CN115212227B - Application of lithioalga soil as effective component in preparing nerve injury repair product - Google Patents
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Abstract
The application provides an application of lithioalga soil as an effective component in preparing a nerve injury repair product and promoting nerve injury repair for non-therapeutic destinations; as an active ingredient for preparing a nerve injury repair product; is used for inducing the differentiation of the neural stem cells into the neuron cells in vitro and/or in vivo, and inhibiting the differentiation of the neural stem cells into astrocytes; as an active ingredient for preparing a product for inducing differentiation of neural stem cells into neuronal cells and inhibiting differentiation of neural stem cells into astrocytes. The application discovers that the lithium algae soil serving as an active ingredient has the effect of promoting the differentiation of the neural stem cells to the direction of neurons but not the direction of astrocytes for the first time; the hydrogel loaded with the lithiumalga soil can continuously and effectively promote the differentiation of the neural stem cells to neurons, and provides a new method and a new way for the repair and treatment of nerve injury and the clinical transformation.
Description
Technical Field
The application relates to the technical field of biological medicine, in particular to application of lithioalga soil as an effective component in preparing a nerve injury repair product.
Background
Nanotechnology is an emerging and rapidly evolving field of modern science. In the past few years, nanotechnology has been used with high efficiency in biomedical research, providing a new approach to cell imaging, delivery of genes or other small molecules, and preparation of biological scaffolds in tissue engineering.
Rat neural stem cells (Neural stem cells, NSCs) are cells isolated from the cerebral cortex of rats and are widely used in biomedical and regenerative medicine research due to their ability to proliferate indefinitely and their multipotency, so far NSCs are increasingly used in the treatment of clinical diseases such as cerebral infarction, spinal cord injury, parkinson's disease, multiple sclerosis, etc. Neural stem cells can differentiate into neurons, astrocytes, oligodendrocytes, etc., although NSCs transplantation has made a certain progress in spinal cord regeneration, it faces several difficulties: (1) limited sources of neural stem cells; (2) The transplanting survival rate of the neural stem cells is low, (3) the effective differentiation rate of the neural stem cells is low, and less than 13 percent of the surviving neural stem cells are differentiated into neurons. Post-transplantation NSCs, such as those differentiated into astrocytes, can be detrimental to axon regeneration and even affect recovery of neural function.
In the prior art, the neural stem cells are required to be induced to be directionally differentiated into the neuron cells by depending on an external induction factor, the existing external induction factor mostly adopts growth factors such as nerve growth factors, brain-derived neurotrophic factors and vascular endothelial growth factors and/or micromolecular drugs such as all-trans-retinoic acid, and the like, and in the treatment process, the growth factors and the micromolecular drugs have the problems of rapid degradation, easy occurrence of diffusion which is difficult to control, complicated dosage collocation, high cost, high clinical transformation difficulty, biological safety, immunogenicity and the like of the exogenous growth factors synthesized by gene recombination.
The lithium algae soil (Laponite) is a magnesium lithium silicate Na + 0.7 [(Mg 5.5 Li 0.3 )Si 8 O 20 (OH) 4 ] - 0.7 Is a nano lamellar material, has good biocompatibility and is biodegradable into Na + ，Mg 2+ ，Si(OH) 4+ And Li (lithium) + And non-toxic and bioabsorbable byproducts, which can be used as carriers and temporary scaffolds. In the prior art, the lithioalga soil is mostly used as a biological cross-linking agent and a drug carrier in the medical field to improve the mechanical strength of the product and the drug slow release efficiency.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present application aims to provide an application of lithium algae soil as an active ingredient in preparing a nerve injury repair product, which is used for solving the problems of rapid degradation, easy diffusion, excessively complex dosage collocation and high cost of the existing nerve injury repair product due to the adoption of small molecular drugs and/or externally added induction factors.
To achieve the above and other related objects, the present application is achieved by including the following technical means.
The application aims at providing an application of the lithioalga soil as an effective component in preparing a nerve injury repair product.
The application also provides a new application of the lithium algae soil in promoting nerve injury repair at a non-therapeutic destination.
Another object of the present application is to provide a use of lithio for inducing differentiation of neural stem cells into neuronal cells in vitro and/or in vivo, and inhibiting differentiation of neural stem cells into astrocytes.
The application also provides a new application of the lithium algae soil, which is used as an active ingredient for preparing a product for inducing the differentiation of the neural stem cells into the neuron cells and inhibiting the differentiation of the neural stem cells into astrocytes.
Preferably, the particle size of the laponite is 20-30 nm.
Preferably, when the lithium algae clay is used as an active ingredient for preparing a nerve injury repair product, the usage amount of the lithium algae clay is 0.1-1% based on the total weight of the nerve injury repair product.
Preferably, when used as an active ingredient in the preparation of a nerve injury repair product, the composition acts on a nerve injury site to induce differentiation of neural stem cells into neurons through interaction of lithio and the neural stem cells.
Preferably, the method is used for inducing the neural stem cells to differentiate into the neuron cells in vitro and/or in vivo, and when the neural stem cells are inhibited from differentiating into astrocytes, the neural stem cells are inoculated into a lithiozone differentiation medium for culture, and the neural stem cells are induced to differentiate into the neurons, wherein the concentration of the lithiozone in the lithiozone differentiation medium is 1-20 mug/ml.
The application further aims to provide an in-vitro lithium algae soil differentiation medium, wherein each 100ml of in-vitro lithium algae soil differentiation medium comprises the following components in percentage by weight: 92-95 ml of DMEM/F12 culture medium, 1-3 ml of B27, 1-2 ml of N2, 1-2 ml of lithium algae soil stock solution, 0.5-1 ml of fetal calf serum and 1-1.5 ml of double-antibody mixed solution of green streptomycin; the concentration of the lithium algae soil in the in-vitro lithium algae soil differentiation culture medium is 1-20 mug/ml.
Preferably, the solvent of the lithium algae soil stock solution is PBS solution.
The fourth object of the application is to provide a composite gel material with the function of inducing neural stem cells to differentiate into neurons, wherein the composite gel material is formed by mixing a hydrogel, a carrier and a stock solution of lithiogel into gel; the carrier is human serum albumin, and the hydrogel is succinimidyl succinate modified polyethylene glycol.
Preferably, the solvent of the lithium algae soil stock solution is PBS solution, and the concentration of the lithium algae soil in the lithium algae soil stock solution is 0.5-1.5 mg/ml.
Preferably, the volume ratio of the carrier to the lithium algae soil stock solution is 1 (1.0-1.2); the mass volume ratio of the hydrogel to the lithium algae soil stock solution is 100-200 mg/ml.
As described above, the application of the lithioalga soil as an effective component in preparing a nerve injury repair product has the following beneficial effects: the application discovers that the lithium algae soil serving as an active ingredient has the effect of promoting the differentiation of the neural stem cells to the direction of neurons but not the direction of astrocytes for the first time; the composite gel material can continuously and effectively promote the differentiation of the neural stem cells to neurons, and provides a new method and a new path for the aspects of nerve injury repair treatment and nerve regeneration clinical transformation.
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FIG. 1 shows immunofluorescence of MAP2 and GFAP positives in promoting neural stem cell differentiation in soil bodies of lithium algae of different concentrations in examples 1-4 and comparative example.
FIG. 2 is a graph showing the statistical results of the percentage of MAP2 and GFAP positive cells in promoting neural stem cell differentiation in soil bodies of lithium algae in different concentrations in examples 1-4 and comparative example.
FIG. 3 is a graph showing Western blot results of MAP2 and GFAP in the process of promoting differentiation of neural stem cells outside the soil bodies of lithium algae in different concentrations in examples 1-4 and comparative example.
FIG. 4 shows Western blot statistics of MAP2 and GFAP (0.05 or less) in the process of promoting differentiation of neural stem cells outside the soil bodies of lithium algae in different concentrations in examples 1-4 and comparative example.
Fig. 5 shows NF and GFAP immunofluorescence at 8 weeks post-surgery for each group of rats in example 5: the A diagram is an NF and GFAP immunofluorescence staining diagram of each group of rats, and the B diagram is a high-magnification enlarged diagram in a corresponding box in each group of Merge diagrams in the A diagram; graph C shows the results of the fluorescent intensity statistical analysis of NF in the damaged areas of rats of each group (P. Ltoreq.0.05; P. Ltoreq.0.01; P. Ltoreq.0.001; P. Ltoreq.0.0001; scale=500 um).
Fig. 6 shows a graph of the evaluation results of Basso-Beattie-Bresnahan (BBB) scoring (.p.ltoreq.0.01; p.ltoreq.0.001; p.ltoreq.0.0001) for each group of example 5 for the behavioral assessment of motor function after spinal cord injury.
Fig. 7 is a graph showing the effect of using axonal tracking to evaluate the growth promotion of axons in a loam-albumin hydrogel loaded with NSCs in example 5: panel a shows immunofluorescence of each group of axon Tracer, red for Tracer axon (Tracer), green for GFAP immunofluorescence marker, and panel B shows statistical analysis of the number of axons in each group of lesion areas (P. Ltoreq.0.01; P. Ltoreq.0.0001; scale = 500 um).
FIG. 8 shows a graph of differentiation of neural stem cells traced with NSCs-GFP composite Lithite-albumin hydrogel 4 weeks after transplantation into a spinal cord injury area of a rat, with Tuj-1 being red, representing a new neuron, and Green (GFP) being NSCs-GFP cells transplanted into the spinal cord injury area, and co-localization analysis under a confocal microscope, showing that the transplanted NSCs can differentiate into neurons in the injury area.
Detailed Description
Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present application may be used to practice the present application according to the knowledge of one skilled in the art and the description of the present application.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present application employ techniques conventional in the art of molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art.
In view of the problems that in the prior art, small molecular drugs are mostly adopted for nerve injury repair, and the small molecular drugs are easy to degrade, diffuse and complex in dosage collocation and high in cost; in order to solve the problem, the applicant creatively adopts silicon-based salt nano-particles with different concentration gradients to co-culture lithium alginate soil and neural stem cells (NSCs for short), and discovers that lithium alginate soil nano-particles with specific ion concentration can promote NSCs to differentiate after endocytosis by the NSCs and differentiate towards neurons under the comprehensive action of Li and Mg plasma released by the lithium alginate soil nano-particles, so that the proportion of the lithium alginate soil nano-particles to astrocytes is greatly reduced. The inventor of the application has provided the technical scheme of the application through long-term research and a large number of practices, mainly found that the in vitro differentiation culture medium containing specific concentration of lithium alginate soil has the effect of promoting the differentiation of the neural stem cells to the direction of neurons instead of the direction of astrocytes, and proved by in vivo and in vitro experiments to have the direct and effective effect of promoting the differentiation of the neural stem cells to the direction of neurons, the effect of promoting the regeneration of nerve injury by taking the lithium alginate soil as an effective component is confirmed, and meanwhile, the lithium alginate soil-albumin hydrogel loaded with the neural stem cells also provides a new method and path for repairing and treating the nerve injury.
The embodiment of the application provides application of lithioalga soil serving as an effective component in preparing a nerve injury repair product.
The embodiment of the application provides an application of lithioalga soil in promoting nerve injury repair at a non-therapeutic destination.
The embodiment of the application provides an application of lithioalga soil in inducing neural stem cells to differentiate into neuron cells in vitro and/or in vivo and inhibiting the differentiation of the neural stem cells into astrocytes.
The embodiment of the application provides a product which takes lithioalga soil as an effective component and is used for preparing a product which induces neural stem cells to differentiate into neuron cells and inhibits the neural stem cells from differentiating into astrocytes.
In a specific embodiment, the formulation of the nerve injury repair product is selected from one of a solution, suspension, pill, injection, tablet, capsule, granule, gel, controlled release formulation, and sustained release formulation.
In a specific embodiment, the particle size of the laponite is 20 to 30nm, such as 20 to 25nm,25 to 30nm.
In a specific embodiment, when the composition is used as an active ingredient in preparing a nerve injury repair product, the usage amount of the lithium algae soil is 0.1-1%, such as 0.1-0.2%, 0.2-0.25%, 0.25-0.50%, 0.50-0.75%, 0.75-1% based on the total weight of the nerve injury repair product.
In a specific embodiment, when used as an active ingredient in the preparation of a nerve injury repair product, the composition acts on a nerve injury site to induce differentiation of nerve stem cells into neurons through interaction of lithiowere and the nerve stem cells.
In a specific embodiment, when the neural stem cells are used for inducing the neural stem cells to differentiate into the neuron cells in vitro and/or in vivo and inhibiting the neural stem cells from differentiating into astrocytes, the neural stem cells are inoculated into a lithio-differential medium for culture, and the neural stem cells are induced to differentiate into the neurons, wherein the concentration of the lithio in the lithio-differential medium is 1-20 mug/ml, such as 1-5 mug/ml, 5-10 mug/ml, 10-15 mug/ml and 15-20 mug/ml. When the concentration of the lithium algae soil in the lithium algae soil differentiation culture medium is 1-10 mug/ml, the effect of promoting the neural stem cells to differentiate into neurons is obvious, and meanwhile, the differentiation of the neural stem cells into astrocytes is inhibited to the greatest extent.
The embodiment of the application also provides an in-vitro lithium algae soil differentiation medium, wherein each 100ml of in-vitro lithium algae soil differentiation medium comprises the following components in percentage by weight: 92-95 ml of DMEM/F12 culture medium, 1-3 ml of B27, 1-2 ml of N2, 1-2 ml of lithium algae soil stock solution, 0.5-1 ml of fetal calf serum and 1-1.5 ml of double-antibody mixed solution of green streptomycin; the concentration of the lithium algae soil in the in-vitro lithium algae soil differentiation culture medium is 1-20 mug/ml.
In a more specific embodiment, the composition comprises the following contents per 100ml of in vitro lithium algae soil differentiation medium: 94ml of DMEM/F12 medium, 2ml of B27, 1ml of N2, 1ml of lithiowere stock solution (concentration 1 mg/ml), 1ml of Fetal Bovine Serum (FBS) and 1ml of green chain mycin double antibody solution (100X).
In the technical scheme of the application, B27 and N2 are produced by Gibco corporation, are common nutrition additives for serum-free culture medium and common additives for neuron culture medium, and are responsible for providing components such as protein, hormone, trace elements and the like necessary for cell growth. The DMEM/F12 culture medium is rich in nutrient components and contains various microelements on the basis of the DMEM culture medium, and is widely applied to culture of various mammalian cells.
In a specific embodiment, the concentration of the lithium algae soil in the lithium algae soil stock solution is 0.5-1.5 mg/ml, such as specifically 0.5-1.0 mg/ml, 1.0-1.5 mg/ml.
In a specific embodiment, the volume fraction of fetal bovine serum in the in vitro lithio differential medium is 0.5-1.5%, such as specifically 0.5%,0.8%,1%,1.5%.
The embodiment of the application also provides a composite gel material with the effect of inducing the neural stem cells to differentiate into neurons, wherein the composite gel material is formed by mixing hydrogel, a carrier and a lithium algae soil stock solution into gel; the carrier is human serum albumin, and the hydrogel is succinimidyl succinate modified polyethylene glycol.
In the technical scheme, the human serum albumin is used as a carrier, the succinimidyl succinate modified polyethylene glycol is used as hydrogel, and the hydrogel and the lithium alginate soil are synergistic to form the composite gel material with the effect of inducing the neural stem cells to differentiate into neurons, so that the problems of rapid degradation of the induction factors, excessively complex dosage collocation, difficult controlled diffusion, short duration time, high cost and the like in the prior art are solved.
In a specific embodiment, the solvent of the lithium algae soil stock solution is PBS solution, and the concentration of the lithium algae soil in the lithium algae soil stock solution is 0.5-1.5 mg/ml.
In a specific embodiment, the volume ratio of the carrier to the lithium algae soil stock solution is 1 (1.0-1.2), and the mass volume ratio of the hydrogel to the lithium algae soil stock solution is 100-200 mg/ml, such as 100-150 mg/ml, and 150-200 mg/ml.
The embodiment of the application also provides a method for inducing the neural stem cells to differentiate into neurons, which comprises the steps of inoculating the neural stem cells into the in-vitro lithium algae soil differentiation medium for continuous culture; or, transplanting the neural stem cells and the composite gel material into a body together, and inducing the neural stem cells to differentiate into neurons through the interaction of the lithium alginate soil and the neural stem cells.
The proportion of neural stem cells differentiated into neurons was determined by detecting the protein expression and fluorescence intensity of the neuronal markers (TUJ-1, MAP 2) and astrocyte activation marker (GFAP).
In a specific embodiment, the neural stem cells are derived from primary and passaged neural stem cells of the rat embryonic brain hippocampus.
Further advantages and effects of the present application will become apparent to those skilled in the art from the disclosure of the present application, which is described by the following specific examples.
In the following embodiments of the present application, the explanation of the relevant terms is as follows:
TUJ-1: early neuronal markers;
MAP2: mature neuronal markers;
GFAP: glial fibrillary acidic protein, marker protein of astrocytes;
NF: neurofilament, neurofibrillin, neurite regeneration markers;
GFP: green fluorescent protein, green fluorescent protein.
In the following examples of the application, the lithiumalbe is purchased from BYK-Chemie GmbH (Pick chemical Co., ltd.) under the model XLS [ Mg ] 5.34 Li 0.66 Si 8 O 20 (OH) 4 Na 0.66 ]The particle size of the lithium algae soil is 20-30 nm, and the layer thickness of the lithium algae soil is 0.92nm.
In the following embodiments of the present application, the preparation method of the lithium algae soil stock solution is as follows: adding the lithium algae soil into 0.1M PBS solution (namely phosphate buffer solution), mixing, magnetically stirring for 4 hours, and vibrating in an ultrasonic cytoclasis instrument for 1Filtering for 5 minutes by using a 0.22 micron filter to obtain a lithium algae soil stock solution; the mass volume ratio of the lithium alga soil in the lithium alga soil stock solution to the PBS solution is 0.5-1.5 mg/ml. The preparation method of the 0.1M PBS solution comprises the following steps: 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 And 0.24g KH 2 PO 4 Adding into a beaker, adding distilled water, adding 800 milliliters, and stirring by using a magnetic stirrer; hydrochloric acid was then added to adjust the pH to 7.4, and distilled water was then added to volume 1000 ml.
Example 1
The embodiment provides an application of the lithiowere to induce the directional differentiation of the neural stem cells into the neuron cells in vitro and to inhibit the differentiation of the neural stem cells into astrocytes.
The embodiment also provides an in-vitro lithium algae soil differentiation medium, wherein each 100ml of in-vitro lithium algae soil differentiation medium comprises the following components in percentage by weight: 94ml of DMEM/F12 medium, 2ml of B27, 1ml of N2, 1ml of lithiowere stock solution (1 mg/ml concentration, 1M PBS solution as solvent), 1ml of Fetal Bovine Serum (FBS) and 1ml of a mixture of green streptomycin diabodies (100X). The concentration of the lithium alga soil nano particles in the lithium alga soil differentiation culture medium is 10 mug/ml, and the volume fraction of fetal calf serum is 1%.
The embodiment also provides a method for inducing the neural stem cells to differentiate into neurons by adopting the in-vitro lithiowere differentiation medium, which comprises the following steps:
placing coverslips pre-treated with polylysine in culture plates to obtain primary or passaged neural stem cells derived from rat embryonic brain Hippocampus at 1×10 7 And (3) inoculating the cells/mL into culture holes containing nerve stem cell lithium algae soil differentiation culture medium respectively, replacing the new lithium algae soil differentiation culture medium every other day, continuously culturing for 7 days, observing under a microscope, taking out a cover glass after the morphological development of the cells is mature, and performing immunofluorescence staining to identify the differentiated cell types. Lithium algae soil differentiation culture medium: and removing bFGF and EGF in the neural stem cell complete culture solution, and adding a proper amount of lithium algae soil nano particles and fetal bovine serum to promote the differentiation of the neural stem cells into neurons.
Examples 2 to 4
Examples 2 to 4 differ from example 1 in that the concentration of lithium alginate in the in vitro lithium alginate differentiation medium was different, 0.1. Mu.g/ml, 1. Mu.g/ml and 20. Mu.g/ml, respectively, and the rest of the process was identical.
Comparative example 1
Comparative example 1 was different from example 1 in that conventional adherent differentiation culture was used, and the rest of the process was exactly the same.
The specific procedure for examples 1-4 and comparative example 1 is:
1) Differentiation culture of neural stem cells:
lithium algae soil differentiation media with different lithium algae soil concentrations (0.1 mug/ml, 1 mug/ml, 10 mug/ml and 20 mug/ml) were prepared before the experiment, and the control group was a differentiation medium without lithium algae soil (comparative example 1). At the beginning of the experiment, the cultured rat primary neural stem cells were centrifuged (300 g,5 min), digested, re-centrifuged, and then the cells were resuspended in medium containing 1% fetal bovine serum, added to 96-well plates, and about 1 ten thousand cells per well was cultured overnight in a cell incubator. After the cells are attached to the wall the next day, the cell culture medium is changed into a differentiation culture medium containing different lithium algae soil concentrations, and liquid is changed every 3 days in the culture process.
2) Identification of neural stem cell differentiation in vitro by Lithite:
after one week, the 96-well plate with the cells attached is taken out, the culture solution is sucked and removed, PBS is used for cleaning for 2-3 times, 4% paraformaldehyde is added for fixing for 30min, PBS is used for cleaning for 2-3 times, 0.8% Triton X-100 membrane rupture is carried out for 5min, PBS is used for cleaning for 2-3 times, 5% bovine serum albumin is added for sealing for 1h, primary antibody is incubated at 4 ℃ overnight, PBS is used for cleaning for 3 times the next day, secondary antibody is used for incubation for 1h at room temperature, PBS is used for cleaning for 3 times, and the immunofluorescence staining process is completed. Cells were photographed under a fluorescence microscope and the ratio of MAP2 positive cells to GFAP positive cells was counted.
Comparing the neural stem cells differentiated and cultured by the lithium alginate soil nanoparticles with the neural stem cells differentiated and cultured by the conventional adherence:
immunofluorescence experiments of mature neuron marker MAP2 and astrocyte marker GFAP were performed by grouping the coverslips obtained in comparative example 1 and examples 1 to 4, respectively;
randomly selecting N Zhang Pianzi as samples, randomly taking N visual fields from each sample system, wherein N is a natural number, taking fluorescent markers as positives, and calculating the percentage of each platelet of neural stem cells differentiated into neuron cells according to the following formula:
neuronal cell fraction= (number of fluorescent-labeled positive cells/total number of cells) ×100%;
the results of the immunofluorescence graphs of MAP2 and GFAP positivity in the process of promoting the differentiation of the neural stem cells outside the soil body of the lithium algae with different concentrations in comparative example 1 and examples 1-4 are shown in FIG. 1, and it can be seen from FIG. 1 that 0.1-20 mug/mL of lithium algae soil can promote the differentiation of the neural stem cells into the neuron cells in vitro.
The statistics of the percentages of MAP2 and GFAP positive cells in the process of promoting the differentiation of neural stem cells outside the soil body of lithium algae with different concentrations in comparative example 1 and examples 1-4 are shown in FIG. 2, and semi-quantitative analysis results show that the proportion of MAP2 positive cells in the control group, the group with 0.1 mug/ml, the group with 1 mug/ml, the group with 10 mug/ml and the group with 20 mug/ml. Compared with the control group, the lithium algae soil can obviously promote the improvement of the differentiation proportion of the neural stem cells to the neurons, wherein the concentration is about 1-10 mug/ml and is the optimal concentration. At the same time, when the lithio concentration is greater than 20. Mu.g/ml, inhibition of differentiation of NSCs into neurons and/or astrocytes begins to be exhibited. This suggests that the specific concentration range of the lithiowere effective in promoting differentiation of neural stem cells toward neurons, not glial cells.
Western blot results of MAP2 and GFAP in the process of promoting neural stem cell differentiation outside the lithium alga soil bodies with different concentrations in comparative example 1 and examples 1-4 are shown in figure 3, and Western blot statistical analysis results are shown in figure 4, and it can be seen from figures 3-4 that the optimal concentration range of in vitro lithium alga soil for promoting NSCs to neuron differentiation is 1-10 mug/mL, and the concentration range inhibits NSCs to astrocyte differentiation.
Example 5
The embodiment provides application of the lithioalga soil serving as an effective component in preparing a nerve injury repair product.
The embodiment also provides an in-vitro lithium algae soil differentiation medium, wherein each 100ml of in-vitro lithium algae soil differentiation medium comprises the following components in percentage by weight: 94ml of DMEM/F12 medium, 2ml of B27, 1ml of N2, 1ml of lithiowere stock solution (1 mg/ml concentration, 1M PBS solution as solvent), 1ml of Fetal Bovine Serum (FBS) and 1ml of a mixture of green streptomycin diabodies (100X). The concentration of the lithium alga soil nano particles in the lithium alga soil differentiation culture medium is 10 mug/ml, and the volume fraction of fetal calf serum is 1%.
The embodiment provides a composite gel material with the effect of inducing the neural stem cells to differentiate towards neurons, 50mg of succinimidyl succinate modified polyethylene glycol (PEG- (SS) 2) is dissolved in 0.5ml of lithium alginate soil stock solution with the final concentration of 1mg/ml, and the solution is mixed with 0.5ml of human serum albumin in equal volume, and the mixture is automatically gelled within 10-20 minutes, so as to obtain the composite gel material (lithium alginate soil-albumin hydrogel) with the effect of inducing the neural stem cells to differentiate towards neurons.
The embodiment also provides a method for inducing the neural stem cells to differentiate into neurons by adopting the composite gel material, which comprises the following steps of
1) Constructing a rat spinal cord half-cut injury model: female Sprague-Dawley rats weighing 220-250g for in vivo experiments were purchased from Hangzhou medical institute (China, zhejiang) and fed in a controlled temperature and humidity environment; after anesthetizing the rat (4 mL/Kg) with 1% sodium pentobarbital, laminectomy was performed at the T9 vertebrae, and then incision was made layer by layer through skin, subcutaneous tissue and fascia to isolate the outside muscle tissue; thereafter, all tissues of the dorsal side of the spinal cord, which were 1mm deep and 2mm long, were cut and removed using iris scissors and ophthalmic forceps to prepare a rat Spinal Cord Injury (SCI) model;
2) Performing secondary operation one week after injury of the rat, adding the composite Gel material with the effect of inducing the neural stem cells to differentiate into neurons, mixing NSCs, and marking as a laponite-albumin hydrogel loaded NSCs group (cell+gel); the method comprises the following steps: after anesthesia, connective tissue in the lesion site is first removed and the graft mixture is placed into the lesion site and the wound is sutured for subsequent function scoring, axonal tracking and immunohistological experiments.
Referring to the above procedure, the following control groups were designed simultaneously: a lithiogel-albumin hydrogel group (Gel group), an albumin hydrogel-loaded NSCs (Cell group), and a lesion untreated group (Ctrl group); the NF and GFAP immunofluorescence and statistical analysis of each group of rats 8 weeks post-surgery are shown in fig. 5: the A diagram is an NF and GFAP immunofluorescence staining diagram of each group of rats, and the B diagram is a high-magnification enlarged diagram in a corresponding box in a Merge diagram of each group in the A diagram; panel C is a graph of the results of statistical analysis of fluorescence intensity of NF in the injured area of each group of rats. As can be seen from fig. 5, it is shown that the loam-albumin hydrogel loaded NSCs Cell group (cell+gel group) has the best effect on axon regeneration, and has statistical significance; the effects of albumin hydrogel on NSCs (Cell group) and lithiumalginate-albumin hydrogel group (Gel group) were similar (P. Ltoreq.0.05; P. Ltoreq.0.01; P. Ltoreq.0.001; P. Ltoreq.0.0001; scale=500 um).
The results of the baso-Beattie-Bresnahan (BBB) scoring of the motor functional behavioural assessment after spinal cord injury for each group of rats are shown in fig. 6: as can be seen from fig. 6, the post-injury motor function scores of the lithange-albumin hydrogel-loaded NSCs (cell+gel group) and the albumin hydrogel-loaded NSCs (Cell group) were significantly higher than those of the lithange-albumin hydrogel (Gel group) and the untreated injury group (Ctrl group), wherein Ctrl group had the worst functional recovery (P0.01; P0.001; P0.0001; cell+gel group and Ctrl group).
The results of the 8-week post-operation axon tracing for each group of rats are shown in fig. 7, wherein the graph a is an immunofluorescence graph of the axon tracing for each group, the red is a tagged axon (tracker), the green is a GFAP immunofluorescence marker, and the graph B is a statistical analysis result of the axon number in each group of damaged areas. From fig. 7, it can be seen that the axon regeneration condition after injury of the rat in the loam-albumin hydrogel-loaded NSCs group (cell+gel group) is optimal, and the loam-albumin hydrogel group (Gel group) has better axon regeneration than the single albumin hydrogel-loaded NSCs (Cell group), and the recovery effect of the injured untreated group (Ctrl group) is poor (P is less than or equal to 0.01; P is less than or equal to 0.001; P is less than or equal to 0.0001; scale=500 um).
Differentiation of NSCs in the lithange-albumin hydrogel-loaded NSCs (cell+gel) and albumin hydrogel-loaded NSCs (Cell) was followed using GFP-labeled NSCs, and the results of TUJ-1 immunofluorescent staining of the two groups were 4 weeks post-operatively, as shown in FIG. 8, FIG. 8 shows that NSCs-GFP in the damaged areas of the lithange-albumin hydrogel-loaded NSCs (cell+gel) were highly expressed as TUJ-1, while NSCs in the damaged areas of the albumin hydrogel-loaded NSCs (Cell) were hardly expressed as TUJ-1. The results indicate that the loam-albumin hydrogel can promote survival and differentiation of transplanted NSCs-GFP into neurons in vivo.
In summary, the application discovers that the lithio with specific concentration range has the effect of promoting the differentiation of the neural stem cells to the direction of neurons, but not the direction of astrocytes for the first time; the hydrogel loaded with the lithium algae soil can continuously and effectively promote the differentiation of the neural stem cells to the neurons, has the characteristics of definite nerve induction differentiation effect, controllable ion slow release, long duration of action, low cost and the like, provides a new method and a new path for repairing and treating the nerve injury, effectively overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (8)
1. The application of the lithium algae soil is characterized in that: the particle size of the lithium algae soil is 20-30 nm;
as an active ingredient for preparing a nerve injury repair product; when the lithium algae soil is used as an active ingredient for preparing a nerve injury repair product, the usage amount of the lithium algae soil is 0.1-1% based on the total weight of the nerve injury repair product.
2. The application of the lithium algae soil is characterized in that: the particle size of the lithium algae soil is 20-30 nm;
is used for inducing the neural stem cells to differentiate into neurons in vitro and inhibiting the differentiation of the neural stem cells into astrocytes; when the method is used for inducing the neural stem cells to differentiate into the neuron cells in vitro and inhibiting the neural stem cells from differentiating into astrocytes, the neural stem cells are inoculated into a lithium algae soil differentiation culture medium for culture, and the concentration of the lithium algae soil in the lithium algae soil differentiation culture medium is 1-20 mug/ml.
3. The application of the lithium algae soil is characterized in that: the particle size of the lithium algae soil is 20-30 nm;
as an active ingredient for preparing a product for inducing differentiation of neural stem cells into neurons and inhibiting differentiation of neural stem cells into astrocytes; when the product which is used as an active ingredient and is used for inducing the differentiation of the neural stem cells into neurons and inhibiting the differentiation of the neural stem cells into astrocytes is prepared, the usage amount of the lithiumalga soil is 0.1-1% based on the total weight of the product which is used for inducing the differentiation of the neural stem cells into neurons and inhibiting the differentiation of the neural stem cells into astrocytes.
4. Use according to claim 1, characterized in that: when used as an active ingredient for preparing a nerve injury repair product, the composition acts on nerve injury parts, and the differentiation of nerve stem cells to neurons is induced through the interaction of the lithium algae soil and the nerve stem cells.
5. The use according to claim 2, characterized in that it comprises the following components in the following amounts per 100ml of in vitro lithio differential medium: 92-95 ml of DMEM/F12 culture medium, 1-3 ml of B27, 1-2 ml of N2, 1-2 ml of stock solution of lithium algae soil, 0.5-1 ml of fetal bovine serum and 1-1.5 ml of double-antibody mixed solution of green streptomycin.
6. Use according to claim 5, characterized in that: the solvent of the stock solution of the lithium algae soil is PBS solution.
7. A composite gel material for inducing differentiation of neural stem cells into neurons, characterized by: the composite gel material is formed by mixing hydrogel, a carrier and stock solution of lithium algae soil into gel; the carrier is human serum albumin, and the hydrogel is succinimidyl succinate modified polyethylene glycol; the solvent of the stock solution of the lithium algae soil is PBS solution, and the concentration of the lithium algae soil in the stock solution of the lithium algae soil is 0.5-1.5 mg/ml; and the usage amount of the lithium algae soil is 0.1-1% based on the total mass of the composite gel material.
8. The composite gel material of claim 7, wherein: the volume ratio of the carrier to the stock solution of the lithium algae soil is 1 (1.0-1.2); the mass volume ratio of the hydrogel to the stock solution of the lithium algae soil is 100-200 mg/ml.
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