KR20170091891A - Composition for promoting the differentiation of neural cells comprising silver nanoparticle and retinoic acid - Google Patents
Composition for promoting the differentiation of neural cells comprising silver nanoparticle and retinoic acid Download PDFInfo
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Abstract
Description
The present invention relates to a composition for promoting differentiation of neurons comprising silver nanomaterials and retinoic acid, and more particularly, to a culture medium composition for promoting differentiation into neurons in a neural stem cell line by treating silver nanomaterials and retinoic acid, To a method for promoting differentiation into neural cells.
Silver is known as a natural antibiotic that is harmless to the human body and has excellent sterilizing effect and is not resistant. Specifically, silver has excellent antimicrobial ability to kill more than 650 kinds of pathogens, and is a perfect natural antibiotic material. It has no side effects and sterilizes all harmful bacteria and absorbs harmful wavelengths such as electromagnetic waves and snakes. . Particularly, silver nanoparticles of nanometer size have a large surface area per unit mass of the particles and can easily penetrate the skin.
Silver nanoparticles have a silver ion release and a slow oxidizing ability. They bind to the bacterial cell membrane and induce penetration into bacteria, and have a strong antibacterial ability to kill bacterial cells as well as damage. Silver ions Silver plays an important role in the biological mechanism of nanomaterials. Silver nanomaterials have been used for 120 years in medical and health fields such as antibacterial, antiviral, antifungal and anti-fungal cystitis suppression, antiinflammatory agents, angiogenesis and wound healing. With these effects, detergents, antibacterial sprays, Nanomaterials are included in the back. Silver nanomaterials are widely used medically, but high concentrations of silver nanomaterials are known to be detrimental to health.
The process of neural differentiation consists of the growth of cells, the elongation of cells, and the formation of neurites, which are connected with the surrounding neurons to form a neural network. In this process, the cells undergo neuronal differentiation, And mature neurogenesis is induced by biochemical changes such as an increase in the expression of the mature neurons. Therefore, neural differentiation and neurite growth are essential for nerve injury recovery.
Recent studies have shown that silver nanomaterials stimulate bone differentiation in stem cells and have been applied to promote neurite growth by coating nanomaterials on glass. Recently, studies have been conducted to confirm the biochemical ability of silver nanomaterials in neuronal differentiation. However, the mechanism of action of silver nanomaterials has not been clarified so far. Specifically, detailed mechanism of silver nanoparticle neurogenesis has been controversial .
Therefore, the inventors of the present invention induced neurogenesis using nanomaterials and retinoic acid, and found that when treated with retinoic acid after pretreatment of silver nanomaterials rather than silver nanomaterial or retinoic acid alone, .
The present invention aims to provide a composition for promoting differentiation of neurons including a silver nanomaterial and retinoic acid.
The present invention also relates to a silver nanomaterial pretreatment step; And a method for promoting differentiation of neurons including a step of treating retinoic acid.
In order to achieve the above object, the present invention provides a composition for promoting differentiation of neurons comprising silver nanomaterials and retinoic acid.
In one embodiment of the present invention, the silver nanomaterial may be contained at a concentration of 0.1 to 10 μM.
In one embodiment of the present invention, the retinoic acid may be contained at a concentration of 1 to 10 μM.
Also, in one embodiment of the present invention, the silver nanomaterial may have a size of 10 to 100 nm.
Further, in one embodiment of the present invention, the composition may be a medium composition.
In one embodiment of the present invention, the medium is at least one kind selected from the group consisting of antibiotics, growth factors, amino acids, inhibitors, fetal calf serum (FCS) and fetal bovine serum (FBS) . ≪ / RTI >
The present invention also relates to (a) a nanomaterial pretreatment step; And (b) a retinoic acid treatment step.
Also, in one embodiment of the present invention, the silver nanoparticle pretreatment of step (a) may be performed for 12 to 24 hours before the retinoic acid treatment of step (b).
The present invention exhibits an effect of promoting differentiation of neurons and can be usefully used for the treatment of neurodegenerative diseases.
1 is a schematic diagram showing an outline of an experimental procedure.
Figure 2 shows the effect of silver nanomaterials treatment on the production of reactive oxygen species (ROS), mitochondrial function, and cytotoxicity.
Figure 3 shows the effect of silver nanomaterials on neuronal differentiation, expression of DUSPs, and kinase phosphorylation.
FIG. 4 shows the effect of N-acetylcysteine (NAC), which is an active oxygen scavenger, on the production of active oxygen induced by nanomaterials or neuronal differentiation.
Figure 5 shows the effect of silver nanomaterial pretreatment on neuronal differentiation efficiency.
Hereinafter, a composition for promoting neuronal differentiation promotion comprising a silver nanomaterial and a retinoic acid according to an embodiment of the present invention, and (a) a nanomaterial pretreatment step; And (b) a step of treating retinoic acid, will be described in detail.
Hereinafter, terms used in the present invention are defined.
The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the present invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. As used herein, the term " and / or " includes each and every combination of one or more of the components mentioned.
The terms " comprises, " and / or " comprising ", as used herein, are intended to encompass the components, steps, operations and / Or < / RTI >
As used herein, the term " neural cell " means neurons and / or glias of the central nervous system or peripheral nervous system, such as astrocytes, glial cells and / or schwann cells, Differentiation into cells was confirmed using morphological and molecular biological features.
As used herein, the term "pre-treat" refers to a pretreatment by chemical action prior to a basic reaction or processing. For example, the present invention means that the silver nanomaterial is preliminarily treated and then the retinoic acid is post-treated.
The pretreatment of the silver nanomaterial according to the present invention can be performed 12 to 24 hours before the treatment with retinoic acid, preferably 18 to 24 hours before treatment with the retinoic acid.
The composition according to the present invention may contain silver nanomaterials at a concentration of 0.1 to 10 μM, preferably 0.1 to 0.4 μM, more preferably 0.1 to 0.2 μM.
The composition according to the present invention may contain retinoic acid at a concentration of 1 to 10 μM, preferably at a concentration of 1 to 5 μM.
The silver nanomaterial of the present invention may be synthesized biologically, and the size of the silver nanomaterial may be 10-100 nm, preferably 20-30 nm.
In the culture according to the present invention, DMEM (Dulbecco's Modified Eagle's Medium), DMEM / F-12, F-12, McCoy's 5A, RPMI1640, Williams' medium E or IMDM Dulbecco's Medium medium) may be used, and serum-free DMEM / F-12 is preferred in the present invention.
The medium may be supplemented with antibiotics, growth factors, amino acids, inhibitors or the like, as well as fetal calf serum (FCS) or fetal bovine serum (FBS). In the present invention, , Albumin, hydrocortisone, and insulin.
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.
[ Example 1] Materials and methods
1. Silver Nanomaterial Synthesis and cell culture
Silver nanomaterials were biologically synthesized and silver nanomaterials were characterized via WPA Biowave II UV-vis spectroscopy (Biochrom). The size and shape of the silver nanoparticles were confirmed by transmission electron microscopy (JEM-1200EX, EM Lab Services), and the particle size in the aqueous solution was analyzed by a nanoparticle analyzer using a Zetasizer Nano ZS90 (Malvern Instruments).
SH-SY5Y cells, a human neural stem cell host, were cultured in DMEM (Hyclone) containing a high amount of glucose, supplemented with 10% fetal bovine serum (FBS, Hyclone), 50 u / mL penicillin, And streptomycin (Invitrogen) at 50 μg / mL were added to the culture medium and cultured in an incubator at 37 ° C and 5% carbon dioxide environment.
2. Identification and differentiation of cytotoxicity
Was measured using EZ-Cytox (Daeil Lab Service), which is a cell activity confirmation kit, in order to confirm the cell activity after treatment with nanomaterials and retinoic acid. SH-SY5Y cells were cultured in a 96-well plate in an incubator at 1x10 4 cells / well, 0.1 mu M of silver nanomaterial or 1 mu M of retinoic acid. After 24 hours, 48 hours, and 72 hours of incubation for each sample, the medium was changed to a culture medium containing 10% EZ-Cytox capable of measuring cytotoxicity and cultured in a dark environment incubator for 3 hours. The optical density was measured at a wavelength of 480 nm using an x-MarkTM spectrophotometer (Bio-RAD). Cell viability was assessed as compared to treatment with silver nanomaterials or retinoic acid, with arbitrarily set to 100% without any treatment.
3. Immunocyte staining
SH-SY5Y cells were cultured in a 35 mm High IbiTreat dish (ibidi GmbH) at a density of 4? 104 cells / dish per day in an incubator, treated with nanomaterials or retinoic acid for 1 week, and then immunocytochemically stained. Conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Labs) was added to the culture supernatant after incubation with a specific primary antibody after preventing the binding of non-specific antibodies with normal goat serum (10% Cells were further treated by treatment. Nuclear staining was carried out using TO-PRO-3 (Molecular Probes), and then photographs were taken using a confocal laser microscope (Leica Microsystems).
4. Western Blot
SH-SY5Y cells were treated with nanomaterials and retinoic acid, and the soluble proteins were centrifuged. Only the supernatant was recovered and the protein was quantified using Bradford Protein Assay Reagent (Bio-Rad) Respectively. The same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes.
In this experiment, the primary antibodies were p-AKT, AKT, p-ERK1 / 2, and β-Actin (Santa Cruz) and ERK1 / 2 (Cell Signaling) After using HRP or anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology), protein signals were confirmed using ECL kit (enhanced chemiluminescence kit, Amersham Bioscience).
5. Silver Nanomaterial Pretreatment and co-treatment
In order to compare the differentiation efficiency of human neural stem cell lines with silver nanomaterials and retinoic acid, the pre-treatment of silver nanomaterials and the co-treatment of silver nanomaterials with retinoic acid, Method. In the pretreatment method, the silver nanomaterials were treated for one day, the next day, nanomaterials were removed, and the new culture medium was supplemented with retinoic acid for 4 days. In the co-treatment method, silver nanomaterials and retinoic acid It was done by way of processing. In order to confirm the differentiation efficiency of the two methods, immunocyte staining and gene expression quantification were performed by PCR.
[ Example 2] Nanomaterial Intracellular reactive oxygen ( ROS ) Production and cytotoxicity
In the present invention, experiments were conducted using silver nanomaterials having an average size of about 30 nm through biologically synthesized transmission electron microscopy and nanoparticle analyzer. First, the extent of active oxygen production in the cells was analyzed by treatment with silver nanomaterials or retinoic acid, and the intensity of reactive oxygen signals was confirmed by fluorescence photographs. The results are shown in FIG.
FIG. 2 shows that the production of reactive oxygen species increases more in the cells treated with silver nanomaterials than in the cells treated with retinoic acid. Particularly, in the result after 3 hours after exposure to silver nanomaterials, It was confirmed that the production of oxygen was increased more.
In addition, the effect of silver nanomaterials and retinoic acid on the activity of SH-SY5Y cells at 24, 48, and 72 hours was confirmed, and the results are shown in FIG.
2, silver nanomaterials showed some cytotoxicity, but 0.1 μM showed lower cytotoxicity than nanomaterials when treated with 1 μM retinoic acid.
[ Example 3] Nanomaterial Effects on Neuronal Differentiation and Kinase Signals
In order to examine the effect of nanomaterials on neuron differentiation, neuronal stem cell lines were exposed to nanomaterials, and then the appearance of the nanomaterials was observed and Western blotting was performed. The results are shown in FIG.
3, when the neural stem cell line was exposed to the silver nanomaterial for 5 days, the SH-SY5Y cells treated with the silver nanomaterial or retinoic acid exhibited similar morphological changes such as the growth of the neurite, Β-tubulin III expression was higher than that of β-tubulin III. The expression of MAP-2, β-tubulin III, synaptophysin, Gap-43, and neurogenin-1 in neurons was significantly increased in SH-SY5Y cells treated with silver nanomaterials, Lt; RTI ID = 0.0 > SY5Y < / RTI > cells.
In addition, we compared the expression levels of phosphorylated AKT and ERK with the seven genes coding for DUSP1, 2, 3, 4, 6, 7, and 9 DUSPs after neuronal differentiation with silver nanomaterials or retinoic acid. The results are shown in Fig. 3, expression of DUSP genes was decreased in cells treated with silver nanomaterials but increased in cells treated with retinoic acid. The expression of phosphorylated ERK and AKT is increased in both SH-SY5Y cells treated with silver nanomaterials and retinoic acid. However, in the cells treated with silver nanomaterials, the levels of ERK phosphorylation and low AKT phosphorylation were higher than those of cells treated with retinoic acid.
[ Example 4] Nanomaterial Removal of reactive oxygen species in neuronal differentiation
(N-acetylcysteine, N-acetylcysteine, N-acetylcysteine, N-acetylcysteine, N-acetylcysteine, and N-acetylcysteine) -acetylcysteine (NAC) was pretreated to study the role of silver nanomaterials induced by neurogenesis in SH-SY5Y cells. When 0.1 mM silver nanoparticles were treated, the amount of active oxygen was significantly increased, but the production of silver nanocrystal - induced reactive oxygen species was significantly inhibited in the group pretreated with NAC compared with the group not treated with NAC.
As a result of immunocytochemical staining, the pretreatment of NAC decreased the growth of neurites induced by silver nanomaterials and the expression of β-tubulin III, which is a neural differentiation marker, but treated with retinoic acid At the same time, the production of reactive oxygen species increased only slightly, and NAC pretreatment did not change retinoic acid induced neurogenesis. The growth of silver nanoparticle-induced neurites blocked by NAC and the expression of β-tubulin III are different depending on the amount of NAC, and when 10 mM NAC is pretreated, the differentiation of neurons by silver nanomaterials It was confirmed that it was completely blocked. However, in SH-SY5Y cells exposed to retinoic acid, the pretreatment effect of NAC was very small.
[ Example 5] Nanomaterial Effects of Pretreatment on Neurite Outgrowth and Neuronal Differentiation
In the present invention, silver nanomaterials and retinoic acid were combined for pretreatment or co-treatment in order to find a way to more effectively increase the degree of neuronal differentiation induced by silver nanomaterials or retinoic acid. The results are shown in FIG. 5 . In the pretreatment method, the silver nanomaterials were treated for one day, the next day, nanomaterials were removed, and the new culture medium was supplemented with retinoic acid for 4 days. In the co-treatment method, silver nanomaterials and retinoic acid It was done by way of processing.
5, when neurogenesis was induced by a method of treating nanomaterials one day before treatment with retinoic acid, the length of the neurites increased significantly compared with the treatment with only the retinoic acid, and immunocytochemistry and neurogenic markers MAP-2 and β-tubulin III, respectively. On the other hand, when the silver nanomaterials and retinoic acid were co-treated in combination, it was not confirmed that the growth of the neurite or the expression of neurogenic markers was highly increased.
From the above results, it can be seen that a composition comprising silver nanomaterial and retinoic acid, and (a) silver nanomaterial processing step; And (b) retinoic acid treatment step. In particular, the efficiency of differentiation of neurons in the case of co-treatment of silver nanomaterials and retinoic acid was increased, but the efficiency of differentiation of neurons in the case of pretreatment of silver nanomaterials and posttreatment of retinoic acid was remarkably superior I could confirm that
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| CN115094039A (en) * | 2022-07-01 | 2022-09-23 | 济南大学 | Retinoic acid-calcium nano sustained release agent and application thereof in promoting stem cells to differentiate towards neurons |
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| CN115094039B (en) * | 2022-07-01 | 2023-11-21 | 济南大学 | Retinoic acid-calcium nanometer sustained release agent and application thereof in promoting differentiation of stem cells to neurons |
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