RESUMO
The development of synthetic carriers for small interfering RNA (siRNA) and plasmids is crucial for effective gene therapy. In this study, we synthesized magnetic graphene oxide nanoflakes as carriers for siRNA delivery, with the goal of knockdown specific genes such as the green fluorescence protein (GFP). Our approach combined magnetically reduced graphene oxide with polyethylenimine (PEI) crosslinked to its surface using carbonyl diimidazole. To evaluate the adsorption capacity of the PEI-modified nanocomposite, we investigated its ability to bind two types of nucleic acids: short-hairpin (sh)RNA plasmids and siRNA targeting GFP. The nanocomposite exhibited significant adsorption, with maximum capacities of 426 ng/µg for shRNA and 71 ng/µg for siRNA, respectively. Simultaneous delivery of siRNA and shRNA using our designed nanocomposites was successfully achieved in human hepatoma and prostate cancer cells. Under magnetic guidance, the knockdown efficiencies reached 73.5 % in hepatoma cells for dual delivery of siRNA and shRNA. Our findings revealed that the nanocomplexes were internalized by the cells through a caveolae-dependent endocytosis mechanism. The demonstrated ability of the nanoflakes to efficiently transport siRNA and shRNA, with high loading capacity, controlled release, and magnetic targeting, resulted in effective GFP knockdown in vitro. These findings highlight the potential of magnetic graphene oxide nanoflakes as promising carriers for siRNA delivery and gene knockdown in therapeutic applications.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Masculino , Humanos , Técnicas de Silenciamento de Genes , Próstata , Neoplasias Hepáticas/genética , RNA Interferente Pequeno/genética , Fenômenos Magnéticos , PolietilenoiminaRESUMO
Vascular endothelial growth factor (VEGF) is the principal growth factor responsible for the retinal neovascularization in the pathogenesis of retinopathy of prematurity (ROP). Current therapies for ROP include laser ablation and intravitreal anti-VEGF injection. However, these treatments either destroy the peripheral retina or associate with problems of persistent peripheral avascular retina or later recurrence of ROP. In the present study we investigated a new therapeutic approach by exploring the potential role of a specific microRNA, miR-126, in regulating VEGFA expression and retinal neovascularization in a rat oxygen-induced retinopathy (OIR) model. We demonstrated that miR-126 mimic and plasmid effectively suppresses VEGFA mRNA expression in both human and rat retinal pigment epithelium cell lines, quantified with qRT-PCR. Animal experiments on rat OIR model revealed that intravitreal injection of miR-126 plasmid efficiently downregulated VEGFA expression in the intraocular fluid and retinal tissues measured by ELISA, and significantly suppressed retinal neovascularization, which was confirmed by calculating sizes of neovascularization areas on fluorescence microscopic images of flat mounted retina stained with Alexa Fluor 594-conjugated isolectin B4 to visualize blood vessels. Together, these results showed that intravitreal injection of miR-126 plasmid could inhibit retinal neovascularization by down-regulating VEGFA expression, suggesting a potential therapeutic effect for ROP.
Assuntos
MicroRNAs/uso terapêutico , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Humanos , Oxigênio , Plasmídeos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Retina/patologia , Vasos Retinianos/patologia , Retinopatia da Prematuridade/induzido quimicamente , Retinopatia da Prematuridade/metabolismoRESUMO
Curcumin is a powerful scavenger of reactive oxygen species and could prevent the corneal cells from oxidative damage. However, the clinical efficacy of curcumin is limited by its low aqueous solubility and stability, leading to poor bioavailability. ß-cyclodextrin, with a hydrophilic surface and a hydrophobic cavity and self-assembling properties, can form inclusion complexes with lipophilic drugs such as curcumin for ocular delivery. We synthesized ethylene diamine (EDA)-modified ß-cyclodextrin and prepared the curcumin complexation using the solvent evaporation method. The EDA-ß-cyclodextrin provided a better thermodynamic stability and higher complex yield for curcumin complexes, compared to ß-cyclodextrin, which were demonstrated on the analysis of their van't Hoff plots and phase solubility diagrams. We characterized EDA-ß-cyclodextrin curcumin nanoparticles and determined that the EDA modified ß-cyclodextrin is a more suitable carrier than parental ß-cyclodextrin, using FT-IR, XRD, TEM, and analyses of solubility and storage stability. In addition, the curcumin-EDA-ß-cyclodextrin nanoparticles had better in vitro corneal penetration and 3 -h cumulative flux in a porcine cornea experiment, and displayed an improved biocompatibility, confirmed by the histological examination of porcine corneas and cell viability of bovine corneal epithelial cells. These results together revealed a role of EDA modification in the ß-cyclodextrin carrier, including the improvement of curcumin complex formation, thermodynamic properties, cytotoxicity, and the in vitro corneal penetration. The EDA-ß-cyclodextrin inclusion can provide curcumin a higher degree of aqueous solubility and corneal permeability.
Assuntos
Córnea/química , Curcumina/farmacocinética , Sistemas de Liberação de Medicamentos , Etilenodiaminas/farmacocinética , Nanopartículas/química , beta-Ciclodextrinas/farmacocinética , Animais , Córnea/metabolismo , Curcumina/química , Etilenodiaminas/química , Tamanho da Partícula , Solubilidade , Propriedades de Superfície , Suínos , beta-Ciclodextrinas/químicaRESUMO
Cationic macromolecules condense DNA into small nanoparticles and form polyplex. The composition of the polyplex determines the endocytic process, the intracellular routing and the fate of the polyplex. Previously, oligochitosan-modified vectors with different protein moieties are used as gene delivery vector and the types of protein moiety can influence the endosome escape ability and transfection efficiency. Among the modified vectors, oligochitosan-modified bovine serum albumin (BSA) showed 90% transfection efficeincy compared to the modified zein and ovalbumin. These data encouraged us to investigate the mechanism of internalization involved in the superior transfection efficiency of modified BSA/ plasmid polyplex. The effect of specific endocytic inhibitors was studied in two adherent cell lines. The caveolae-mediated and lipid-mediated pathways play a significant role in the polyplex internalization. Next, a colocation of polyplex with lysosome was investigated in the presence of LysoTracker using confocal microscopy. Up to 70% of polyplex successfully escaped the lysosome without degradation. Four non-adherent cell lines showed above than 60% transfection efficiency at an optimized vector/plasmid ratio. Moreover, no significant hemolytic effect was observed up to 500⯵g/mL of cationic BSA, indicating no detectable cell membrane disruption. Overall, the hybrid biomacromolecule showed good intracellular delivery and safety in a mice model.
Assuntos
Quitina/análogos & derivados , DNA/química , DNA/metabolismo , Portadores de Fármacos/química , Endocitose , Plasmídeos/genética , Soroalbumina Bovina/química , Animais , Células CHO , Quitina/química , Quitina/toxicidade , Quitosana , Cricetulus , DNA/genética , Portadores de Fármacos/toxicidade , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos , Lisossomos/química , Teste de Materiais , Oligossacarídeos , Soroalbumina Bovina/toxicidade , TransfecçãoRESUMO
OBJECTIVES: The relationships of manipulation of culture temperature and medium circulation rate on the metabolic parameters were regressed by multiple linear regression analysis in hollow fiber bioreactors (HFB). RESULTS: The high circulation rate could significantly enhance the oxygen consumption of the hybridoma cells and the medium's oxidation-reduction potential. A mildly hypothermic condition of 36 °C and a circulation rate of 182.5 mL/min could support the hybridoma had the maximal antibody titer of 60.75 µg/mL for 20 days. When the ammonium ion was 65 ppm or lactate close to 2.6 g/L, the medium was replaced to maintain the stable and healthy cells at the high cell concentration of 3.33 × 108/mL for continuous antibody production. Two serum-free media could be successfully applied to this perfusion system and maintain hybridoma growth and antibody production. CONCLUSION: The single-use HFBs could provide the advantages including high cell density, low shear stress, and continuous antibody production.
Assuntos
Anticorpos/metabolismo , Reatores Biológicos , Contagem de Células , Hibridomas/metabolismo , Anticorpos/genética , Meios de Cultura/química , Análise Multivariada , Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Oligochitosan-modified proteins have gained attention as efficient non-viral vectors for gene delivery. However, little information exists if protein moieties can serve as an important role for internalization and endosome escape ability of the genetic material. To explore this issue, we designed two cationic oligochitosan-modified vectors that consist of different proteins, namely a hydrophobic plant protein (zein) and a hydrophilic animal protein (ovalbumin (OVA)) to deliver pDNA to epithelial cell line CHO-K1 and HEK 293 T. These cationic vectors were systematically characterized by molecular weight, infrared (IR) structural analysis, transmission electron microscopy (TEM) morphology, and surface charge. A remarkable impact of protein moieties was observed on physiochemical properties of the developed vectors. Oligochitosan-modified zein containing hydrophobic protein exhibited high buffering capacity and excellent DNA binding ability compared to the oligochitosan-modified OVA. The data on transfection in the presence of endocytic inhibitors indicated that the caveolae-mediated pathway (CvME) played a key role in the internalization of the zein-based polyplex. However, the OVA-based polyplex was internalized in CHO-K1 cells via CvME and in HEK 293 T cells via the lipid-mediated pathway. Moreover, oligochitosan-modified zein exhibited lower cytotoxicity, greater lysosomal escape ability, better plasmid stability, and better transfection efficiency than the oligochitosan-modified OVA. This study offers a facile procedure for the synthesis of cationic vectors and elucidates the relationship that exists between protein moieties and transfection activity, thus providing an alternative, non-viral platform for the gene delivery.
Assuntos
Quitina/análogos & derivados , Técnicas de Transferência de Genes , Ovalbumina/química , Zeína/química , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quitina/química , Quitina/farmacologia , Quitosana , Cricetulus , Células HEK293 , Humanos , Oligossacarídeos , Tamanho da Partícula , Transfecção , Zea mays/químicaRESUMO
Chitosan and its derivatives have shown to be potential gene carriers with biocompatiblility and safety. However, their practical delivery is far from being ideal because of the low transfection efficiency. The present work describes the potential of a natural protein, bovine serum albumin (BSA), conjugated with a natural oligosaccharide, oligochitosan (OC), as a considerable promising approach for a safe and efficient non-viral gene delivery vector. The FTIR spectra proved the effective conjugation of BSA with OC through covalent bond. The condensation ability of plasmid DNA (pDNA) with a BSA-OC biopolymer was analyzed by gel retardation assay, competition binding assay, and dynamic light scattering used to measure the nanoparticle size. In addition, the BSA-OC biopolymer showed the protection of pDNA from enzymatic degradation by DNase I and showed good stability when exposed to 50% fetal bovine serum. The transfection efficiency was evaluated in the presence of 10% serum-supplemented media or serum-free media on three kinds of mammalian cells. Our results showed that the BSA-OC biopolymer is a good non-viral vehicle for gene delivery. We investigated the parameters such as the pDNA payload, temperature, incubating duration, and biopolymer/pDNA ratio on the transfection efficiency. This hybrid vehicle had the ability to transfect 90% of cells and to maintain 80% of cell viability. The aforementioned results suggest that the facile synthesis of the BSA-OC biopolymer could overcome the cytotoxicity problem and transfection barriers during in vitro gene delivery.
Assuntos
Quitosana/análogos & derivados , Albumina Sérica/química , Transfecção/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/metabolismo , Vetores Genéticos/genética , Células HEK293 , HumanosRESUMO
PURPOSE: To determine the systemic impact of intravitreal injection of bevacizumab (IVB), an anti-vascular endothelium growth factor antibody, in newborn rabbits. MATERIALS AND METHODS: We used four groups of rabbits. Group 1 rabbits received a single injection of IVB starting from the age of 6 weeks. Group 2 rabbits received a single injection of balanced salt solution (BSS, 0.025 ml) and served as controls for group 1. Group 3 rabbits received two consecutive injections of IVB at the ages of 6 and 10 weeks. Group 4 rabbits received two consecutive injections of BSS at the ages of 6 and 10 weeks and served as controls for group 3. During the experiment, a complete blood count (CBC), clinical biochemistry, weight gain, food intake, body temperature, blood pressure, pulse, and mortality were measured in the animals. Two months after IVB injection, the animals were sacrificed, and histology of the major organs was checked. Immunohistochemistry was assessed to explore the neurons in the central nervous system (CNS). RESULTS: We found there were no morphological or functional changes in the eyes following IVB injection. Furthermore, there were no differences in CBC, biochemistry, or other measured parameters among the four groups of animals. We checked the histology of the major organs and neurons in the CNS and they did not reveal significant differences among the four groups of animals. CONCLUSIONS: Conclusively, IVB of either one or two injections (0.625 mg) in newborn rabbit eyes is well tolerated and does not cause noticeable systemic organ pathology.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Bevacizumab/administração & dosagem , Animais , Animais Recém-Nascidos , Olho/anatomia & histologia , Olho/efeitos dos fármacos , Injeções Intravítreas , Fenômenos Fisiológicos Oculares/efeitos dos fármacos , CoelhosRESUMO
As pathogens steadily develop resistance to widely used antibiotics, new methodologies for their efficient inactivation must be developed. Photodynamic therapy is an upcoming technique that provides an alternative option for treating pathogenic infections. The efficiency of photodynamic therapy has been limited by the use of aqueous mediums for dispersing photosensitising agents. Toluidine Blue O (TBO) was chosen for this study as a cationic photosensitiser to inhibit Gram-negative bacterium Pseudomonas aeruginosa. Enhanced delivery of the photosensitiser was ensured by utilising an essential oil-based microemulsion. The efficiency of photodynamic therapy was further improved by the use of a chemical penetration enhancer to improve permeability of the bacterial outer membrane. TBO accumulation patterns in neonate pig skin were studied using confocal laser scanning microscopy. The physicochemical properties of the TBO loaded microemulsion, including UV-vis absorbance, size distribution and zeta potential, were analysed to understand the enhanced antimicrobial activity. Confocal laser scanning microscopy confirmed the formation of a TBO reservoir in the skin by the TBO-loaded microemulsions. TBO (5 µg/mL) in the vehicles significantly inhibited the growth of P. aeruginosa. All these efforts resulted in inhibition obtained at a drug concentration and light intensity much lower than what is reported by the works of previous investigators.
Assuntos
Luz , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos da radiação , Cloreto de Tolônio/farmacologia , Animais , Animais Recém-Nascidos , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Emulsões/química , Hibridomas , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Pele/metabolismo , Espectrofotometria , Suínos , Cloreto de Tolônio/química , Cloreto de Tolônio/farmacocinéticaRESUMO
PURPOSE: To investigate the rate of lens subluxation following plasmin and/or SF6 injections in eyes, and whether a subsequent elevated level of vascular endothelial growth factor (VEGF) and vitreous tap would aggravate subluxation. METHODS: Four groups of rabbits were used. Group 1 received an intravitreal injection (IVI) of plasmin and SF6 in the right eye; group 2 received an IVI of plasmin in the right eye; group 3 received an IVI of SF6 in the right eye; and group 4 received an IVI of balanced salt solution in the right eye. After treatment, IVIs of VEGF were given and vitreous tap was performed three times, followed by clinical observation of lens subluxation and scanning electronic microscope evaluation of the zonular fibers. RESULTS: After IVIs of plasmin and SF6, and VEGF and vitreous tap had been performed one to three times, lens subluxation was noted in 0%, 43%, 71%, 71%, and 86% of the eyes in group 1. After IVIs of plasmin, VEGF, and vitreous tap had been performed one to three times, lens subluxation was noted in 11%, 22%, 44%, 44%, and 67% of the eyes in group 2. The eyes in group 3 and 4 did not show signs of lens subluxation after VEGF IVIs and vitreous tap. Histology confirmed zonular fiber damage in the eyes treated with plasmin. CONCLUSIONS: The incidence of lens subluxation increased following plasmin injections in the eyes, and this was aggravated by the subsequent high VEGF level in the eyes and vitreous tapping. Zonular fibers were disrupted following plasmin treatment. These effects should be kept in mind when using plasmin enzymes in patients with vitreoretinal abnormalities.
Assuntos
Fibrinolisina/farmacologia , Subluxação do Cristalino/patologia , Cristalino/efeitos dos fármacos , Hexafluoreto de Enxofre/farmacologia , Animais , Injeções Intravítreas , Subluxação do Cristalino/metabolismo , Cristalino/patologia , Microscopia Eletrônica de Varredura , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Bacteriophytochrome infrared fluorescent protein (IFP) has a long emission wavelength that is appropriate for detecting pathophysiological effects via near-infrared (NIR) based imaging. However, the brightness and photostability of IFP are suboptimal, although an exogenous supply of biliverdin (BV) IXα is able to enhance these properties. In this study, we fused a far red mPlum fluorescent protein to IFP 1.4 via a linker deoxyribonucleic acid (DNA) sequence encoding eight amino acids. The brightness of mPlum-IFP 1.4 fusion protein at the IFP emission channel was comparable to that of native IFP 1.4 protein when fusion protein and IFP 1.4 were excited by 543 and 633 nm using confocal microscopy, respectively. Visualization of IFP 1.4 fluorescence by excitation of mPlum in mPlum-IFP 1.4 fusion protein is likely to be associated with Förster resonance energy transfer (FRET). The FRET phenomenon was also predicted by acceptor photobleaching using confocal microscopy. Furthermore, the expression of mPlum-IFP 1.4 fusion protein could be detected in cell culture and in xenograft tumors in the absence of BV using in vivo imaging system, although the BV was still essential for detecting native IFP 1.4. Therefore, this innovative-fluorescent fusion protein would be useful for NIR-based imaging in vitro and in vivo.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Genes Reporter/genética , Células HEK293 , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genéticaRESUMO
Microemulsions have received great attention for applications in transdermal drug delivery. The use of curcumin for treating various skin diseases like scleroderma, psoriasis, and skin cancer was extensively reported. The solubility of curcumin in various oils, surfactants, and cosurfactants was studied herein in order to find the optimal components for a transdermal delivery vehicle. Microemulsion systems composed of eucalyptol, polysorbate 80, ethanol, and water were developed as transdermal delivery vehicles for curcumin. Effects of the microemulsion composition on transdermal curcumin delivery were studied using Franz diffusion cells. The transdermal curcumin flux, permeability coefficient, and enhancement ratio were analyzed to evaluate the effects of eucalyptol/water ratios in the microemulsions. Pseudo-ternary phase diagrams of the eucalyptol microemulsions with various surfactant/cosurfactant ratios (1:1-1:3) were constructed to investigate their phase behaviors. Conductivity, interfacial tension, size, and viscosity data of the microemulsions were used to characterize the physicochemical properties of transdermal vehicles. The influence of the microemulsions on skin histology and on the delivery route was analyzed using hematoxylin/eosin staining and confocal laser scanning microscopy. In conclusion, microemulsions were successfully developed for transdermal curcumin delivery after screening various components and adjusting the oil/water ratios. The curcumin permeation rate of the microemulsion developed was 15.7-fold higher than that of the control (eucalyptol only). These results indicate that an eucalyptol microemulsion system is a promising tool for the percutaneous delivery of curcumin.
Assuntos
Curcumina/administração & dosagem , Curcumina/química , Cicloexanóis/administração & dosagem , Cicloexanóis/química , Sistemas de Liberação de Medicamentos/métodos , Monoterpenos/administração & dosagem , Monoterpenos/química , Absorção Cutânea/efeitos dos fármacos , Administração Cutânea , Animais , Animais Recém-Nascidos , Curcumina/farmacocinética , Cicloexanóis/farmacocinética , Emulsões , Eucaliptol , Eucalyptus , Monoterpenos/farmacocinética , Absorção Cutânea/fisiologia , Solubilidade/efeitos dos fármacos , SuínosRESUMO
PURPOSE: To investigate the clearance of vascular endothelial growth factor (VEGF) after the induction of posterior vitreous detachment by plasmin and/or SF(6). METHODS: The study design included four groups of rabbits: group 1 received an intravitreal injection of plasmin and SF(6) in the right eye, group 2 received an intravitreal injection of plasmin in the right eye, group 3 received an intravitreal injection of SF(6) in the right eye, and group 4 received an intravitreal injection of balanced salt solution in the right eye. Intravitreal injection of human VEGF (50 µL, 10 ng/µL) was performed in study eyes and control eyes 1 month after plasmin and/or SF(6) injection. Serum and vitreous samples were collected on days 1, 3, and 7 after VEGF injection to determine the serum and vitreous concentrations of VEGF. RESULTS: One day after VEGF injection, residual human VEGF concentration in the vitreous cavity was significantly lower in the plasmin- and SF(6)-treated eyes (group 1) and the plasmin-treated eyes (group 2) when compared with the control eyes (group 4) (P = 0.047 and 0.027, respectively). Three days after VEGF injection, the residual VEGF concentration in the vitreous cavity was still significantly lower in the plasmin- and SF(6)-treated eyes (group 1) when compared with the control eyes (group 4) (P = 0.025). CONCLUSIONS: Eyes treated with plasmin exhibit a more rapid clearance of exogenous VEGF than control eyes. This finding suggests a novel treatment for retinopathies associated with vitreous traction and VEGF elevation.
Assuntos
Fibrinolisina/farmacologia , Hexafluoreto de Enxofre/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacocinética , Corpo Vítreo/metabolismo , Descolamento do Vítreo/metabolismo , Animais , Combinação de Medicamentos , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Injeções Intravítreas , Microscopia Eletrônica de Varredura , Coelhos , Corpo Vítreo/efeitos dos fármacos , Descolamento do Vítreo/induzido quimicamenteRESUMO
Xanthohumol is one of the main flavonoids in hop extracts and in beer. Very few investigations of xanthohumol have studied hepatocellular carcinoma. In this study, the inhibitory effects of xanthohumol on human hepatocellular carcinoma cell lines were investigated. The IC(50) values of xanthohumol for two hepatocellular carcinoma cell lines and one normal hepatocyte cell line were 108, 166 and 211 microm, respectively. Normal murine hepatocyte cell line had more resistance to xanthohumol than hepatocellular carcinoma cell lines. Besides, the inhibitory effects of xanthohumol on human hepatocellular carcinoma cell lines were attributed to apoptosis as indicated in the results of flow cytometry, fluorescent nuclear staining and electrophoresis of oligonucleosomal DNA fragments. Hop xanthohumol was more efficient in the growth inhibition of hepatocellular carcinoma cell lines than the flavonoids silibinin and naringin from thistle and citrus. It was shown for the first time that xanthohumol from hops effectively inhibits proliferation of human hepatocellular carcinoma cells in vitro.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Humulus/química , Extratos Vegetais/farmacologia , Propiofenonas/farmacologia , Análise de Variância , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Fragmentação do DNA , Flavanonas/farmacologia , Flavonoides , Humanos , Camundongos , Fitoterapia , Silibina , Silimarina/farmacologiaRESUMO
Adenoviruses are the most commonly used vectors in clinical trials for gene therapy. How to efficiently produce abundant and high-quality adenoviral vectors for therapeutic research is a challenge for biochemical engineers. A recombinant adenovirus carrying a green fluorescent protein (GFP) gene together with an anchorage-dependent 293 cell line is used as a model system for evaluating the effects of chemicals on adenoviral production in this study. Our aim is to develop a formulation to be added to an infection medium that could enhance the in vitro production of adenoviral vectors. Eleven ingredients obtained from a literature survey were screened for their stimulatory effects on adenoviral production using the 50% tissue culture infectious dose (TCID(50)) method. Among these ingredients, sucrose and mannitol when supplemented to the infection medium significantly increased adenovirus titer. Central composite design and response surface methodology were also adopted to determine the optimal concentrations of sucrose and mannitol. The formulation developed, which is composed of DMEM/F12 medium plus 0.54 M sucrose and 0.37 M mannitol, can significantly increase adenoviral production by 13-fold that of the control (DMEM/F12 medium).
Assuntos
Adenoviridae/efeitos dos fármacos , Adenoviridae/crescimento & desenvolvimento , Rim/efeitos dos fármacos , Rim/virologia , Manitol/administração & dosagem , Sacarose/administração & dosagem , Cultura de Vírus/métodos , Adenoviridae/isolamento & purificação , Algoritmos , Linhagem Celular , Técnicas de Química Combinatória/métodos , Simulação por Computador , Relação Dose-Resposta a Droga , Modelos Biológicos , Controle de QualidadeRESUMO
There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3-MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.
Assuntos
Metaloproteinases da Matriz/metabolismo , Análise Serial de Proteínas , Inibidores Teciduais de Metaloproteinases/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/isolamento & purificação , Inibidores Teciduais de Metaloproteinases/química , Inibidores Teciduais de Metaloproteinases/isolamento & purificaçãoRESUMO
We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1beta, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3alpha, osteoprotegerin, PARC, PIGF, TGF-beta2, TGF-beta3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-beta1, TNF-alpha, and TNF-beta were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1beta was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1beta promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1beta stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1beta in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.