Application of gold nanoparticles in preparation of products for inducing stem cell differentiation

Abstract

The invention discloses application of gold nanoparticles in preparation of products for inducing stem cell differentiation, and belongs to the technical field of biological medicine. The gold nanoparticles are gold nanoparticles with a layer structure; mixing the gold nanoparticles and the stem cells, standing, placing the stem cells under a rotating magnetic field after the stem cells swallow the gold nanoparticles, and performing intracellular electrical stimulation on the stem cells to realize differentiation of the stem cells to neurons. According to the present invention, the stem cells can phagocytize the multi-shell gold nanoparticles by using the ability of the stem cells to swallow the gold nanoparticles, and the magnetic field is applied to perform electromagnetic induction, such that the gold nanoparticles generate the induced current so as to stimulate the stem cells to differentiate to neurons so as to achieve the intracellular electrical stimulation of the stem cells. The multi-shell gold nanoparticles can control intracellular electrical stimulation of cells, and cannot generate abnormally high-level active oxygen, and the generated active oxygen meets the amount required for promoting stem cell differentiation. The invention provides a new thought for intracellular electrical stimulation of the stem cells to differentiate into neurons.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of gold nanoparticles in the preparation of products for inducing stem cell differentiation. Background Art

[0002] Spinal cord injury (SCI) is a serious and disabling neurological disease that causes permanent and degenerative damage to the central nervous system (CNS). Symptoms include mobility impairment, pain, and autonomic dysfunction, which imposes a heavy economic burden on patients, families, and society, and is the most challenging global medical problem. The rupture of neuronal axons and the death of neurons after SCI lead to dysfunction of neural circuits, resulting in loss of sensory and motor control. Therefore, promoting axon regeneration, myelinating axons, and rebuilding neural circuits are the focus of treatment. Although researchers have developed a variety of new treatments in recent years, there is no effective therapy that can cure or reverse spinal cord injury. Among them, cell therapy and stem cell therapy are considered to be promising candidate therapies for promoting functional recovery. Stem cell therapy mainly relies on self-renewal and differentiation capabilities, among which neural stem cells stand out among many types of stem cells. However, during the differentiation process in vivo after neural stem cell transplantation, they also tend to differentiate into more astrocytes rather than neurons, causing gliosis and further hindering axon regeneration.

[0003] Studies have shown that the electrical activity of cells can play a vital role in the proliferation, migration and differentiation of early nerves by activating cell membrane receptors, ion transport channels or intracellular communication. In addition, in vivo electrical stimulation delivered by external power supplies and percutaneous wires has been shown to significantly promote the regeneration of peripheral nerves or the recovery of other diseases. Traditional electrical stimulation involves implanting power supplies and wires connected to external power supply devices, which may cause additional risks of injury, inflammation and infection. Moreover, it is difficult to accurately apply electrical stimulation to individual cells to control and promote the differentiation direction and speed of neural stem cells. Therefore, in order to avoid the use of external wires in tissues, it is necessary to achieve wireless stimulation. Wireless stimulation can be divided into three types: piezoelectric stimulation, photoelectric stimulation, and electromagnetic induction stimulation. However, the penetration depth of light in biological tissues in photoelectric stimulation is limited, and the sharp attenuation of ultrasound waves in piezoelectric stimulation at the interface between different tissues or components limits its application in the treatment of spinal cord injury. In contrast, the interaction between magnetic fields and biomolecules is weak and can penetrate deep into the human body without attenuation. For this reason, truly non-invasive and remote in situ electrical stimulation can be achieved to regulate the differentiation direction of neural stem cells. At the same time, studies have shown that it has the potential to promote axon regeneration and nerve function recovery in SCI repair. However, the effect of extracellular electrical stimulation will be weakened by interference from external factors such as culture medium resistance and membrane capacitance. Intracellular electrical signals more effectively improve the neural differentiation effect of stem cells, and accelerate the electrophysiological function maturation of differentiated neurons by increasing the activity of voltage-dependent ion channels. However, giving cells intracellular electrical stimulation will inevitably cause cells to produce reactive oxygen species. The production of abnormally high levels of reactive oxygen species will cause neurotoxicity and dysfunction in neural stem cells. However, ROS produced during normal aerobic metabolism can also promote the differentiation of neural stem cells as a molecular regulator. Therefore, how to give cells intracellular electrical stimulation and control the level of ROS so that it is within the normal range to promote cell differentiation and differentiation into neurons is a problem that needs to be solved. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of gold nanoparticles in the preparation of products for inducing stem cell differentiation. The present invention utilizes the ability of stem cells to phagocytose gold nanoparticles (MS-Au NPs), so that stem cells can phagocytize multi-shell gold nanoparticles, and when a magnetic field is applied for electromagnetic induction, the gold nanoparticles generate an induced current to stimulate the stem cells to differentiate into neurons, thereby achieving intracellular electrical stimulation of stem cells. The multi-shell gold nanoparticles used in the present invention can control the intracellular electrical stimulation of cells, and will not produce abnormally high levels of reactive oxygen species. The reactive oxygen species produced meet the amount required to promote stem cell differentiation. The present invention provides a new idea for intracellular electrical stimulation of stem cells to differentiate into neurons.

[0005] To achieve the above object, the present invention adopts the following technical solution: The present invention provides an application of gold nanoparticles in preparing a product for inducing stem cell differentiation. The gold nanoparticles are gold nanoparticles with a layered structure. The gold nanoparticles and stem cells are mixed and allowed to stand, and after the stem cells engulf the gold nanoparticles, the stem cells are placed in a rotating magnetic field to perform intracellular electrical stimulation on the stem cells, thereby achieving differentiation of the stem cells into neurons.

[0006] Preferably, the gold nanoparticles with a layer structure are multi-shell gold nanoparticles; the multi-shell gold nanoparticles are prepared by the following method: (1) Usually, distilled water and melamine are mixed and heated and stirred, and HAuCl is added 4 •4H 2 O solution and stirred until the transparent solution turned yellow, centrifuged to obtain a yellow product, washed and dried to obtain colloidal spheres; (2) Disperse the colloidal spheres into the mixed solution, add sodium poly(p-styrene sulfonate), and finally quickly add NaBH 4 The aqueous solution is stirred, centrifuged, washed and dried to obtain multi-shell gold nanoparticles.

[0007] Preferably, in step (1), the distilled water, melamine and HAuCl 4 •4H 2 The ratio of the added amount of O solution was 100 mL:50 mg:6 mL; the HAuCl 4 •4H 2 The concentration of the O solution is 0.024 M; the heating temperature is 70°C.

[0008] Preferably, in step (2), the mixed solution is obtained by mixing ethylene glycol and water in a volume ratio of 10:3; the colloidal spheres, the mixed solution, sodium poly(p-styrene sulfonate) and NaBH 4 The ratio of the amount of aqueous solution added is 10 mg:78 mL:20 mg:2 mL; the NaBH 4 The concentration of the aqueous solution was 25 mg / mL.

[0009] Preferably, the gold nanoparticles and stem cells are mixed by adding the gold nanoparticles to a differentiation medium to obtain a gold nanosolution, adding the stem cells to a proliferation medium, and then adding them to a culture dish, and after the stem cells adhere to the wall, removing the proliferation medium and adding the gold nanosolution.

[0010] Preferably, the concentration of the gold nanoparticle solution is 20-80 μg / mL.

[0011] Preferably, the concentration of the gold nanoparticle solution is 40 μg / mL.

[0012] Preferably, the rotation speed of the rotating magnetic field is 400-600 rpm; the magnetic field strength of the rotating magnetic field is 0.1-1 Tesla; the induced electromotive force generated by the gold nanoparticles induced by the rotating magnetic field is 0.1-10 mV.

[0013] Preferably, the rotation speed of the rotating magnetic field is 500 rpm; the induced electromotive force generated by the rotating magnetic field-induced gold nanoparticles is 2-4 mV.

[0014] Preferably, the standing time is ≥4h.

[0015] Preferably, the stem cells are neural stem cells; and the differentiation of the stem cells into neurons is differentiation of stem cells into dopaminergic neurons and acetylcholinergic neurons.

[0016] Preferably, the application includes accelerating the neural differentiation of stem cells and increasing the proportion of stem cells differentiating into neurons.

[0017] Preferably, the induced stem cell differentiation products include products that induce stem cells to differentiate into neurons, spinal cord injury repair drugs, and neurodegenerative disease repair drugs.

[0018] Beneficial effects of the present invention: (1) The present invention utilizes the ability of stem cells to phagocytose gold nanoparticles, allowing stem cells to phagocytize multi-shell gold nanoparticles. When a magnetic field is applied for electromagnetic induction, the gold nanoparticles generate an induced current, which stimulates the stem cells to differentiate into neurons, thereby achieving intracellular electrical stimulation of stem cells. The multi-shell gold nanoparticles used in the present invention can control intracellular electrical stimulation of cells and will not produce abnormally high levels of reactive oxygen species. The reactive oxygen species produced meet the amount required to promote stem cell differentiation. The present invention provides a new idea for intracellular electrical stimulation of stem cells to differentiate into neurons.

[0019] (2) When treating spinal cord injury, the present invention utilizes electromagnetic induction to generate induced current, thereby avoiding the adverse effects such as secondary injury or infection caused by the use of external wires, and promoting functional recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :(a)SEM image of MS-Au NPs, scale bar is 1 μm;(b)SEM image of MS-Au NPs, scale bar is 500 nm;(c)TEM of MS-Au NPs;(d)HRTEM image of MS-Au NPs;(e)EDS image of MS-Au NPs;(f)Distribution of Au in MS-Au NPs;(g)XRD image of MS-Au NPs;(h)Voltage of MS-Au NPs at different rotation speeds; Figure 2:(a)TEM image of NSCs after incubation with MS-Au NPs for 24 h;(b)Representative fluorescence image of NSCs after incubation with 40 μg / mL MS-Au NPs for 24 h;(c)ROS fluorescence images of AuNPs and MS-AuNPs with different concentrations under rotating magnetic field conditions;(d)Live / dead staining experiment of NSCs after culture with different concentrations of MS-Au NPs for 2 days, live cells and dead cells were stained green and red, respectively;(e)ImageJ analysis of the average fluorescence intensity of the live / dead staining experiment;(f)The viability of NSCs after culture with different concentrations of MS-Au NPs for 1, 2 and 3 days; Figure 3 :(a) Immunofluorescence staining images of Tuj1 and GFAP expression in NSCs after 5 days of treatment in each group;(b) Immunofluorescence staining images of MAP2 and GFAP expression in NSCs after 7 days of treatment in each group, red fluorescence represents Tuj1 and MAP2 expression, green fluorescence represents GFAP expression, and blue fluorescence represents DAPI-stained cell nuclei;(c) Quantitative analysis of the average fluorescence intensity of Tuj1 at 5 days;(d) Quantitative analysis of the average fluorescence intensity of MAP2 at 7 days;(e) Effects of different magnetic field rotation speeds on Tuj1 and MAP2 mRNA expression; Figure 4 :(a) Immunofluorescence staining images of Tuj1 and GFAP expression in NSCs after 7 days of treatment in each group;(b) Immunofluorescence staining images of MAP2 and GFAP expression in NSCs after 10 days of treatment in each group, red fluorescence represents Tuj1 and MAP2 expression, green fluorescence represents GFAP expression, and blue fluorescence represents DAPI-stained cell nuclei;(c) Quantitative analysis of the average fluorescence intensity of Tuj1 at 7 days;(d) Quantitative analysis of the average fluorescence intensity of MAP2 at 10 days;(e) The percentage of Tuj1-positive cells according to the results of immunofluorescence staining;(f) The percentage of MAP2-positive cells according to the results of immunofluorescence staining;(g) Representative images of two-dimensional neurons reconstructed according to the expression of MAP2 protein in neurons;(h) Sholl analysis was performed to quantify the complexity of neuronal processes by calculating the intersection points between neurites and concentric circles centered on the cell body and with a specific radius; Figure 5:(a) RT-qPCR analysis results of NSCs-related marker mRNA after 5 days of treatment in each group;(b) RT-qPCR analysis results of NSCs-related marker mRNA after 7 days of treatment in each group;(c) WB bands of NSCs-related marker proteins after 7 days of treatment in each group;(d) Relative quantitative analysis of Tuj1, GAP43 and GFAP protein bands;(e) WB bands of NSCs-related marker proteins after 7 days of treatment in each group;(f) Relative quantitative analysis of MAP2, PSD95, NeuN and SYP protein bands;(g) WB bands of neuron marker proteins related to 5-day Ctrl, 40 μg / mL-RMF and 10-day Ctrl;(h) Relative quantitative analysis of MAP2, Tuj1, SYP and GAP43 protein bands; Figure 6 :(a)WB bands of multiple functional neuron-specific marker proteins in NSCs cultured with 40 μg / mL-RMF for 7 days;(b)Relative quantitative analysis of TH, CHAT, GABA and GAD65 protein bands;(c)Fluorescence image of NSCs treated with 40 μg / mL-RMF after stimulation with dopamine neurotransmitter;(d)Analysis of representative cell average fluorescence intensity of NSCs treated with 40 μg / mL-RMF after stimulation with dopamine neurotransmitter;(e)Fluorescence image of NSCs treated with 40 μg / mL-RMF after stimulation with acetylcholine neurotransmitter;(f)Analysis of representative cell average fluorescence intensity of NSCs treated with 40 μg / mL-RMF after stimulation with acetylcholine neurotransmitter; Figure 7 :(a) The BMS score on the grid was used to evaluate the motor recovery of the right hind limb of mice;(b) The reaction time of mice in each group to the 55℃ hot plate. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0022] As introduced in the background technology section, intracellular electrical stimulation will inevitably cause cells to produce reactive oxygen species, and abnormally high levels of reactive oxygen species will cause neurotoxicity and dysfunction in neural stem cells. However, ROS produced during normal aerobic metabolism can act as a molecular regulator to promote the differentiation of neural stem cells.

[0023] Based on this, the purpose of the present invention is to provide the use of gold nanoparticles in the preparation of induced stem cell differentiation products. The multi-shell gold nanoparticles MS-Au NPs of the present invention are prepared according to the method disclosed in "Robust synthesis of Au-based multishell structures as plasmonic catalysts for selective hydrogenation of 4-nitrostyrene" (Shuyan Song, Jian Li, Angewandte Chemie International Edition, 10.1002 / anie.201910836). The study uses multi-shell gold nanoparticles as catalysts to convert nitrobenzene to aminobenzene under light. The present invention uses multi-shell gold nanoparticles as a medium for the first time. After being internalized by neural stem cells, an induced current is generated under a rotating magnetic field. After the induced current is generated in the cell, the intracellular ROS level will increase, causing cell death. The multi-shell gold nanoparticles MS-Au NPs can not only generate an induced current but also reduce the intracellular ROS level, controlling it within a reasonable range to ensure cell survival. The present invention has the dual effects of adjusting the intensity and frequency of electrical signals and regulating the level of ROS by changing the rotation speed of the magnetic field, thereby promoting the differentiation of neural stem cells and the repair of spinal cord injury. In vitro experiments have shown that magnetic field-driven MS-Au NPs can promote the differentiation of NSCs into functional neurons that can respond to neurotransmitters and transmit nerve impulses. In in vivo experiments, transplantation of NSCs combined with MS-Au NPs treatment can improve the neurological function of rats with spinal cord injury. Under the action of a periodic magnetic field, they can effectively differentiate into new functional neurons and promote the repair of damaged tissues, thereby significantly improving the behavioral defects of SCI mice. The present invention may provide new ideas for the treatment of neural injury, and is particularly suitable for the treatment of diseases characterized by oxidative stress, such as spinal cord injury and neurodegenerative diseases.

[0024] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0025] Note: The electromagnetic induction induction device used in the example is prepared with reference to the electromagnetic induction induction device in the patent application number CN 114561357 A "Method for accelerating the differentiation of neural stem cells into neurons based on radio signals generated by electromagnetic induction".

[0026] Rhodamine dye was purchased from ThemorFisher; catalog number: L1392; TRIzol™Plus RNA Purification Kit was purchased from ThemorFisher; catalog number: 12183555; The primary antibody goat anti-mouse Tuj1 was purchased from abcam; catalog number: ab18207; The primary antibody goat anti-rabbit GFAP monoclonal antibody was purchased from Proteintech; catalog number: 16825-1-AP; The primary antibody sheep anti-mouse MAP2 monoclonal antibody was purchased from abcam; catalog number: ab5392; Secondary antibody goat anti-rabbit Alexa Fluor488 was purchased from Proteintech; catalog number: RGAR002; Goat anti-mouse Alexa Fluor594 was purchased from Proteintech; catalog number: RGAM004; Primary antibodies against CHAT, TH, GABA, or GAD65, with catalog numbers: 20747-1-AP, 25859-1-AP, 12410-1-AP, 20746-1-AP, were purchased from Proteintech; The product number of goat anti-mouse IgG / horseradish enzyme-labeled secondary antibody is ZB-2305, and the product number of goat anti-rabbit IgG / horseradish enzyme-labeled secondary antibody is ZB-2301; both were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0027] BCA (Bicinchoninic Acid) protein quantification kit was purchased from Shandong Sikoje Biotechnology Co., Ltd.; catalog number: EC0001-A.

[0028] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1: Preparation of MS-Au NPs (1) Stir 100 mL of distilled water and 50 mg of melamine at 70 °C, add 6 mL of 0.024 M HAuCl 4 •4H 2 O solution, stirred (12h) until the transparent solution turned yellow. Centrifuged to obtain a yellow product, washed with ethanol, and dried at 25 °C to obtain colloidal spheres with a particle size of 400nm.

[0030] (2) Add 50 mg NaBH 4 Add 2 mL of water to obtain NaBH 4Aqueous solution. Disperse 10 mg of colloidal spheres (400 nm) into a mixed solution at 25°C (containing 60 mL of ethylene glycol and 18 mL of water); then add 20 mg of sodium poly(p-styrene sulfonate) (PSS); finally quickly add NaBH 4 The aqueous solution was stirred carefully for 12 h. After centrifugation, the product was washed with ethanol and dried to obtain MS-Au NPs.

[0031] Example 2: Inducing stem cells to differentiate into neurons The composition of the proliferation medium is: 2% B27 supplement + 1% streptomycin penicillin + 1% glutamine + 10ul 100 μg / mL basic fibroblast growth factor + 10μL of 100μg / mL epidermal growth factor + 96% neurobasal basal medium; The composition of the differentiation medium is: 2% B27 supplement + 1% streptomycin penicillin + 1% glutamine + 1% FBS + 95% neurobasal basal medium.

[0032] The MS-Au NPs prepared in Example 1 were added to the differentiation medium to obtain an MS-Au NPs solution. Neural stem cells (NSCs) were added to the proliferation medium and then poured into a culture dish. After the cells adhered to the wall, the proliferation medium was discarded and the MS-Au NPs solution was added and allowed to stand for 4 hours. Observation was performed using a transmission electron microscope. Figure 2 As shown in (a), MS-Au NPs entered the lysosomes after being internalized by NSCs, indicating that MS-Au NPs were phagocytosed by neural stem cells.

[0033] The culture dish was placed in an electromagnetic induction induction device to induce stem cells to differentiate into neurons. Specifically, the culture dish was placed directly below the permanent magnet, 0.8 cm away from the permanent magnet; the power of the rotating motor was turned on, and the rotating motor rotated to drive the rotating rod to rotate, and the rotating rod drove the permanent magnet to rotate. The electromagnetic induction generated by the permanent magnet caused the MS-Au NPs in the stem cells to generate an induced current, and under the stimulation of the induced current, the stem cells differentiated into neurons. The magnetic field strength of the permanent magnet was 0.3 Tesla, and the rotation speed of the permanent magnet was 500 rpm. After 15 minutes per day for 5 days, the increase in the expression of neuron-related markers (Tuj1, MAP2) could be detected.

[0034] Example 3: Characterization of MS-Au NPs like Figure 1 As shown in (a), the SEM image shows that the MS-Au NPs have a vesicle-like morphology, a rough surface, and an average diameter of about 250 nm. Figure 1As shown in (b), the TEM image shows that MS-Au NPs have a clear three-layer shell structure, and the high-resolution transmission electron microscopy (HRTEM) image shows that the lattice distance is 0.258 nm, corresponding to the (200) plane of metallic Au. Figure 1 The EDS image in (c) shows that these multi-shell layers are formed by the fusion of small Au nanoparticles, and gaps with a size of less than 10 nm are distributed throughout the shell. Figure 1 (d) The crystal structure of MS-Au NPs was analyzed using X-ray powder diffractometer (XRD), showing the diffraction peaks of Au corresponding to MS-Au NPs. The diffraction peak signal of Au (PDF#04-0784) is relatively obvious. The diffraction peaks detected at 38.2°, 44.4°, 64.6° and 77.5° correspond to the (111), (200), (220) and (311) crystal planes of Au. Figure 1 In (e), the induced electromotive force generated by MS-Au NPs under the rotating magnetic field of Example 2 was measured to be 2~4mV.

[0035] Experimental Example 1: Gold Nanoparticles Regulate ROS in Neural Stem Cells (1) Neural stem cells were obtained from c57bl / 6J 13-15 day pregnant mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The pregnant mice were euthanized (cervical dislocation). Under sterile conditions, surgical scissors were used to open the abdominal cavity of the mother mouse, the uterus was removed and placed in ice-cold PBS. Using a dissecting microscope, fine forceps were used to gently remove the head of the embryo, cut the skull, remove the intact brain tissue, and remove the meninges to ensure high purity. The brain tissue was removed and placed in 1 mL of proliferation medium (same as the proliferation medium in Example 2). The brain tissue was cut into small pieces of about 1 mm³ using dissecting scissors, and the tissue was aspirated with a sterile pipette until a uniform cell suspension was formed. The cell suspension was passed through a 40 µm cell strainer to remove tissue fragments. The cell density was adjusted to 2×10 5 cells / mL, inoculated cells in T-shaped culture flasks and placed at 37°C, saturated humidity, and 5% CO 2 Suspension culture was performed in an incubator, and cell growth was observed daily, especially changes in the size of neurospheres, and recorded and photographed under an inverted microscope. After about 5-7 days of primary culture, the diameter of the neurospheres was observed to be about 100-200 μm. At this time, the neurospheres were subcultured and NSCs cells were obtained by culture to the second generation. NSCs cells were prepared into a single cell suspension with a density of 1×10 5cells / mL. 1 mL of single cell suspension was added to each well, and the cells were seeded on the coverslip. Rhodamine dye working solution (1 mg of rhodamine powder dissolved in 1 mL of PBS) and 10 mg / mL of MS-Au NPs were used in advance to label MS-Au NPs at a ratio of 200:1, so that they were fluorescent red, and then dispersed in the differentiation medium (differentiation medium of Example 2) to make the MS-Au NPs concentration 40 μg / mL. After the NSCs cells adhered to the wall and spread, the original proliferation medium was removed, the differentiation medium containing MS-Au NPs was added, and the cells were placed in a cell culture incubator for 24 h. After 24 h, the differentiation medium was discarded, the cells were rinsed with PBS three times (5 min / time), Lyso-TrackerGreen staining working solution (1:1000 diluted stock solution) was added, and the cells were incubated in a cell culture incubator for 30 min, the Lyso-Tracker Green staining working solution was removed, and fresh differentiation medium was added. Then, laser confocal microscopy was used to observe, and the lysosomes were observed to be brightly stained with strong fluorescence, while the labeled MS-Au NPs appeared red, and fluorescence images were taken. Figure 2 As shown in (b), the co-staining phenomenon shows that MS-Au NPs and lysosomes co-localize (yellow part), proving that MS-Au NPs enter the lysosomes, indicating that MS-Au NPs have been phagocytosed by NSCs cells.

[0036] (2) Experimental groups: Au NPs group and MS-Au NPs group prepared in Example 1. The Au NPs group used solid gold nanoparticles with the same particle size as the MS-Au NPs used in the MS-Au NPs group; the concentrations of the Au NPs group were 0, 10, 20, 40, 80 and 100 μg / mL, of which 0 μg / mL was Ctrl; the concentrations of the MS-Au NPs group were 0, 10, 20, 40, 80 and 100 μg / mL, of which 0 μg / mL was Ctrl. Both groups were induced with neural stem cells according to the method of Example 2. The permanent magnet was energized and rotated for 15 minutes. At the same time, the gold nanoparticles in the neural stem cells generated an induced current. Under the stimulation of the induced current, a large amount of ROS was generated in the neural stem cells. DCFH-DA was diluted at 1:1000 to a final concentration of 10 μmol / L. All cell culture medium was removed and an appropriate volume of diluted DCFH-DA was added. The volume added should be sufficient to cover the cells, and the cells were incubated in a 37°C cell culture incubator for 20 min. The cells were washed three times with PBS to fully remove the DCFH-DA that did not enter the cells. Fluorescence images were taken in real time using a laser confocal microscope.

[0037] like Figure 2As shown in (c), only trace amounts of green fluorescence signals were detected in the cells of the Au NPs group. It is noteworthy that the Au NPs group exhibited a strong fluorescence signal, indicating that a large amount of ROS was generated. It was found that under the same concentration and magnetic field conditions, the ROS level of NSCs in the MS-Au NPs group was lower than that of NSCs in the Au NPs group, indicating that the ability of MS-Au NPs to scavenge ROS is significantly higher than that of Au NPs, thereby maintaining the activity of cells.

[0038] (3) Experimental groups: The MS-Au NPs group prepared in Example 1 was denoted as MS-Au NPs, and the MS-AuNPs + permanent magnet (RMF) group prepared in Example 1 was denoted as MS-Au NPs-RMF; the concentrations of the MS-Au NPs group were 0, 10, 20, 40, 80 and 100 μg / mL, of which 0 μg / mL was Ctrl; the concentrations of the MS-Au NPs-RMF group were the same as those of the MS-Au NPs group, denoted as 10 μg / mL -RMF, 20 μg / mL -RMF, 40 μg / mL -RMF, 80 μg / mL -RMF and 100 μg / mL-RMF, and 0 μg / mL-RMF was Ctrl-RMF. 20 μL of 10 μg / mL Laminin was added to the 96-well plate and incubated in a cell culture incubator for 4 h. Laminin was aspirated and the second generation NSCs cells were prepared into a single cell suspension with a density of 1×10 5 cells / mL, 200 μL of single cell suspension was added to each well, and the cells were inoculated into the well plate. 10 mg / mL MS-Au NPs were added to the differentiation medium to make the concentration of MS-Au NPs 0, 10, 20, 40, 80 and 100 μg / mL. After the neural stem cells adhered to the wall and spread, the original proliferation medium was removed, and the differentiation medium containing the concentration gradient of MS-Au NPs was added. 18 wells were set for each concentration gradient and placed in a cell culture incubator for more than 4 hours.

[0039] Nine wells of each concentration gradient in the MS-Au NPs-RMF group were placed 0.8 cm below the permanent magnet and a rotating magnetic field was applied for 15 min for 3 days; no magnetic field was applied to the MS-Au NPs group; both groups were replaced with fresh differentiation medium every two days. Three wells in each group were selected every day and 20 μL of cck-8 solution was added, and the cells were incubated in the cell culture incubator for another hour. The absorbance was measured at 450 nm using an ELISA reader to detect the number of live cells. Figure 2As shown in (f), cck-8 was used to detect the effect of different concentrations of MS-AuNPs on the proliferation of NSCs after 1, 2, and 3 days of culture. The number of NSCs increased over 3 days, which was the same as that of Ctrl, proving that the cells were in good condition and that the cells treated with MS-Au NPs and RMF had good viability and proliferation ability. It is worth noting that the number of live cells of NSCs in RMF was generally lower than that of the non-RMF group, indicating that the presence of RMF may promote the differentiation of NSCs, as it has been previously reported that cell proliferation is reduced before differentiation.

[0040] (4) The experimental grouping and neural stem cell treatment were the same as (3). Three wells of each concentration gradient of the MS-Au NPs-RMF group were placed 0.8 cm below the permanent magnet and a rotating magnetic field was applied for 15 min for two days; no magnetic field was applied to the MS-Au NPs group. After 48 h, the culture medium was removed, the cells were washed once with PBS, and 200 μL of Calcein AM / PI detection working solution was added to each well. Incubate at 37°C in the dark for 30 min. After incubation, the cells were washed 3 times with PBS, and the live / dead staining results of the cells were observed using a laser confocal microscope.

[0041] like Figure 2 As shown in (d), the survival rate of NSCs under the conditions of magnetic field and MS-Au NPs concentration gradient is greater than 95%. At the same time, NSCs differentiate into various neural cells due to adhesion and have protrusions, and no longer maintain a round shape. Figure 2 As shown in (e), there was no significant difference in fluorescence intensity after ImageJ analysis, indicating that MS-Au NPs have good biocompatibility.

[0042] The above experiments indicate that MS-Au NPs have good biocompatibility and may promote the differentiation of NSCs.

[0043] Experimental Example 2: Gold nanoparticles promote differentiation of neural stem cells into neurons in vitro Then, we conducted a preliminary exploration on whether MS-Au NPs could promote the differentiation of NSCs through ICs produced in RMF. We used cell immunofluorescence protein staining experiments to verify the expression of neural-specific markers, including the analysis of βⅢ tubulin (Tuj1), glial fibrillary acidic protein (GFAP), and microtubule-associated protein 2 (MAP2). We conducted a preliminary study on the effects of MS-Au NPs and RMF in promoting the differentiation of NSCs and attempted to find the most suitable concentration of MS-Au NPs for NSCs differentiation.

[0044] (1) Experimental grouping and neural stem cell treatment were the same as (3) of Experimental Example 1.

[0045] Each concentration gradient was set up with 12 replicate wells and placed in a cell culture incubator for more than 4 h. Six wells were taken from each concentration gradient of the MS-Au NPs-RMF group and placed 0.8 cm below the permanent magnet, and a rotating magnetic field was applied for 15 min for 7 days; no magnetic field was applied to the MS-AuNPs group. Three wells of cells were taken out of each group on the 5th and 7th days for staining, the culture medium was aspirated, and the cells were rinsed three times with PBS (5 min / time). The slides were covered with 4% paraformaldehyde, the cells were fixed for 30 min, and the cells were rinsed three times with PBS (5 min / time). The membrane was broken with 0.3% Triton X-100 for 5 min, the cells were blocked with 1% BSA for 1 h, and rinsed three times with PBS (5 min / time). Primary goat anti-mouse Tuj1 (1:800) monoclonal antibody and goat anti-rabbit GFAP (1:1000) monoclonal antibody were added to the cells collected on the 5th day; primary goat anti-mouse MAP2 (1:500) monoclonal antibody and goat anti-rabbit GFAP (1:1000) monoclonal antibody were added to the cells collected on the 7th day. The primary antibody was aspirated, and the cells were rinsed with PBS three times (5 min / time). The corresponding secondary antibodies goat anti-rabbit Alexa Fluor 488 (1:1000) and goat anti-mouse Alexa Fluor 594 (1:1000) were added to each group, incubated at room temperature in the dark for 1 hour, the secondary antibody was aspirated, and the cells were rinsed with PBS three times (5 min / time). The nuclear stain DAPI was added, incubated in the dark for 10 min, and rinsed with PBS three times (5 min / time). The staining results were observed with a laser confocal microscope, and the average fluorescence intensity of the captured fluorescence images was analyzed by ImageJ. Figure 3 (a) is a fluorescence image of neural stem cells after 5 days of differentiation. Figure 3 Middle (b) is the fluorescence image after 7 days of differentiation.

[0046] like Figure 3 (a) and Figure 3 As shown in (b), the number of cell nuclei in cells cultured with different concentrations of MS-Au NPs was similar to that of Ctrl cells, which is consistent with the previous cck-8 and cell live / dead staining results that revealed that MS-Au NPs and RMF did not affect the activity of NSCs. In a series of MS-Au NPs concentrations, 20 μg / mL and 40 μg / mL of MS-Au NPs had a more obvious effect on promoting the differentiation of NSCs, especially in the cells placed in RMF at 20 μg / mL and 40 μg / mL, the expression of Tuj1 and MAP2 proteins was the highest, and the axons were longer and more complex, indicating that the differentiated neurons were more numerous and more mature. Figure 3 (c) and Figure 3As shown in (d), the average fluorescence intensity of Tuj1 and MAP2 proteins of no less than 500 cells was analyzed by ImageJ. It was found that the expression of Tuj1 in the MS-Au NPs group at 20 μg / mL and 40 μg / mL on the fifth day was 1.7 times and 2 times that of the Ctrl group, respectively; while the expression of Tuj1 in the MS-Au NPs-RMF group at 20 μg / mL-RMF and 40 μg / mL-RMF was 2.8 times and 4.6 times that of the Ctrl group. On the 7th day, the expression of MAP2 in the MS-Au NPs group at 20 μg / mL and 40 μg / mL was 1.5 times and 2 times that of the Ctrl group, respectively; the expression of MAP2 in the MS-Au NPs-RMF group at 20 μg / mL-RMF and 40 μg / mL-RMF was 1.7 times and 3.3 times that of the CtrlF-RMF group, respectively. This result reveals that MS-Au NPs may have a certain differentiation effect on NSCs, and the 20 μg / mL-RMF and 40 μg / mL-RMF in the MS-Au NPs-RMF group have better differentiation effects, which may be because the induced current (IC) generated by MS-Au NPs in the rotating magnetic field promotes the expression of Tuj1 and MAP2 in NSCs. However, regardless of whether the induced current exists, 20 μg / mL and 40 μg / mL MS-Au NPs have a significant promoting effect on NSCs differentiation. However, the difference between 20 μg / mL and 40 μg / mL in promoting the differentiation of NSCs is not obvious. Combined with cell live / dead staining and cck-8 experiments, these two concentrations were initially selected for subsequent experiments to verify the role of MS-Au NPs and IC in promoting the differentiation of NSCs and determine the most suitable MS-Au NPs concentration for NSCs differentiation.

[0047] (2) We further evaluated the effect of magnetic field rotation speed on the neuronal differentiation of NSCs.

[0048] Experimental groups: MS-Au NPs 40 μg / mL, the rotation speeds of the applied magnetic field were 0, 400, 500 and 600 rpm, respectively, recorded as 0 rpm group, 400 rpm group, 500 rpm group and 600 rpm group.

[0049] The 6-well plate was incubated with Laminin. After 4 h of incubation, Laminin was removed and the second-generation NSCs cells were prepared into a single cell suspension with a density of 1×10 5cells / mL, 2 mL of single cell suspension was added, and the cells were seeded in 6-well plates. After the neural stem cells adhered to the wall and spread, the original proliferation medium was removed, and the differentiation medium containing 40 μg / mL MS-Au NPs was added. Three wells were set up for each concentration gradient and placed in a cell culture incubator for more than 4 hours. The well plate was placed 0.8 cm below the permanent magnet and a rotating magnetic field was applied for 15 min for 5 days. The permanent magnet speeds of the 0 rpm group, 400 rpm group, 500 rpm group and 600 rpm group were 0, 400, 500 and 600 rpm, respectively. The cells were collected on the 5th day and every 10 7 Each cell was dissolved in 600 μL lysis buffer, and total cell RNA was isolated and extracted using the TRIzol™ Plus RNA purification kit. The integrity of RNA was detected by agarose gel electrophoresis, and the concentration and purity of RNA were calculated using a NanoDrop 1000 spectrophotometer. RNA was reverse transcribed into cDNA, which was then amplified by qPCR, and the Cq value of each gene in each group was analyzed.

[0050] Figure 3 (e) shows the RT-qPCR results of NSCs with 40 μg / mL MS-Au NPs exposed to a magnetic field for 5 days at rotation speeds of 0, 400, 500, and 600 rpm. Compared with the 0 rpm group, the Tuj1 expression of NSCs cultured under the magnetic field in the 400, 500, and 600 rpm groups was upregulated by 2.18, 6.85, and 5.7 times, respectively, while the MAP2 expression was upregulated by 1.3, 4.7, and 1.9 times, respectively. The above results indicate that a higher magnetic field rotation speed can better induce the mRNA expression of Tuj1 and MAP2 in NSCs, but the rotation speed of 600 rpm has a lower differentiation-promoting effect on NSCs than 500 rpm. Considering that a higher rotation speed may cause NSCs to produce more ROS, a rotation speed of 500 rpm was used when applying the magnetic field in subsequent experiments. (3) To verify the above conclusions, NSCs were cultured for a longer period of time and the relative expression levels of Tuj1 and MAP2 proteins were detected by immunofluorescence staining experiments after 7 days of treatment.

[0051] The experimental groups were: MS-Au NPs-RMF group and MS-Au NPs group. The concentrations of the MS-Au NPs group were set to 0, 20, and 40 μg / mL, of which 0 μg / mL was Ctrl. The concentrations of the MS-Au NPs-RMF group were the same as those of the MS-Au NPs group, recorded as 20 μg / mL -RMF, 40 μg / mL -RMF, and 0 μg / mL -RMF as Ctrl-RMF. The experimental process was the same as that of Experiment 2 (1), except that the magnetic field was applied for 7 days and 10 days, respectively. The staining results were observed using a laser confocal microscope, and the average fluorescence intensity analysis and cell counting of the captured fluorescence images were performed using ImageJ.

[0052] like Figure 4 (a) and Figure 4 As shown in (b), after the MS-Au NPs-RMF group and the MS-Au NPs group treated NSCs for 7 and 10 days, compared with 0 μg / mL (0 μg / mL-RMF), other concentrations expressed more Tuj1 and MAP2 positive cells at similar cell numbers, and showed longer axons and more complex neural networks compared with the 5-day induction in Experimental Example 2 (1), indicating that the MS-Au NPs-RMF group can stably and efficiently promote the differentiation of NSCs. Figure 4 (c) and Figure 4As shown in (d), the average fluorescence intensity of Tuj1 and MAP2 proteins in no less than 2000 cells was analyzed by ImageJ. It was found that on the 7th day, the expression levels of Tuj1 in the MS-Au NPs group at 20 μg / mL and 40 μg / mL were 1.3 times and 1.7 times that of the Ctrl, respectively, and the expression levels of Tuj1 in the MS-AuNPs-RMF group at 20 μg / mL-RMF and 40 μg / mL-RMF were 1.6 and 2 times that of the Ctrl-RMF, respectively; on the 10th day, the expression levels of MAP2 in the MS-Au NPs group at 20 μg / mL and 40 μg / mL-RMF were 1.2 times and 1.5 times that of the Ctrl, respectively, and the expression levels of MAP2 in the MS-Au NPs-RMF group at 20 μg / mL-RMF and 40 μg / mL-RMF were 1.6 times and 3 times that of the Ctrl-RMF, respectively, while the expression levels of Tuj1 in the MS-Au NPs-RMF group at 20 μg / mL-RMF and 40 μg / mL-RMF were The expression of MAP2 in the 20 μg / mL-RMF and 40 μg / mL-RMF groups was 1.3 times and 2 times that in the MS-AuNPs group. In addition, the relative expression levels of Tuj1 and MAP2 at different days were compared. The relative expression levels of Tuj1 in the 20 μg / mL-RMF and 40 μg / mL-RMF groups at 7 days were 1.2 times and 1.8 times that in Experiment 2 (1) at 5 days; the relative expression levels of MAP2 in the 20 μg / mL-RMF and 40 μg / mL-RMF groups at 10 days were 1.5 times and 1.5 times that in the 7 days, indicating that IC and MS-Au NPs can continuously and stably promote the differentiation of NSCs. It is worth noting that there was no significant difference in the expression of Tuj1 on day 7 or MAP2 on day 10 in the 0 μg / mL MS-Au NPs group and the 0 μg / mL-RMF in the MS-Au NPs-RMF group, indicating that RMF alone could not promote the differentiation of NSCs. The percentage of Tuj1 positive cells after 7 days of culture and MAP2 positive cells after 10 days of culture in all cells was further calculated based on the results of immunofluorescence staining. Figure 4 Middle (e) and Figure 4As shown in (f), on the 7th day, among all NSCs, the proportion of Ctrl cells differentiating into early neuronal cells was 7%, while the proportion of 40μg / mL-RMF-differentiated into early neuronal cells in the MS-Au NPs-RMF group was 24%. On the 10th day, the proportion of NSCs cultured in Ctrl cells differentiating into mature neuronal cells was 16%, while the proportion of NSCs cultured in 40μg / mL-RMF-differentiated into mature neuronal cells was 48%. In order to more comprehensively evaluate the morphology of neurons, Sholl analysis was performed by calculating the intersection points between neurites and concentric circles centered on the cell body and with a specific radius as the unit to quantify the complexity of neuronal processes. As shown in Figure 4 Medium (g) and Figure 4 As shown in (h), 40 μg / mL-RMF in the MS-Au NPs-RMF group presented higher neurite complexity and greater neurite length than Ctrl-RMF, indicating that it had undergone a higher degree of neural differentiation.

[0053] (4) The experimental grouping was the same as that of Experiment 2 (3). In the MS-Au NPs-RMF group, NSCs were cultured in a culture medium containing 0, 20, and 40 μg / mL MS-Au NPs and placed in RMF for 5 and 7 days. No magnetic field was applied to the MS-Au NPs group. Total RNA of NSCs cultured for 5 and 7 days in each group was extracted, and the expression of neural-specific markers such as nestin, βⅢ tubulin (Tuj1), glial fibrillary acidic protein (GFAP), and microtubule-associated protein 2 (MAP2) was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The promoting effect of MS-Au NPs and RMF on NSC differentiation was semi-quantitatively analyzed. The RNA was reverse transcribed into cDNA, which was then amplified by qPCR, and the Cq value of each gene in each group was analyzed. Figure 5 (a) and Figure 5As shown in (b), the relative expression of Tuj1 in the MS-Au NPs group at 20 μg / mL and 40 μg / mL on the 5th day was comparable to that of the Ctrl group, but the relative expression of Tuj1 in the MS-Au NPs group at 40 μg / mL on the 7th day was significantly higher than that of the Ctrl group, which was 2.3 times that of the Ctrl group. At the same time, the 40 μg / mL-RMF in the MS-Au NPs-RMF group on the 5th day was 3.2 times that of the 40 μg / mL in the MS-Au NPs group, indicating that the combined action of MS-Au NPs and RMF promoted NSCs to express more Tuj1. Analysis may be that MS-Au NPs can promote the differentiation of NSCs into neurons after treating NSCs, but the promotion effect is relatively slow, and the effect is not different until the 7th day. RMF treatment on the basis of MS-Au NPs can make NSCs express more Tuj1 on the 5th day. In addition, the GFAP expression in the 0 μg / mL MS-Au NPs group and the 0 μg / mL-RMF MS-Au NPs-RMF group was lower than that in the other concentrations on the fifth day, and similar results were found on the seventh day, indicating that MS-Au NPs and MS-Au NPs-RMF may inhibit the differentiation of NSCs into astrocytes and promote the differentiation of NSCs into neurons. In the MS-Au NPs-RMF group, compared with Ctrl-RMF, the relative expression level of MAP2 mRNA showed a significant difference only in 40 μg / mL-RMF, which was 2.3 times that of Ctrl-RMF on the 5th day and 3.9 times that of Ctrl-RMF on the 7th day. There was no significant difference in the expression of MAP2 in 20 μg / mL, 40 μg / mL of MS-Au NPs group and in Ctrl-RMF and 20 μg / mL-RMF of MS-Au NPs-RMF group, indicating that MS-Au NPs-RMF played a role in promoting nerve repair. This indicates that MS-Au NPs treatment alone, RMF treatment alone (Ctrl-RMF in the MS-Au NPs-RMF group) and 20 μg / mL-RMF in the MS-Au NPs-RMF group cannot promote the expression of mature neuron marker MAP2, revealing that 40μg / mL-RMF (i.e., concentration of 40μg / mL) in the MS-Au NPs-RMF group is the best condition for promoting NSCs differentiation. In addition, the expression level of NSCs treated with Ctrl-RMF in the MS-Au NPs-RMF group is almost the same as that of Ctrl in the MS-AuNPs group, indicating that the application of magnetic field alone cannot significantly promote the differentiation of NSCs.In summary, although adding MS-Au NPs to the NSCs culture medium promotes the expression of NSCs Tuj1 and inhibits the expression of GFAP, which promotes the differentiation of NSCs into neurons to a certain extent, the effect is not obvious, and it cannot increase the expression of MAP2 and promote nerve repair. In contrast, 40μg / mL-RMF in the MS-Au NPs-RMF group has a more obvious effect on promoting the differentiation of NSCs into neurons, indicating that in addition to MS-Au NPs, the role of RMF is more critical, which also confirms the previous assumption.

[0054] (5) To further confirm that MS-Au NPs can promote the differentiation of NSCs into neurons, Western Blot experiments were used to analyze the expression levels of neuron-specific proteins in NSCs treated with MS-Au NPs and RMF for 7 days, including nestin, βⅢ tubulin (Tuj1), glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), synaptophysin (SYP), growth-associated protein 43 (GAP43), neuron-specific nuclear protein (NeuN), and postsynaptic density protein (PSD).

[0055] The experimental grouping was the same as that of Experimental Example 2 (4). In the MS-Au NPs-RMF group, NSCs were cultured in a culture medium containing 0, 20, and 40 μg / mL MS-AuNPs and placed in RMF for 7 days. In the MS-Au NPs group, no magnetic field was applied. On the 7th day, the cells were lysed using RIPA lysis buffer to extract total protein. The protein concentration was determined using a BCA (Bicinchoninic Acid) protein quantification kit. The membrane was transferred to a polyvinylidene fluoride membrane (PVDF) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with primary antibodies against MAP2, Tuj1, or GFAP (the same antibodies as those in Experimental Example 2 (1)) at 4°C overnight. After washing three times with PBS, goat anti-mouse IgG / horseradish peroxidase-labeled secondary antibodies were added and incubated at room temperature for 1 hour at 37°C. The relevant proteins were detected using an enhanced chemiluminescence reagent. Figure 5 (c) and Figure 5The Western Blot results shown in (e) are similar to the results of immunoglobulin staining, in which the expression levels of Tuj1 and GAP43 proteins, which are early markers of neuronal differentiation, and MAP2 and NeuN, which are markers of mature neurons, were the highest in the 40 μg / ml-RMF in the MS-Au NPs-RMF group. Similarly, SYP, a marker of mature synapses, and PSD95, which forms mature synaptic networks, were significantly increased only in the 40 μg / ml-RMF in the MS-AuNPs-RMF group. This indicates that only 40 μg / mL of MS-Au NPs and RMF treatment can promote the differentiation of NSCs into mature neurons on the 7th day. Figure 5 (d) and Figure 5As shown in (f), in order to quantitatively evaluate protein expression, the grayscale values ​​of the protein bands of the Western Blot results were further analyzed using ImageJ software. The results showed that the expression level of Tuj1 in the MS-Au NPs-RMF group at a concentration of 40 μg / ml-RMF was 1.8 times that of the Ctrl-RMF in the MS-Au NPs-RMF group, and the expression level of MAP2 protein was 2.2 times that of the Ctrl-RMF in the MS-Au NPs-RMF group, further proving that the MS-AuNPs-RMF group showed the best performance in promoting NSCs neural differentiation at a concentration of 40 μg / mL. The expression levels of SYP, GAP43, NeuN, and PSD proteins, which also play an important regulatory and marker role in the differentiation of neural stem cells into neurons and promote the development and functional maturation of neurons, increased to varying degrees in the 40 μg / mL-RMF group of MS-Au NPs-RMF, which were 1.5 times, 1.5 times, 1.3 times, and 1.2 times of Ctrl-RMF in the MS-Au NPs-RMF group, respectively. This indicates that NSCs treated with MS-AuNPs-RMF can not only significantly promote the expression of neuronal marker proteins Tuj1 and MAP2, but also enhance the expression of key proteins such as SYP, PSD, NeuN, and GAP43, indicating that MS-Au NPs-RMF can promote the differentiation and maturation of neurons in many aspects, verifying its potential application value in neurobiological research. Moreover, due to the characteristics that SYP, PSD and GAP43 are only expressed on synapses and protrusions, the increase of these proteins usually means the enhancement and development of synaptic function. The increase in their expression levels shown by WB also indirectly confirms the previous analysis of synaptic complexity. In addition, WB results show that the expression of GFAP in NSCs treated with MS-Au NPs and applied magnetic field (RMF) and MS-Au NPs alone decreased to varying degrees. At the same time, there was no difference in the expression of Ctrl in the MS-Au NPs group and the expression of Ctrl-RMF in the MS-Au NPs-RMF group, which confirmed the RT-qPCR results. In order to further detect the rate of acceleration of NSC differentiation under the treatment of MS-Au NPs plus RMF, the expression of neuron-specific proteins in NSCs at different culture times under the same conditions was detected. Figure 5 (g) and Figure 5As shown in (h), the NSCs cultured in 40 μg / mL-RMF in the MS-Au NPs-RMF group on day 5 and the NSCs cultured in Ctrl in the MS-Au NPs group on day 10 had almost the same expression levels of Tuj1, MAP2, SYP and GAP43, and there was no significant difference after grayscale value analysis by ImageJ. This indicates that after treatment with MS-Au NPs-RMF at a concentration of 40 μg / mL, NSCs differentiated at a rate nearly twice that of natural conditions.

[0056] Experimental Example 3: Gold nanoparticles promote the differentiation of NSCs into dopaminergic and acetylcholinergic neurons (1) As mentioned above, MS-Au NPs+RMF can promote the differentiation of NSCs into neurons by upregulating the expression of neuronal marker proteins and genes. Next, the type of neurons derived from NSCs was further determined by analyzing the specific biomarkers of functional neurons. In this study, the expression levels of GAD65, DBH, CHAT, and TH proteins in each group on day 7 were detected by WB experiments to verify the neuronal subtypes differentiated from cultured NSCs.

[0057] The experimental grouping was the same as that of Experimental Example 2 (5). In the MS-Au NPs-RMF group, NSCs were cultured in a culture medium containing 0, 20, and 40 μg / mL MS-AuNPs and placed in RMF for 7 days. In the MS-Au NPs group, no magnetic field was applied. On the 7th day, cells were lysed using RIPA lysis buffer to extract total protein; protein concentration was determined using a BCA (Bicinchoninic Acid) protein quantification kit and transferred to a polyvinylidene fluoride membrane (PVDF) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with primary antibodies against CHAT, TH, GABA, or GAD65 at 4°C overnight. After washing three times with PBS, goat anti-rabbit IgG / horseradish enzyme-labeled secondary antibodies were added and incubated at room temperature for 1 hour at 37°C. The relevant proteins were detected using an enhanced chemiluminescence reagent. Figure 6 (a) and Figure 6As shown in (b), WB results show that at 40 μg / ml-RMF in the MS-Au NPs-RMF group, the expression levels of CHAT and TH proteins among the four functional neuron marker proteins increased significantly. The grayscale values ​​of the protein bands in the Western Blot results were analyzed using ImageJ software, which were 1.62 times and 1.48 times that of Ctrl-RMF in the MS-Au NPs-RMF group, respectively; while the expression levels of DBH and GAD65 proteins showed a downward trend, indicating that MS-Au NPsRMF promotes the differentiation of NSCs into acetylcholinergic neurons and dopaminergic neurons.

[0058] (2) To verify the above conclusions, a calcium ion fluorescent probe (Fluo-4 AM) was used to evaluate the responsiveness of cells to neurotransmitters in the MS-Au NPs-RMF group at a concentration of 40 μg / mL after 7 days of culture. A sterile round coverslip was placed in a 24-well plate, 10 μg / mL of Laminin was added to the coverslip, and the cells were incubated in a cell culture incubator for 4 h. Laminin was aspirated, and the second-generation NSCs cells were prepared into a single cell suspension with a density of 1×10 5 cells / mL, 1 mL of single cell suspension was added to each well, and the cells were seeded into the well plate. MS-Au NPs were added to the differentiation medium to a concentration of 40 μg / mL. After the neural stem cells adhered to the wall and spread, the original proliferation medium was removed, and the differentiation medium containing MS-Au NPs was added. Three wells were set up for replication and placed in a cell culture incubator for more than 4 hours. The well plate was placed 0.8 cm below the permanent magnet and a rotating magnetic field was applied for 15 min for 7 days. On the 7th day, the culture medium was removed and washed three times with PBS. Fluo-4 AM working solution (obtained by diluting neurobasal 1000 times) was added, and the amount of solution was based on sufficient coverage of the cells. Incubate at 37°C for 30 minutes to load the fluorescent probe. Subsequently, the cells were washed three times with PBS, dopamine neurotransmitters and acetylcholine neurotransmitters were used to stimulate the cells, and fluorescence was detected in real time at each time point using a laser confocal microscope to determine changes in intracellular calcium ion concentration. The results are shown in Figure 6 (c) and Figure 6 As shown in (e), most cells respond quickly to stimulation by neurotransmitters such as acetylcholine and dopamine. Figure 6 (c) and Figure 6 In (e), 5 cells were selected for fluorescence intensity analysis. Figure 6 Middle (d) and Figure 6(f) shows the corresponding fluorescence intensity analysis of 5 representative cells that responded to acetylcholine neurotransmitters and dopamine neurotransmitters. These results confirm that neurons differentiated from MS-Au NPs-RMF-treated NSCs are able to respond to neurotransmitters such as acetylcholine and dopamine. In addition, after neurotransmitter stimulation, calcium sparks appeared sequentially and directional in adjacent cells, indicating that there is a calcium spark network between cells that enables nerve impulses to be transmitted. In summary, intracellular Ca 2+ The imaging results confirmed that MS-Au NPs-RMF could not only induce NSCs to differentiate into specific types of neurons, but also enable NSCs to differentiate into mature neurons that could perform their normal functions.

[0059] Test Example 4: Animal Model Test Establishment of spinal cord injury model in mice: Male C57BL / 6J mice (8-10 weeks old, n = 125) were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, Shandong; No.: SCXK(LU)2022-0006). The mice were randomly divided into 6 groups: sham operation (Sham), spinal cord injury (SCI), cells (NSCs), cells + RMF (NSCs-RMF), cells + MS-Au NPs (NSCs+MS-Au NPs), and cells + MS-Au NPs + RMF (NSCs+MS-Au NPs-RMF). The sham operation group was normal mice without treatment. The mice in the other groups were first anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). Tissue scissors were used to fully expose the T9-T11 vertebrae, and the T10 vertebrae were removed. After the T10 spinal cord was completely exposed, the KW-ZJ fully automatic craniocerebral spinal cord impactor was used to impact the spinal cord at a force of 70 kD. For both the cell group and the cell+RMF group, NSCs (5 × 10 6 The cells were implanted into the subarachnoid space of the spinal cord and maintained on the surface of the injured spinal cord. For the cell + MS-Au NPs group and the cell + MS-Au NPs + RMF group, the cells were cultured in a differentiation medium containing 40 μg / mL MS-Au NPs one day in advance; the NSCs that had internalized the MS-Au NPs were centrifuged the next day to obtain a cell pellet, which was then implanted into the subarachnoid space of the spinal cord and maintained on the surface of the injured spinal cord, and then the muscles, subcutaneous tissues and skin were sutured in layers. For the cell + RMF group and the cell + MS-Au NPs + RMF group, a changing magnetic field provided by a permanent magnet on a rotator (500 rpm) was applied to the mice every day at a distance of 1 cm for 30 minutes each time for two weeks. At the same time, to prevent the mice from moving, the mice were placed in an animal fixture when the magnetic field was used to treat the mice. In addition, the magnet was kept 1 cm away from the mouse to avoid fluctuations in magnetic treatment. Mice in each group urinated and ate every day.

[0060] The recovery of hindlimb motor function was systematically assessed by Basso Mouse Scale (BMS) scoring and slope test. Choose an experimental environment with good lighting and no distractions, usually an open observation field. Allow the mice to adapt to the observation field for a few minutes to ensure natural behavior. Observe for 4-5 minutes and score the mice based on their hindlimb movement. Figure 7 (a) shows the BMS scores of mice every week after surgery. In the sham group, the scores of mice were stable at around 9 because their motor function was not impaired. For mice in the SCI group, cell group, cell+RMF group, and cell+MS-Au NPs group, although their BMS scores increased significantly from day 1 to day 28, the scores at week 4 were still below 2. In contrast, the BMS scores of mice in the cell+MS-Au NPs+RMF group increased from 0 on day 1 to 1.9 on day 7, 3.6 on day 14, 4.4 on day 21, and 5.3 on day 28, which were much higher than those of mice in other groups.

[0061] The hot plate test was used to detect the sensitivity of mice with spinal cord injury to thermal stimulation. The thermal stimulation temperature was fixed at 55°C, and the timing was started after each group of mice were placed on the hot plate. The recording was stopped when the mice showed high-frequency paw lifting movements. Figure 7 As shown in (b), the reaction time of mice in the cells+MS-Au NPs+RMF group was approximately 0.9 seconds, and the reaction time of mice in the SCI group was approximately 5.8 seconds.

[0062] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of gold nanoparticles in the preparation of induced stem cell differentiation products, characterized in that: The gold nanoparticles are gold nanoparticles with a layered structure. The gold nanoparticles and stem cells are mixed and allowed to stand, and after the stem cells engulf the gold nanoparticles, the stem cells are placed in a rotating magnetic field to perform intracellular electrical stimulation on the stem cells to achieve differentiation of the stem cells into neurons.

2. The use according to claim 1, characterized in that: The gold nanoparticles with layer structure are multi-shell gold nanoparticles; the multi-shell gold nanoparticles are prepared by the following method: (1) Usually, distilled water and melamine are mixed and heated with stirring, HAuCl4•4H2O solution is added and stirred until the transparent solution turns yellow, and the yellow product is obtained by centrifugation, which is then washed and dried to obtain colloidal spheres; (2) The colloidal spheres are dispersed in the mixed solution, sodium poly(p-styrene sulfonate) is added, and finally, a NaBH4 aqueous solution is quickly added and stirred, and then centrifuged, washed, and dried to obtain multi-shell gold nanoparticles.

3. The use according to claim 2, characterized in that: In step (1), the ratio of the added amounts of distilled water, melamine and HAuCl4•4H2O solution is 100 mL:50 mg:6 mL; the concentration of the HAuCl4•4H2O solution is 0.024 M; and the heating temperature is 70°C.

4. The use according to claim 2, characterized in that: In step (2), the mixed solution is obtained by mixing ethylene glycol and water in a volume ratio of 10:3; the added amount ratio of the colloidal spheres, the mixed solution, sodium polystyrene sulfonate and the NaBH4 aqueous solution is 10 mg:78 mL:20 mg:2 mL; and the concentration of the NaBH4 aqueous solution is 25 mg / mL.

5. The use according to claim 1, characterized in that: The gold nanoparticles and stem cells are mixed by adding the gold nanoparticles to a differentiation medium to obtain a gold nano solution, adding the stem cells to a proliferation medium, and then adding them to a culture dish, and after the stem cells adhere to the wall, removing the proliferation medium and adding the gold nano solution.

6. The use according to claim 5, characterized in that: The concentration of the gold nanoparticle solution is 20-80 μg / mL.

7. The use according to claim 6, characterized in that: The concentration of the gold nanoparticle solution is 40 μg / mL.

8. The use according to claim 1, characterized in that: The rotation speed of the rotating magnetic field is 400-600 rpm; the magnetic field strength of the rotating magnetic field is 0.1-1 Tesla; the induced electromotive force generated by the gold nanoparticles induced by the rotating magnetic field is 0.1-10 mV.

9. The use according to claim 1, characterized in that: The stem cells are neural stem cells; the differentiation of the stem cells into neurons includes: differentiation of the stem cells into dopaminergic neurons and acetylcholinergic neurons.

10. The use according to claim 1, characterized in that: The induced stem cell differentiation products include products that induce stem cells to differentiate into neurons, spinal cord injury repair drugs, and neurodegenerative disease repair drugs.

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