional medicinal product consisting of an oleoresin (a combination of macromolecules of carbohydrates and proteins) exuded from the rhizome of the plant Ferula foetida (asafoetida gum) and evaluated its biological properties. Materials and Methods: The silver nanoparticles synthesized using asafoetida gum (As-AgNPs) were characterized using UV/Vis spectroscopy, fourier infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), X-ray diffraction (XRD) and transmission electron microscopy (TEM) and EADX. The cytotoxicity and antimicrobial activity As-AgNPs were evaluated against MCF-7 cell lines and selected microbial pathogens, respectively. Results: The synthesized silver nanoparticles were crystalline in nature with a spherical shape. The average particle size was 5.6-8.6 nm. The cytotoxicity of the synthesized As-AgNPs was evaluated against MCF-7 cell lines, and the As-AgNPs were found to be effective in inhibiting the multiplication of cancer cells. The As-AgNPs exhibited significant antimicrobial activity towards E. coli, K. pneumoniae and C. albicans. The MIC of the synthesized As-AgNPs was 7.80 µg/mL for E. coli ATCC 25922, Salmonella sp. WS50- and S. typhi; 15.60 µg/mL for S. typhimurium and S. aureus WS10, and 31.20 µg/mL for K. pneumoniae and S. aureus ATCC 43300-MRSA. In addition, MIC values of 15.60 µg/mL for C. albicans ATCC8436 and 31.20 µg/mL for C. krusei ATCC6258 were obtained. Conclusion: As asafoetida is a good traditional medicine, its involvement in the synthesis of AgNPs led the silver nanoparticles to exhibit good cytotoxic and antimicrobial effects.


Assuntos
Anti-Infecciosos/farmacologia , Ferula/química , Nanopartículas Metálicas/química , Gomas Vegetais/química , Prata/farmacologia , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Células MCF-7 , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Extratos Vegetais/química , Staphylococcus aureus/efeitos dos fármacos , Difração de Raios X
7.
Int J Nanomedicine ; 15: 4471-4481, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606689

RESUMO

Background: Ineffective integration has been recognized as one of the major causes of early orthopedic failure of titanium-based implants. One strategy to address this problem is to develop modified titanium surfaces that promote osteoblast differentiation. This study explored titanium surfaces modified with TiO2 nanotubes (TiO2 NTs) capable of localized drug delivery into bone and enhanced osteoblast cell differentiation. Materials and Methods: Briefly, TiO2 NTs were subjected to anodic oxidation and loaded with Metformin, a widely used diabetes drug. To create surfaces with sustainable drug-eluting characteristics, TiO2 NTs were spin coated with a thin layer of chitosan. The surfaces were characterized via scanning electron microscopy, atomic force microscopy, and contact angle measurements. The surfaces were then exposed to mesenchymal bone marrow stem cells (MSCs) to evaluate cell adhesion, growth, differentiation, and morphology on the modified surfaces. Results: A noticeable increase in drug release time (3 days vs 20 days) and a decrease in burst release characteristics (85% to 7%) was observed in coated samples as compared to uncoated samples, respectively. Chitosan-coated TiO2 NTs exhibited a considerable enhancement in cell adhesion, proliferation, and genetic expression of type I collagen, and alkaline phosphatase activity as compared to uncoated TiO2 NTs. Conclusion: TiO2 NT surfaces with a chitosan coating are capable of delivering Metformin to a bone site over a sustained period of time with the potential to enhance MSCs cell attachment, proliferation, and differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Quitosana/química , Liberação Controlada de Fármacos , Metformina/farmacologia , Nanotubos/química , Osteoblastos/citologia , Titânio/química , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanotubos/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Osteogênese/efeitos dos fármacos , Ratos Wistar , Molhabilidade
8.
Proc Natl Acad Sci U S A ; 117(27): 16027-16034, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571946

RESUMO

Puzzle-shaped pavement cells provide a powerful model system to investigate the cellular and subcellular processes underlying complex cell-shape determination in plants. To better understand pavement cell-shape acquisition and the role of auxin in this process, we focused on the spirals of young stomatal lineage ground cells of Arabidopsis leaf epidermis. The predictability of lobe formation in these cells allowed us to demonstrate that the auxin response gradient forms within the cells of the spiral and fluctuates based on the particular stage of lobe development. We revealed that specific localization of auxin transporters at the different membranes of these young cells changes during the course of lobe formation, suggesting that these fluctuating auxin response gradients are orchestrated via auxin transport to control lobe formation and determine pavement cell shape.


Assuntos
Arabidopsis/metabolismo , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Proteínas de Arabidopsis , Transporte Biológico , Epiderme Vegetal/metabolismo , Folhas de Planta/metabolismo , Estômatos de Plantas/metabolismo
9.
Int J Nanomedicine ; 15: 3695-3716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547023

RESUMO

Purpose: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity. Materials and Methods: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice. Results: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed. Conclusion: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.


Assuntos
Apoptose/efeitos dos fármacos , Nanopartículas Metálicas/química , NADPH Oxidase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/parasitologia , Prata/farmacologia , Toxoplasmose/patologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Fase G1/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos
10.
PLoS One ; 15(6): e0234641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574164

RESUMO

Chondrocytes, comparable to many cells from the connective tissue, dedifferentiate and end up in a similar fibroblastoid cell type, marked by the loss of the specific expression pattern. Here, chondrocytes isolated from osteoarthritic (OA) patients were investigated. The OA chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of known cartilage markers, such as SOX5, SOX6, SOX9, aggrecan and proteoglycan 4, remained unchanged. Since chondrocytes from OA and healthy tissue show different DNA methylation patterns, the underlying mechanisms of cartilage marker maintenance were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of dedifferentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2´-deoxycytidine (5-aza-dC) displayed no considerable impact on the maintenance of marker gene expression observed in the dedifferentiated state, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment for four weeks with 1 µM 5-aza-dC. In conclusion, 5-aza-dC affects the differentiation capacity of OA chondrocytes, while the global DNA methylation level remains stable. Furthermore, dedifferentiated chondrocytes isolated from late-stage OA patients represent a reliable cell source for in vitro studies and disease models without the need for additional alterations.


Assuntos
Condrócitos/patologia , Decitabina/farmacologia , Osteoartrite/patologia , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Biomarcadores/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Forma Celular/efeitos dos fármacos , Forma Celular/genética , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteoartrite/genética , Osteogênese/efeitos dos fármacos , Osteogênese/genética
11.
PLoS One ; 15(5): e0232759, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453737

RESUMO

SUMMARY: Reprogramming autologous adult cells to pluripotent cells allows for relatively safe cell replacement therapy. This can be achieved by nuclear transfer, cell fusion, or induced pluripotent stem cell technology However, the epigenetic memory of the cell is considered as a great challenge facing the complete reprograming of cells by these methods. Introducing oocyte-specific factors into differentiated cells may present a promising approach by mimicking cellular reprogramming during fertilization. METHODS: Human bone marrow mesenchymal stromal cells (hBM-MSCs) were cultured with different concentrations of human metaphase II (M II) oocyte extract (0.1, 1, 5, 10, 30 ng/µl). Reprogramming was assessed at various exposure times (1, 4, 7 days). Cells were tested for their proliferation rate, morphological changes, expression of pluripotency markers, expression of mesenchymal to epithelial transition markers, and mitochondrial rejuvenation. (mitochondrial localization, morphological changes, bioenergetics, transmembrane potential, and levels of reactive oxygen species, ROS). RESULTS: Treatment of human BM-MSCs with 10 ng/µl oocyte extract resulted in increased cell proliferation, which was associated with the upregulation of the pluripotency genes OCT-4, NANOG, and SOX-2 and a concomitant downregulation of mesenchymal-specific genes. MSCs exhibited small, immature round mitochondria with few swollen cristae localized proximal to the cell nucleus. This was accompanied by morphological cell changes, a metabolic shift towards oxidative phosphorylation, a high mitochondrial membrane potential, and increased ROS production. CONCLUSION: These data show that treatment with 10 ng/µl human MII-phase oocyte extract induced genetic and mitochondrial reprogramming of human BM-MSCs to a more embryonic phenotype.


Assuntos
Extratos Celulares/farmacologia , Reprogramação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oócitos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fatores de Tempo
12.
Int J Nanomedicine ; 15: 3281-3290, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32440124

RESUMO

Introduction: Cells exhibit high sensitivity and a diverse response to the nanotopography of the extracellular matrix, thereby endowing materials with instructive performances formerly reserved for growth factors. This finding leads to opportunities for improvement. However, the interplay between the topographical surface and cell behaviors remains incompletely understood. Methods: In the present study, we showed nanosurfaces with various dimensions of nanopits (200-750 nm) fabricated by self-assembling polystyrene (PS) nanospheres. Human adipose-derived stem cell behaviors, such as cell morphology, adhesion, cytoskeleton contractility, proliferation, and differentiation, were investigated on the prepared PS nanopit surface. Results: The osteogenic differentiation can be enhanced by nanopits with a diameter of 300-400 nm. Discussion: The present study provided exciting new avenues to investigate cellular responses to well-defined nanoscale topographic features, which could further guide bone tissue engineering and stem cell clinical research. The capability to control developing biomaterials mimicking nanotopographic surfaces promoted functional tissue engineering, such as artificial joint replacement, bone repair, and dental applications.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Nanoestruturas/química , Osteogênese , Poliestirenos/farmacologia , Células-Tronco/citologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nanoestruturas/ultraestrutura , Osteogênese/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura
13.
Sci Rep ; 10(1): 6716, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317746

RESUMO

Synthetic biodegradable polymers including poly(lactic acid) (PLA) are attractive cell culture substrates because their surfaces can be micropatterned to support cell adhesion. The cell adhesion properties of a scaffold mainly depend on its surface chemical and structural features; however, it remains unclear how these characteristics affect the growth and differentiation of cultured cells or their gene expression. In this study, we fabricated two differently structured PLA nanosheets: flat and microgrooved. We assessed the growth and differentiation of mouse primary cultured cortical neurons on these two types of nanosheets after pre-coating with poly-D-lysine and vitronectin. Interestingly, prominent neurite bundles were formed along the grooves on the microgrooved nanosheets, whereas thin and randomly extended neurites were only observed on the flat nanosheets. Comparative RNA sequencing analyses revealed that the expression of genes related to postsynaptic density, dendritic shafts, and asymmetric synapses was significantly and consistently up-regulated in cells cultured on the microgrooved nanosheets when compared with those cultured on the flat nanosheets. These results indicate that microgrooved PLA nanosheets can provide a powerful means of establishing a culture system for the efficient and reproducible differentiation of neurons, which will facilitate future investigations of the molecular mechanisms underlying the pathogenesis of neurological disorders.


Assuntos
Diferenciação Celular , Polaridade Celular , Neurônios/citologia , Poliésteres/farmacologia , Tecidos Suporte/química , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nanopartículas/química , Nanopartículas/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Células PC12 , Polilisina/farmacologia , Análise de Componente Principal , Ratos , Vitronectina/farmacologia
14.
Nat Methods ; 17(6): 587-593, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341544

RESUMO

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.


Assuntos
Citometria de Fluxo/métodos , Microfluídica/métodos , Actinas/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Processamento de Imagem Assistida por Computador , Tiazolidinas/administração & dosagem
15.
Arch Microbiol ; 202(7): 1677-1685, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32285166

RESUMO

Triterpenoid compounds are important bioactivity materials. Morchella is an abundant medicinal fungi found worldwide. In this study, we optimised the isolation and purification of triterpenoid compounds from Morchella mycelium fermentation. The results showed that the triterpenoid compounds yield was 35.22 mg/g, and we also identified two triterpenoid compounds using high-performance liquid chromatography. In addition, we evaluated the anti-tumour and antioxidant activity of the products, and the results showed that triterpenoid compounds from Morchella mycelium fermentation showed good bioactivity. The IC50 values of four cancer cell lines treated with the triterpenoid compounds for 48 h were 7.20, 14.96, 4.41, and 13.43 mg/mL, respectively. Morphological changes associated with the apoptosis of PC-3 cells were observed using confocal scanning laser microscopy after treatment with triterpenoid compounds for 48 and 72 h. The triterpenoid compounds also exhibited DPPH radical, hydroxyl, and ABTS-free radical scavenging activities in vitro. These results suggest that triterpenoid compounds from Morchella mycelium fermentation, which are found in functional foods and used in the field of pharmacology, might be excellent products for the treatment of cancer and age-related illnesses.


Assuntos
Ascomicetos/química , Micélio/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fermentação , Humanos , Radical Hidroxila/química , Concentração Inibidora 50 , Células PC-3
16.
Biochem Biophys Res Commun ; 526(4): 920-926, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32279993

RESUMO

Ovarian cancer G protein-coupled receptor 1 (OGR1), also known as GPR68, is a proton-sensing G protein-coupled receptor (GPCR) coupling to Gq/11/phospholipase C/Ca2+ signaling pathways. The specific histidine residues at the extracellular surface of OGR1 are suggested to be involved in the proton sensing. Later, some metal ions, including nickel ion (Ni2+), are also indicated to be OGR1 ligands. OGR1 polymorphic variants have recently been found in three families with amelogenesis imperfecta, which suggested that OGR1 is required for the process of dental enamel formation. One of these families possesses a missense mutation from leucine to proline at 74 (L74P) of OGR1. In the present study, we characterized HEK293 cells with L74P OGR1 (L74P-OGR1) and hemagglutinin (HA)-tag, as compared with cells with wild-type OGR1 (WT-OGR1) and HA-tag. We found that either acidic pH or NiCl2 induced intracellular Ca2+ mobilization and morphological change in WT-OGR1-transfected cells; however, the extracellular stimulus-induced actions were severely damaged in L74P-OGR1-transfected cells. We further confirmed that either WT-OGR1 or L74P-OGR1 is localized mainly in the surface of the cells, but only WT-OGR1 is internalized in response to acidification or NiCl2. Thus, the L74P-OGR1 protein may be distributed in the plasma membranes but severely damaged in the receptor functions. We speculate that L74P in the second transmembrane domain in OGR1 may result in conformational changes in the receptor, thereby disturbing the sensing extracellular signals, i.e., protons or metal ions, and/or transducing them to the intracellular signaling machinery through G proteins.


Assuntos
Amelogênese Imperfeita/genética , Amelogênese Imperfeita/metabolismo , Mutação de Sentido Incorreto/genética , Receptores Acoplados a Proteínas-G/genética , Sinalização do Cálcio , Forma Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Lisofosfolipídeos/farmacologia , Níquel/toxicidade , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo
17.
Nat Neurosci ; 23(4): 533-543, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203497

RESUMO

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. These include fetal alcohol spectrum disorders (FASDs) with a wide range of cognitive deficiencies, including impaired motor skill development. Although these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells altered gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlated with motor learning deficits in a mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a new intervention for learning disabilities in FASD.


Assuntos
Comportamento Animal/efeitos dos fármacos , Transtornos do Espectro Alcoólico Fetal/tratamento farmacológico , Deficiências da Aprendizagem/tratamento farmacológico , Aprendizagem/efeitos dos fármacos , Córtex Motor/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Animais , Forma Celular/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Modelos Animais de Doenças , Deficiências da Aprendizagem/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Córtex Motor/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Venenos de Escorpião/uso terapêutico , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
18.
Soft Matter ; 16(13): 3325-3337, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32196025

RESUMO

Surface tension governed by differential adhesion can drive fluid particle mixtures to sort into separate regions, i.e., demix. Does the same phenomenon occur in confluent biological tissues? We begin to answer this question for epithelial monolayers with a combination of theory via a vertex model and experiments on keratinocyte monolayers. Vertex models are distinct from particle models in that the interactions between the cells are shape-based, as opposed to distance-dependent. We investigate whether a disparity in cell shape or size alone is sufficient to drive demixing in bidisperse vertex model fluid mixtures. Surprisingly, we observe that both types of bidisperse systems robustly mix on large lengthscales. On the other hand, shape disparity generates slight demixing over a few cell diameters, a phenomenon we term micro-demixing. This result can be understood by examining the differential energy barriers for neighbor exchanges (T1 transitions). Experiments with mixtures of wild-type and E-cadherin-deficient keratinocytes on a substrate are consistent with the predicted phenomenon of micro-demixing, which biology may exploit to create subtle patterning. The robustness of mixing at large scales, however, suggests that despite some differences in cell shape and size, progenitor cells can readily mix throughout a developing tissue until acquiring means of recognizing cells of different types.


Assuntos
Caderinas/genética , Adesão Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Caderinas/química , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Humanos , Propriedades de Superfície
19.
Soft Matter ; 16(13): 3234-3244, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32163061

RESUMO

Vesicles composed of diblock copolymers, or polymersomes, have proven to possess numerous applications ranging from drug delivery to catalytically driven nano-motors. The shape of a polymersome can be responsive to external stimuli, such as light or solvent. Molecular dynamics simulations reveal that the shape change upon the contraction of the inner volume of a polymersome vesicle occurs in two separate regimes-a stretching regime and a bending regime. The barrier is shown to be dependent on the solvent environment. These results suggest that tailoring the bending modulus of polymer membranes can be used as a design methodology to engineer new stimuli-responsive vesicles.


Assuntos
Sistemas de Liberação de Medicamentos , Vesículas Extracelulares/química , Simulação de Dinâmica Molecular , Polímeros/química , Forma Celular/efeitos dos fármacos , Microambiente Celular/genética , Polimerização , Solventes/química
20.
Biochem Biophys Res Commun ; 525(3): 633-638, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32122651

RESUMO

Mesenchymal stem cell therapy has drawn much attention as a promising therapeutic option for the treatment of different diseases. Due to insufficient cell population derived from freshly isolated tissues, in vitro propagation is required prior to clinical use. However, reduced cell viability of aging mesenchymal stem cell (MSCs) with repeated propagations has yet not be fully investigated, especially for the biological characteristics of immunoregulatory ability and paracrine factors. In this study, we compared the biological properties of human umbilical cord-MSCs (hUC-MSCs) at different passages, especially for immunomodulatory ability and secretions. Our results showed that hUC-MSCs at early passage (P2) and late passage (P8) exhibited similar morphology and surface marker expression, but hUC-MSCs at P8 displayed reduced proliferation and differentiation potential, immunoregulatory and secretory ability. In particular, hUC-MSCs at P2 and P5 could significantly suppress the population of proinflammatory Th1 and Th17 cell subsets and upregulate Treg cells, but not with hUC-MSCs at P8. For paracrine mechanism, higher level of secretions such as growth factors, cell adhesions, anti-inflammatory factors of hUC-MSCs were observed at P2 and P5 compared to that at P8. Therefore, it is essential to verify and validate the biological characteristics of hUC-MSCs that possess a good vitality before they are released for clinical use. Altogether, this study provides a rationale and two important parameters for how to select appropriate passage and vitality of MSCs for cell therapy.


Assuntos
Senescência Celular , Imunomodulação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Cordão Umbilical/citologia , Adipogenia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Imunomodulação/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Solubilidade
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