Composition of protein for treatment of nerve injury and biological material and use thereof

Abstract

Provided is use of insulin-like growth factor binding protein 2 in nerve injury, specifically use of insulin-like growth factor binding protein 2 in preparing a formulation. The formulation is used for (i) treating nerve injury, (ii) promoting migration, proliferation, and / or activation of glial cells; and / or (iii) promoting neuron growth. Provided is a product. The product comprises (a) insulin-like growth factor binding protein 2; and (b) a matrix gel. Insulin-like growth factor binding protein 2 can promote glial cell activation and / or neuron growth, thereby treating nerve injury.

Description

A protein and biomaterial composition for treating nerve damage and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a protein and biomaterial composition for treating nerve damage and applications thereof. Background Art

[0002] Neurological damage, including damage to the brain, spinal cord, and peripheral nerves, can result from a variety of causes, including trauma, stroke, infection, metabolic disorders, and toxins. Common symptoms include pain, numbness, tingling, muscle weakness, poor coordination, vision or hearing loss, speech impairment, difficulty swallowing, and difficulty urinating or defecating. Central nervous system damage in adults is often permanent and irreversible.

[0003] Spinal cord injury (SCI) refers to damage to the spinal cord that causes temporary or permanent functional changes. It is characterized by high morbidity, high treatment costs, high disability rates, and a low age of onset. Depending on the cause, pathophysiology, and severity of the injury, SCI can be categorized as traumatic or non-traumatic, acute SCI can be divided into primary and secondary injuries, and complete and incomplete injuries. Globally, the incidence of SCI varies, ranging from approximately 239 to 1,009 cases per million people. In China, over one million people suffer from SCI, and the number of patients is growing at a rate of 120,000 per year.

[0004] The treatment and prognosis of nerve injury depend on the type and severity of the injury. Taking spinal cord injury (SCI) as an example, the current treatment drugs and methods are very limited, and the symptom improvement and functional recovery achieved by patients through treatment are also very limited.

[0005] Currently, clinical research on the treatment of spinal cord injury mainly focuses on new drug development, cell therapy, biomaterial transplantation, and physical regulation. The only clinical drug approved by the U.S. Food and Drug Administration (FDA) for spinal cord injury is methylprednisolone (MP). A large amount of clinical evidence shows that the use of large doses of MP can produce harmful side effects and may increase the incidence of complications such as infection, gastrointestinal bleeding, hyperglycemia, and steroid-induced myopathy. Issues such as the poor efficacy of stem cell therapy, unclear time window, associated tumorigenic risks, immune response after transplantation, and cell survival rate in the human body are all issues that need to be carefully considered.

[0006] Therefore, there is an urgent need in this field to develop a nerve injury drug with good therapeutic effect.

[0007] Summary of the Invention

[0008] The object of the present invention is to provide a method for treating and / or preventing nerve damage.

[0009] Another object of the present invention is to provide use of insulin-like growth factor binding protein 2 for treating or preventing nerve damage.

[0010] The first aspect of the present invention provides a use of insulin-like growth factor binding protein 2 for preparing a preparation or a pharmaceutical composition, wherein the preparation or the pharmaceutical composition is used for

[0011] (i) treatment of nerve damage,

[0012] (ii) promoting the proliferation and / or activation of glial cells;

[0013] (iii) inhibiting neuronal degeneration and / or death;

[0014] (iv) promoting axonal growth;

[0015] (v) promoting recovery of sensory function after nerve injury; and / or

[0016] (vi) Treatment of neurodegenerative diseases.

[0017] In another preferred embodiment, the insulin-like growth factor binding protein 2 is derived from human or mouse.

[0018] In another preferred embodiment, the insulin-like growth factor binding protein 2 includes wild-type and / or mutant insulin-like growth factor binding protein 2.

[0019] In another preferred embodiment, the insulin-like growth factor binding protein 2 has the amino acid sequence shown in SEQ ID NO: 1.

[0020] In another preferred embodiment, the preparation or pharmaceutical composition is used to treat acute nerve injury.

[0021] In another preferred embodiment, the glial cells include astrocytes.

[0022] In another preferred embodiment, the glial cells are primary glial cells.

[0023] In another preferred embodiment, the glial cells are primary astrocytes.

[0024] In another preferred embodiment, the nerve injury includes spinal cord injury.

[0025] In another preferred embodiment, the spinal cord injury includes complete spinal cord transection injury and / or hemi-spinal cord transection injury.

[0026] In another preferred embodiment, the spinal cord injury is selected from the group consisting of spinal cord trauma, spinal cord compression, spinal cord inflammation, or a combination thereof.

[0027] In another preferred embodiment, the nerve damage is caused by trauma, stroke, neurodegenerative disease, metabolic disorder, immune abnormality and / or infection.

[0028] In another preferred embodiment, the neurons are from cortical neurons.

[0029] In another preferred embodiment, the preparation or pharmaceutical composition promotes the growth of nerve axons.

[0030] In another preferred embodiment, the growth of nerve axons is promoted by activating glial cells.

[0031] In another preferred embodiment, the average length of the longest neurite L1 of neurons treated with the preparation or pharmaceutical composition is compared with the average length of the longest neurite L0 of neurons not treated with the preparation or pharmaceutical composition, and the two ratios (L1 / L0) are ≥1.2, preferably ≥1.3, and more preferably ≥1.4.

[0032] In another preferred embodiment, the promoting motor recovery after nerve injury comprises:

[0033] (u1) Improve fine motor accuracy after neurological injury; and / or

[0034] (u2) Improve overall motor coordination after nerve injury.

[0035] In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, spinocerebellar ataxia, multiple sclerosis, optic atrophy, peripheral neuropathy and other nervous system diseases characterized by neuronal degeneration and / or death.

[0036] In another preferred embodiment, the preparation or pharmaceutical composition is administered to a subject selected from the group consisting of a rodent or a primate.

[0037] In another preferred embodiment, the subject suffers from a disease related to nerve damage.

[0038] In another preferred embodiment, the subject is a human or non-human mammal.

[0039] In a second aspect, the present invention provides a pharmaceutical composition comprising:

[0040] (i) an active ingredient comprising insulin-like growth factor binding protein 2; and

[0041] (ii) a pharmaceutically acceptable carrier.

[0042] In another preferred embodiment, the pharmaceutical composition is a gel preparation.

[0043] In another preferred embodiment, the pharmaceutically acceptable carrier comprises matrix gel.

[0044] In another preferred embodiment, the matrix gel is a thermosensitive gel.

[0045] In another preferred embodiment, the matrix gel is selected from the group consisting of matrix gel, hydrogel, gelatin, composite material, or a combination thereof.

[0046] In another preferred embodiment, the pharmaceutical composition is used for

[0047] (i) treatment of nerve damage,

[0048] (ii) promoting the proliferation and / or activation of glial cells;

[0049] (iii) inhibiting neuronal degeneration and / or death;

[0050] (iv) promoting axonal growth;

[0051] (v) promoting recovery of sensory function after nerve injury; and / or

[0052] (vi) Treatment of neurodegenerative diseases.

[0053] In another preferred embodiment, the pharmaceutical composition activates glial cells to promote the growth of nerve axons.

[0054] In another preferred embodiment, the glial cells include astrocytes.

[0055] In another preferred embodiment, the glial cells are primary glial cells.

[0056] In another preferred embodiment, the nerve injury includes spinal cord injury.

[0057] In another preferred embodiment, the spinal cord injury includes complete spinal cord transection injury and hemi-spinal cord transection injury.

[0058] In another preferred embodiment, the spinal cord injury is selected from the group consisting of spinal cord trauma, spinal cord compression, spinal cord inflammation, or a combination thereof.

[0059] In another preferred embodiment, the nerve damage is caused by trauma, stroke, neurodegenerative disease, metabolic disorder, immune abnormality and / or infection.

[0060] In another preferred embodiment, the pharmaceutical composition promotes the growth of nerve axons.

[0061] In another preferred embodiment, the neurons are from cortical neurons.

[0062] In another preferred embodiment, the promoting motor recovery after nerve injury comprises:

[0063] (u1) Improve fine motor accuracy after neurological injury; and / or

[0064] (u2) Improve overall motor coordination after nerve injury.

[0065] In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, spinocerebellar ataxia, multiple sclerosis, optic atrophy, peripheral neuropathy and other nervous system diseases characterized by neuronal degeneration and / or death.

[0066] In another preferred embodiment, the preparation or pharmaceutical composition is administered to a subject selected from the group consisting of a rodent or a primate.

[0067] In another preferred embodiment, the subject suffers from a disease related to nerve damage.

[0068] In another preferred embodiment, the subject is a human or non-human mammal.

[0069] In a third aspect, the present invention provides a method for activating glial cells in vitro, comprising the steps of:

[0070] Insulin-like growth factor binding protein 2 is overexpressed in glial cells to activate glial cells.

[0071] In another preferred embodiment, the method comprises the steps of:

[0072] Insulin-like growth factor binding protein 2 was overexpressed in glial cells cultured in vitro, thereby activating glial cells.

[0073] In another preferred embodiment, the method comprises the steps of:

[0074] Insulin-like growth factor binding protein 2 is brought into contact with glial cells cultured in vitro, thereby activating the glial cells.

[0075] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0076] In another preferred embodiment, the glial cells include astrocytes.

[0077] In another preferred embodiment, the glial cells are primary glial cells.

[0078] In another preferred embodiment, the glial cells are primary astrocytes.

[0079] In a fourth aspect, the present invention provides a method for promoting neuronal growth in vitro, comprising the steps of:

[0080] Bringing insulin-like growth factor binding protein 2 into contact with neurons, thereby promoting neuronal growth.

[0081] In another preferred embodiment, the method comprises the steps of:

[0082] Neurons are cultured under the condition of 125 ng / mL to 1000 ng / mL of insulin-like growth factor binding protein 2, thereby promoting neuronal growth. In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0083] In another preferred embodiment, the neurons are cortical neurons.

[0084] A fifth aspect of the present invention provides a method for treating nerve damage and / or nerve degeneration, comprising the steps of:

[0085] Administer a safe and effective amount of the pharmaceutical composition according to the second aspect of the present invention to a subject in need.

[0086] In another preferred embodiment, the administration includes oral administration, injection, or a combination thereof.

[0087] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0088] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0089] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIG1 shows the results of the effect of insulin-like growth factor binding protein 2 (IGFBP2) levels on the proliferation ability of astrocytes.

[0091] FIG2 shows the results of the effect of insulin-like growth factor binding protein 2 (IGFBP2) levels on the morphology of astrocyte reactivity.

[0092] FIG3 shows the results of the effect of insulin-like growth factor binding protein 2 (IGFBP2) levels on the reactivity and migration ability of astrocytes.

[0093] FIG4 shows the result that the culture medium supernatant containing secretions of astrocytes overexpressing insulin-like growth factor binding protein 2 (IGFBP2) can promote the growth of nerve axons.

[0094] FIG5 shows the result that directly adding insulin-like growth factor binding protein 2 (IGFBP2) to neuronal culture medium can promote the growth of nerve axons.

[0095] FIG6 shows the results that matrix gel treatment containing insulin-like growth factor binding protein 2 (IGFBP2) can reduce the degeneration and loss of neurons caused by spinal cord injury.

[0096] FIG7 shows the results that matrix gel containing insulin-like growth factor binding protein 2 can promote the recovery of sensory function after spinal cord injury (von Frey Hair test).

[0097] FIG8 shows the results that matrix gel treatment containing insulin-like growth factor binding protein 2 (IGFBP2) can promote motor function recovery after spinal cord injury (BMS score experiment).

[0098] FIG9 shows the results that matrix gel treatment containing insulin-like growth factor binding protein 2 (IGFBP2) can promote the recovery of fine motor function after spinal cord injury (irregular ladder experiment).

[0099] FIG10 shows the results of the matrix gel treatment containing insulin-like growth factor binding protein 2 (IGFBP2) that can promote the recovery of motor coordination after spinal cord injury (gait analysis experiment).

[0100] Figure 11 shows a summary diagram of the multi-layered and multi-level regulatory effects of insulin-like growth factor binding protein 2 on astrocytes and neurons, which jointly promote the rapid recovery of neurological function after injury. DETAILED DESCRIPTION

[0101] After extensive and in-depth research, numerous experiments, and screening, the present inventors unexpectedly discovered for the first time that insulin-like growth factor binding protein 2 (IGFBP2) can promote the proliferation and / or activation of glial cells, as well as promote axonal growth, delay neurodegeneration, and reduce neuronal loss, thereby treating nerve damage, particularly spinal cord injury. This invention was completed on this basis.

[0102] the term

[0103] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0104] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., is understood to include the stated elements or components but does not exclude other elements or components.

[0105] Insulin-like growth factor binding protein 2

[0106] Insulin-like growth factor binding protein 2 (IGFBP2) is one of six different IGF (insulin-like growth factor) binding proteins. Previous studies have suggested that IGF-binding proteins primarily regulate IGF's biological activity in the body by binding to it, enhancing or inhibiting its effects, thereby regulating cell growth, differentiation, and metabolism. IGFBP2 is the second most abundant IGF-binding protein in the systemic circulation and the most abundant in cerebrospinal fluid. In addition to regulating the biological activity and stability of IGF, IGFBP2 has also been found to participate in the regulation of various physiological and pathological processes, including embryonic development and metabolic regulation.

[0107] The IGFBP2 of the present invention has the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0108] The amino acid sequence of human IGFBP2 is as follows:

[0109] The amino acid sequence of mouse IGFBP2 is as follows:

[0110] The IGFBP2 of the present invention also includes the amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 or 2 while maintaining the protein function.

[0111] The IGFBP2 of the present invention can be obtained by the following methods:

[0112] (a) Commercially available;

[0113] (b) Endogenous overexpression through viral infection or other means.

[0114] preparation

[0115] The formulation provided herein preferably contains 0.1-99 wt% IGFBP2. If desired, one or more pharmaceutically acceptable carriers may be added to the formulation. Such carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, and the like.

[0116] The preparations provided by the present invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or infusion.

[0117] The various dosage forms of the preparation of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the formulation generally contains 0.05-1000 mg of the IGFBP2 of the present invention, preferably 1-500 mg of the IGFBP2 of the present invention.

[0118] The preparations of the present invention can be used clinically in mammals, including humans and animals, and can be administered via the oral, nasal, dermal, pulmonary, or gastrointestinal routes. Oral administration is most preferred. The most preferred daily dose is 0.01-400 mg / kg body weight taken as a single dose, or 0.01-200 mg / kg body weight taken in divided doses. Regardless of the route of administration, the optimal individual dose will depend on the specific treatment being used. Typically, a low dose is started and gradually increased until the most suitable dose is found.

[0119] The preparations of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation, such as introduction into the body into muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or can be mixed or encapsulated with other substances and introduced into the body.

[0120] Typically, the IGFBP2 of the present invention or a preparation containing it can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0121] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0122] The IGFBP2 of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems.

[0123] In order to prepare the IGFBP2 of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropyl alcohol, etc.; adhesives can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0124] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0125] To prepare the dosing unit as a capsule, the active ingredient IGFBP2 of the present invention can be mixed with a diluent and a glidant, and the mixture can be placed directly into a hard or soft capsule. Alternatively, the active ingredient can be first mixed with a diluent, a binder, and a disintegrant to form granules or pellets, which are then placed into a hard or soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.

[0126] To prepare the IGFBP2 of the present invention as an injection, water, ethanol, isopropanol, propylene glycol, or a mixture thereof can be used as the solvent, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. Examples of solubilizers or cosolvents include poloxamer, lecithin, and hydroxypropyl-β-cyclodextrin; pH adjusters include phosphates, acetates, hydrochloric acid, and sodium hydroxide; and osmotic pressure regulators include sodium chloride, mannitol, glucose, phosphates, and acetates. For lyophilized powder injections, mannitol, glucose, and the like can also be added as support agents.

[0127] Furthermore, if necessary, coloring agents, preservatives, perfumes, flavoring agents or other additives may be added to the preparation.

[0128] The IGFBP2 or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0129] When the IGFBP2 of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0130] Pharmaceutical composition

[0131] The pharmaceutical composition of the present invention comprises: (i) an active ingredient, wherein the active ingredient comprises insulin-like growth factor binding protein 2; and (ii) a pharmaceutically acceptable carrier.

[0132] The pharmaceutical composition of the present invention is a gel preparation.

[0133] In another preferred embodiment, the pharmaceutically acceptable carrier comprises matrix gel.

[0134] In another preferred embodiment, the matrix gel is selected from the group consisting of matrix gel, hydrogel, gelatin, composite material, or a combination thereof.

[0135] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for treating nerve damage.

[0136] In another preferred embodiment, the pharmaceutical composition is used for (i) treating nerve damage, (ii) promoting the proliferation and / or activation of glial cells; (iii) reducing neuronal degeneration and / or death; and / or (iv) promoting axon growth.

[0137] In another preferred embodiment, the pharmaceutical composition is used as a glial cell activator.

[0138] In another preferred embodiment, the pharmaceutical composition activates glial cells to promote neuronal growth.

[0139] In another preferred embodiment, the glial cells include astrocytes.

[0140] In another preferred embodiment, the glial cells are primary glial cells.

[0141] In another preferred embodiment, the nerve injury includes spinal cord injury.

[0142] In another preferred embodiment, the spinal cord injury includes complete spinal cord transection injury and hemi-spinal cord transection injury.

[0143] In another preferred embodiment, the spinal cord injury is selected from the group consisting of spinal cord trauma, spinal cord compression, spinal cord inflammation, or a combination thereof.

[0144] In another preferred embodiment, the nerve damage is caused by trauma, stroke, neurodegenerative disease, metabolic disorder, immune abnormality and / or infection.

[0145] In another preferred embodiment, the pharmaceutical composition is used as a promoter of neuronal survival.

[0146] In another preferred embodiment, the pharmaceutical composition promotes the growth of nerve axons.

[0147] In another preferred embodiment, the neurons are from cortical neurons.

[0148] In another preferred embodiment, the pharmaceutical composition also has one or more of the following uses: (u1) reducing neuronal degeneration and / or death; (u2) promoting recovery of sensory function after nerve injury; (u3) promoting recovery of motor function after nerve injury; (u4) improving the accuracy of fine movements after nerve injury; and / or (u5) improving overall motor coordination after nerve injury.

[0149] When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the active ingredient comprising insulin-like growth factor binding protein 2 can be reasonably determined based on the weight, age, sex, and symptom severity of each patient to be treated.

[0150] The pharmaceutical compositions of the present invention are suitable for use in the acute phase of nerve damage. They are administered by injection, preferably intraspinal injection. The dosage and frequency of administration of the pharmaceutical compositions of the present invention can be appropriately determined based on the weight, age, sex, and severity of symptoms of each patient being treated. For example, the dosage and frequency of administration are determined by the physician and are generally based on the disease / condition being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to practitioners.

[0151] In another preferred embodiment, the pharmaceutical composition of the present invention is administered once during the acute phase of nerve damage, and then administered again after an interval of 1, 2 or 3 months.

[0152] The main advantages of the present invention include:

[0153] (1) The IGFBP2 of the present invention promotes the proliferation and / or activity of astrocytes.

[0154] (2) The IGFBP2 of the present invention can promote the axon growth of primary cortical neurons.

[0155] (3) The IGFBP2 of the present invention can reduce neuronal degeneration and / or death after spinal cord injury.

[0156] (4) The IGFBP2 of the present invention can improve sensory function, motor function, fine motor accuracy and overall motor coordination after spinal cord injury.

[0157] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0158] Example 1 IGFBP2 protein level affects astrocyte proliferation

[0159] 1.1 Method

[0160] 1.1.1 Plasmid construction

[0161] The expression plasmids used in this example are pCDH-FLAG and pCDH-Igfbp2-FLAG. The plasmid construction steps are as follows: spinal cord tissue of healthy mice was obtained, total RNA was extracted, and after reverse transcription into cDNA, the mouse IGFBP2 protein (protein coding sequence is shown in SEQ ID NO. 2) was extracted from the cDNA by PCR and inserted into the pCDH plasmid (constructed by Steven Artandi et al., see Addgene plasmid #167463; http: / / n2t.net / addgene:167463; RRID:Addgene_167463) by homologous recombination using the ClonExpress™ II One Step Cloning Kit (Vazyme). The insertion sites are EcoRI and BamHI.

[0162] Mouse mIGFBP2 protein coding sequence:

[0163] The FLAG tag was added to the primers, and the primer sequences were as follows:

[0164] IGFBP2 forward primer (F-terminal primer):

[0165] 5'-CTCCATAGAAGATTCTAGAGCTAGCGAATTCATGCTGCCGAGATTGGGC-3'(SEQ ID NO:4)

[0166] IGFBP2 downstream primer (reverse primer, R-terminal primer):

[0167] 5'-GATCCTTCGCGGCCGCGGATCCTTACTTGTCATCGTCGTCCTTGTAATCCTGCACACTTTGGGCATGG-3'(SEQ ID NO:5)

[0168] 1.1.2 Lentiviral packaging

[0169] Transfect 293T cells with the target plasmid constructed in 1.1.1 and the viral packaging plasmids psPAX2 and pMD2.G using Lipofectamine 2000 (Invitrogen, 11668). The total amount of transfected plasmid is 2 μg / mL, the target plasmid, psPAX2, and pMD2.G are used in a 1:4:3 ratio, and the ratio of transfection reagent to transfected plasmid is 1:2 (w / v). Seed the 293T cells 12-18 hours in advance and begin transfection when the cell plating rate reaches approximately 90%. About 6 hours after transfection, the cell medium was changed, and the cell supernatant (containing the target virus) was collected 48 hours later. The cell supernatant was then filtered with a 0.45 μm filter membrane and concentrated using a virus concentrate Lenti-X Concentrator (Clontech, 631232) (the ratio of cell supernatant to virus concentrate was 3:1, and the mixture was thoroughly mixed). The mixture was placed in a 4°C refrigerator overnight and then centrifuged at 1500 g for 45 minutes at 4°C. Finally, the mixture was resuspended with pre-cooled PBS, aliquoted, and frozen in a -80°C refrigerator.

[0170] 1.1.3 Primary astrocyte culture

[0171] Two-day-old C57BL / 6J mice were decapitated and brains were removed. The cerebellum, olfactory bulbs, and meninges were removed. The remaining tissue was minced and digested sequentially with 0.05% DNase I (Sigma, DN-25) for 5 minutes, followed by 0.05% trypsin (Gibco, 15090-046) at room temperature for 20 minutes. The cells were then filtered through a 70 μm cell strainer (FALCON, 352350) to remove undissociated tissue and debris. The filtrate was centrifuged at 150 g for 10 minutes, the supernatant discarded, and the cell pellet resuspended. The resuspended cells were seeded into T-75 culture flasks (Thermo) coated with 50 μg / mL poly-L-lysine (Sigma) and cultured in a 37°C, 5% CO2 incubator.

[0172] The cell culture medium was changed every other day. The composition of the culture medium was Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Biowest) and 2% penicillin-streptomycin (Gbico). After 15 days of culture, the cells were allowed to confluent and astrocytes were purified. The culture flask was shaken to remove loosely attached cells, and then digested with 0.05% DNase I (Sigma) at 37°C for 5 minutes to remove oligodendrocyte progenitor cells. The remaining adherent cells were then digested with 0.25% trypsin for 5 minutes. The cell suspension was mixed with an equal volume of growth medium to inactivate the trypsin and centrifuged to collect the cell pellet. Afterwards, the resuspended cells were incubated in bacterial-grade plates at 37°C for two rounds of incubation for 20 minutes and 90 minutes, respectively, to effectively remove microglia, as microglia are more easily attached to the plate than astrocytes. Finally, the supernatant was transferred to a new T-75 culture flask and cultured for about two weeks until the primary astrocytes grew to confluence, and then subsequent experimental operations could be performed.

[0173] 1.1.4 Cell proliferation count

[0174] Purified astrocytes were seeded at a density of 100,000 cells in 12-well plates pre-coated with 50 μg / mL poly-L-lysine. One day after seeding, the cells were infected with the target virus for four days to overexpress the target protein, IGFBP2. Fresh culture medium was replaced before counting. Cell proliferation was recorded using a Leica DMi8 on day 5 after seeding. Six non-overlapping areas were randomly selected for each group at each time point. Cell counts were performed using ImageJ.

[0175] 1.2 Results

[0176] As shown in Figure 1, overexpression of IGFBP2 protein in astrocytes cultured in vitro can promote cell proliferation. It can be seen that the number of cells per unit area in the control group was 22.7±1.3 / mm 2 , while the number of astrocytes per unit area in the IGFBP2 overexpression group was 38.0±2.0 / mm 2 The proliferation ability of astrocytes overexpressing IGFBP2 protein was nearly doubled compared with that of ordinary astrocytes.

[0177] The results showed that overexpression of IGFBP2 protein in astrocytes can promote cell proliferation.

[0178] Example 2 IGFBP2 protein levels affect astrocyte activation

[0179] 2.1 Methods

[0180] 2.1.1 Plasmid construction

[0181] The expression plasmids used in this example are pCDH-FLAG, pCDH-Igfbp2-FLAG, pCDH-HA, pCDH-Igfbp2-HA, pLKO.1-sh-Ctrl (scrambled shRNA) or pLKO.1-sh-Igfbp2. The construction steps of pCDH-FLAG, pCDH-Igfbp2-FLAG, pCDH-HA, and pCDH-Igfbp2-HA are the same as above.

[0182] For pLKO.1-sh-Igfbp2, the sh-Igfbp2 sequence was designed according to the website and ligated into the pLKO.1 plasmid (constructed by Moffat et al., see Addgene plasmid #10879; http: / / n2t.net / addgene:10879; RRID:Addgene_10879) with insertion sites at EcoRI and AgeI. The pLKO.1-sh-scramble plasmid was constructed by Sarbassov et al., see Addgene plasmid #1864; http: / / n2t.net / addgene:1864; RRID:Addgene_1864).

[0183] The primer sequences for pLKO.1-sh-Igfbp2 are as follows:

[0184] Sh-Igfbp2 forward primer (F-terminal primer):

[0185] 5'-CCGGACTGTGACAAGCATGGCCGGTCTCGAGACCGGCCATGCTTGTCACAGTTTTTTG-3'(SEQ ID NO.4)

[0186] Sh-Igfbp2 downstream primer (reverse primer, R-terminal primer):

[0187] 5'-AATTCAAAAAACTGTGACAAGCATGGCCGGTCTCGAGACCGGCCATGCTTGTCACAGT-3'(SEQ ID NO.5)

[0188] In this example, the virus packaging process and primary astrocyte culture were as described in Example 1.

[0189] 2.1.2 Immunofluorescence

[0190] The cells treated in 2.1.1 above were washed once with PBS, fixed with 4% paraformaldehyde (Bioss) for 20 minutes, and permeabilized with 0.5% Triton-X100 (Sigma) for 30 minutes. Finally, the cells were blocked in PBS buffer (PBST) containing 3% goat serum (Beyotime) and 0.1% Triton-X100 (Sigma) for 1 hour at room temperature. Antibodies (rabbit anti-HA (CST) and rat anti-GFAP (Thermo)) were added and incubated overnight at 4°C. The cells were washed three times with PBST at room temperature and labeled with fluorescent secondary antibodies (goat anti-rabbit-Alexa Fluor 488 (Thermo) and goat anti-rat-Alexa Fluor 568 (Thermo)) for 1 hour at room temperature. The cells were washed three more times with PBST at room temperature and mounted with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories). The signals were observed and collected under a spinning disk confocal microscope (Andor) at 40×.

[0191] 2.1.3 Morphological analysis

[0192] To evaluate the morphology of astrocytes, ImageJ (Skeletonize3D) was used to extract the astrocyte skeleton image, and the AnalyzeSkeleton plug-in was used to obtain the morphological characteristics of astrocytes and quantify the number of their branches and intersections.

[0193] 2.1.4 Gene expression signature analysis

[0194] After complete spinal cord transection in mice, spatial transcriptome sequencing was performed using the 10×Visium spatial transcriptome platform. A type of astrocytes with high expression of IGFBP2 that appeared after injury was identified and discovered. Differential gene expression analysis was performed between them and astrocytes before injury, and GO analysis was performed on the upregulated genes.

[0195] 2.2 Results

[0196] Figure 2A: Representative confocal images of astrocytes (left) and corresponding cytoskeleton images (right). Confocal images demonstrate the morphological characteristics of primary mouse astrocytes cultured in vitro. Cytoskeleton images demonstrate the characteristics of the cell structure.

[0197] Figure 2B shows the morphological analysis results of the total number of astrocyte branches (top) and process intersections (bottom). The number of branches and the number of cell branch intersections for each astrocyte are shown, as well as their statistical data.

[0198] Activated astrocytes can be characterized by their morphology and motility.

[0199] As shown in Figure 2 , the wild-type astrocytes in the control group had an average of 10.5±1.0 branches and 4.8±0.5 bifurcation points, while the astrocytes overexpressing IGFBP2 had an average of 26.4±4.3 branches and 12.4±2.1 bifurcation points. This shows that overexpression of IGFBP2 can promote the morphology of astrocytes to have more branches and more branch intersections.

[0200] The results showed that overexpression of IGFBP2 protein in astrocytes can induce the morphology of astrocytes to change from an inactive state (slender and few branches) to an activated state (hypertrophic and many branches).

[0201] As shown in Table 1, the expression of genes representing functional activation was significantly upregulated in astrocytes with high IGFBP2 expression. These genes are mainly involved in the following cellular functions:

[0202] (1) Regulate cell differentiation, promote cell movement and migration, and / or respond to damage, etc., mainly including genes: S100a6, Serpina3n, Cd44, Clu, Cd9, Ctsb, H2-D1, B2m, Cd63, C4b, Olfml3;

[0203] (2) responding to cytokines and external stimuli, including the following genes: Ccl2, H2-K1, Lgals3bp, Csf1, Ctsl, Fgfr1;

[0204] (3) Regulates ion transmembrane transport, neurotransmitter uptake, sodium ion transport, etc., and the main genes involved are Vegfa, Gpr37l1, and Fgfr3.

[0205] Table 1

[0206] In addition, other upregulated characteristic genes in astrocytes with high IGFBP2 expression include: Ndrg2, Aldoc, Ckb, Ldhb.Atp1a2, Ndufc1, Pmm1, Uqcr10, Cpe, Sparcl1, Pfkm, Idh3b, Camk2g, Abat, Slc1a2, Gpr37l1, Ttyh1, Aldh1l1, Scg3, Pygb, Ank2, Agpat 3,Sdc3,Lrrc8a,Phyhipl,Acsl6,Glud1,Ndufa12,Nckap1,Ntrk2,Kidins220,Fndc4,Atp1b2,Acsbg 1,Pla2g7,Pgk1,Bcan,Ddt,Uqcrfs1,Aco2,Ahcyl1,Tmx2,Suclg1,Atp2a2,Dclk1,Pfkp,Enah,Htra1, Tubb2a,Higd1a,Slc20a1,Tsc22d3,Slc6a1,Cxcl14,Ntm,Ntsr2,Lgi1,Enho,Igfbp2,Chil1,Fgfr3,Paqr7,Slc38a3,Tri l, Kcnk1, Gjb6, Slc7a10, Cldn10, Lsamp, Arxes2, Trim9, Slc1a4, Slc6a11, Itih3, Gpld1, Slc25a18, Trpm3, Baalc, Hpgd, etc.

[0207] As shown in Figures 2 and 3 and Table 1, IGFBP2 can induce astrocytes to exhibit activated morphological characteristics and increase the expression levels of genes representing the activation of astrocyte functions, thereby promoting their transformation into activated astrocytes.

[0208] Example 3 IGFBP2 protein level affects astrocyte migration

[0209] 3.1 Methods

[0210] The plasmid construction, virus packaging process, and primary astrocyte culture in this example are as described in Examples 1 and 2.

[0211] Purified astrocytes were seeded at a density of 7.5 million cells in 12-well plates pre-coated with 5 μg / mL fibronectin (Sigma, F2006). After 4-5 days of growth, the cells were infected with the target virus for 6 days to overexpress or knockdown the target protein IGFBP2. Before wounding, the cells were pretreated with 10 μg / mL mitomycin (Sigma) for 2 hours to eliminate the influence of cell proliferation on migration. After pretreatment, fresh culture medium was replaced, and a 20 μL pipette tip was used to create a uniform scratch from bottom to top. Cell migration in the scratched area was recorded using a Leica DMi8 at 0, 24, and 48 hours after the scratch. To quantify the gap size during cell migration, the scratch area was measured using ImageJ. Six non-overlapping areas were randomly selected for each group at each time point, and the percentage of gap area was calculated using the following formula: relative gap size (%) = S / S0 × 100%, where S represents the number of pixels in the cell-free area of ​​the sample under that condition, and S0 is the number of pixels in the same sample at 0 h after scratching.

[0212] 3.2 Results

[0213] Figures 3A and 3B show the in situ migration of astrocytes in the control group (lentivirally infected with pCDH-FLAG) and the experimental group (lentivirally infected with pCDH-Igfbp2-FLAG) at 0, 24, and 48 hours after wounding. Figures 3C and 3D show the in situ migration of astrocytes in the control group (lentivirally infected with pLKO.1-sh-Ctrl (scrambled shRNA)) and the experimental group (lentivirally infected with pLKO.1-sh-Igfbp2) at the corresponding time points after wounding. All experiments in Figure 3 were repeated three times, and approximately 100 cells were counted in each group. Statistical significance was determined by Student's t-test. No significant differences were found between the two unlabeled groups; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Same below.

[0214] As shown in Figure 3, the relative scratch area of ​​astrocytes in the overexpression control group 48 hours after scratching was 35.3%±2.4% of the original, while the scratch area of ​​astrocytes in the IGFBP2 overexpression group was only 15.3%±1.7% of the original. Overexpression of IGFBP2 significantly promoted the migration of astrocytes.

[0215] In contrast, the relative scratch area of ​​astrocytes in the knockdown control group 48 hours after scratching was 31.6%±1.8% of the original, while the scratch area of ​​astrocytes in the IGFBP2 knockdown group was 46.1%±2.0% of the original, indicating that knockdown of IGFBP2 significantly inhibited astrocyte migration.

[0216] As shown in Figure 3 , IGFBP2 protein levels affected the dynamic migration process of astrocytes.

[0217] Exogenous overexpression of IGFBP2 protein in astrocytes can promote cell migration.

[0218] Examples 1, 2 and 3 show that the protein expression level of IGFBP2 can affect the proliferation and activation of astrocytes.

[0219] Example 4: Culture medium supernatant containing secretions from astrocytes overexpressing IGFBP2 protein promotes axonal growth

[0220] 4.1 Methods

[0221] The expression plasmid, virus packaging and primary astrocyte culture used in this example are the same as those in Examples 1 and 2.

[0222] 4.1.1 Primary cortical neuron culture

[0223] Mouse cortical neurons were isolated from E16-E18 mouse embryos as follows: After embryonic stage E16-E18 mice were removed from their mothers and placed in cold HANKs (Sigma), the mice were decapitated, the brains removed, and the cortex isolated. The isolated cortex was digested with DNase (Sigma, DN-25) and papain (Worthington, LS003126) at 37°C for 20 minutes. After digestion, the isolated cortical neurons were resuspended and cultured in serum-free Neurobasal medium (Invitrogen, 21103049) supplemented with 2% B27 (Invitrogen, 17504044), 1% GlutaMAX (Invitrogen, 35050061), and penicillin-streptomycin (Invitrogen, 10378016), and then plated at the desired experimental density.

[0224] 4.1.2 Preparation of astrocyte-conditioned medium

[0225] Six days after primary astrocytes were infected with the corresponding lentivirus, the supernatant of astrocyte-conditioned medium was collected and concentrated approximately 40-fold using a 10 kDa Amicon Ultra-0.5 centrifugal filter (Millipore, UFC501024). The total protein concentration of the astrocyte-conditioned medium was assessed using a BCA protein assay kit (ThermoFisher, 23225), and the astrocyte-conditioned medium was aliquoted and stored at -80°C until use.

[0226] 4.1.3 Cortical Neuron Neurite Outgrowth Experiment

[0227] After 24 hours of culture of primary mouse cortical neurons, conditioned medium from astrocytes of the corresponding treatment was added. After 24 hours of treatment, 10 fields of view from three independent replicate wells were randomly selected for each independent experiment and imaged using a Leica DMi8. More than 280 cells were collected from three independent replicates for each group. The collected images were used to measure neurite length using the "NeuronJ" function of FUJI software.

[0228] 4.2 Results

[0229] Figure 4 (A) and (B) show the growth status of axons in mouse cortical neurons 24 hours after the addition of culture medium supernatant containing secretions from astrocytes overexpressing IGFBP2-FLAG (experimental group) or expressing FLAG (control group), respectively.

[0230] Figures 4, C and D, show the axonal growth patterns of mouse cortical neurons 24 hours after treatment with culture supernatant containing astrocytes secreted by IGFBP2 knockdown (sh-Igfbp2) or control (sh-Scramble) astrocytes. P values ​​were determined using the Student's t-test, where ** indicates p < 0.01 and *** indicates p < 0.001.

[0231] As shown in Figure 4, 24 hours after adding the conditioned medium of astrocytes overexpressing the control group, the average longest neurite length of neurons cultured in vitro was 152.9±98.0 μm, while 24 hours after adding the conditioned medium of astrocytes overexpressing IGFBP2, the average longest neurite length increased to 200.5±124.3 μm.

[0232] After adding the conditioned medium of astrocytes from the knockdown control group for 24 hours, the average longest neurite length of neurons cultured in vitro was 139.4±78.0 μm, while after adding the conditioned medium of astrocytes overexpressing IGFBP2 for 24 hours, the average longest neurite length was reduced to 126.6±69.4 μm.

[0233] As shown in Example 4, factors secreted and released by activated astrocytes induced by overexpression of IGFBP2 can promote the growth of neuronal axons.

[0234] Example 5 Direct addition of IGFBP2 protein to neuronal culture medium can promote axonal growth

[0235] 5.1 Methods

[0236] The primary cortical neuron cultures used in this example were the same as those used in Example 4. IGFBP2 protein can be expressed by astrocytes and released extracellularly as a secretory protein, thereby acting on surrounding cells, particularly neurons. After 24 hours of in vitro culture of mouse primary cortical neurons, varying amounts of IGFBP2 protein were added to the culture medium, resulting in final IGFBP2 concentrations of 0, 125, 250, 500, and 1000 ng / mL, respectively. After 24 hours of treatment, 10 fields of view from three independent replicate wells in each experiment were randomly selected and imaged using a Leica DMi8. Over 280 cells were collected from three independent replicates for each group. Neurite length was measured using the "NeuronJ" function in Fuji software using the collected images.

[0237] 5.2 Results

[0238] Figure 5 (A) and (B) show the neurite outgrowth of cortical neurons after treatment with different concentrations of IGFBP2 for 24 hours. IGFBP2 treatment significantly promoted neurite outgrowth. Statistical significance was determined by one-way analysis of variance using p-value analysis. ns indicates no statistical difference, and *** indicates p < 0.001.

[0239] As shown in Figure 5, the average longest neurite length of cortical neurons cultured in vitro was 70.2±21.5 μm after 24 hours without the addition of IGFBP2 protein. When the concentration of IGFBP2 protein was 125 ng / mL, the average longest neurite length increased to 89.5±29.4 μm, an increase of 27.5%.

[0240] When the concentration of IGFBP2 protein was further increased to 250, 500, and 1000 ng / mL, an increase in the average longest neurite length of neurons cultured in vitro was also observed, which was 89.1±29.7 μm, 98.0±34.4 μm, and 115.3±41.1 μm, respectively. Compared with the case without IGFBP2 addition, the average longest neurite length of neurons could be increased by 64.2%.

[0241] The results showed that IGFBP2 protein concentrations above 125 ng / mL up to 1000 ng / mL could promote the growth of nerve axons.

[0242] IGFBP2 protein can act directly on neurons to promote neurite growth.

[0243] Example 6 IGFBP2 protein matrix gel treatment reduces neuronal loss caused by spinal cord injury

[0244] 6.1 Methods

[0245] 6.1.1 Spinal Cord Transection Injury and IGFBP2 Protein Matrix Gel Injection

[0246] Spinal cord injury experiments were performed using adult female C57BL / 6J mice aged 8-9 weeks. Mice were first anesthetized with isoflurane, and then the back hair was removed with depilatory cream to expose the cervical and dorsal skin. Scissors were then used to cut the skin, fascia, and muscle, exposing and removing the lamina of the tenth thoracic vertebra (T10) vertebral segment. The spinal cord was then completely severed at T9-T10 using microscissors. To evaluate the effect of IGFBP2 protein on the pathological recovery of spinal cord injury, recombinant mouse IGFBP2 protein (Bio-techne, 797-B2) was prepared in sterile PBS to a 100 μg / mL stock solution and mixed with Matrigel (Corning, 354248) to a final concentration of 5 μg / mL. After spinal cord injury, the wound was cleaned and Matrigel containing IGFBP2 or an equal volume of PBS (for the control group) was immediately injected into the injury site using a 10 μL microsyringe (Hamilton). After the Matrigel solidified at the injury site, the muscles and skin were sutured, and the mice were placed on a heating pad until they regained consciousness. They were then placed in a clean cage with free access to food and water. Lincomycin-lidocaine gel was applied to the wounds of the mice, and they were manually voided at least twice daily for one week after surgery.

[0247] 6.1.2 Spinal cord sampling

[0248] After anesthetizing the mice to be collected, they were perfused through the heart with PBS and then fixed with 4% (w / v) paraformaldehyde (PFA) (Bioss, C2055). Next, the dorsal skin of the mice was exposed, the vertebrae were trimmed with scissors, and then the vertebrae were peeled off one by one with forceps to expose the white spinal cord. The spinal cord tissue 1 cm before and after the injury site was cut with microscissors and fixed with 4% PFA overnight at 4°C. The fixed spinal cord samples were dehydrated and precipitated in PBS containing 20% ​​and 30% (w / v) sucrose (Sangon, A0498). After completion, the surface moisture of the tissue was blotted dry, and then embedded in Tissue-Tek OCT (Sakura, 4583), quickly frozen in liquid nitrogen, and stored at -80°C.

[0249] 6.1.3 Frozen sections

[0250] Sectioning was performed using a cryostat (Leica). Prior to cryosectioning, the sample was equilibrated to -20°C in the cryostat for approximately 30 minutes. The spinal cord was then mounted on a specimen holder and the surface OCT was trimmed with a blade until the injury site was exposed. The spinal cord was then positioned and the handwheel was used to perform cryosectioning, with sections of 20 μm each. The sections were then mounted on slides and stored at -80°C.

[0251] 6.1.4 Tissue immunofluorescence

[0252] The spinal cord sections on the slides were incubated at 37°C for 1 minute, then fixed again with 4% PFA for 15 minutes at room temperature, washed three times with PBS for 10 minutes each, and then permeabilized and blocked with blocking solution (PBS containing 10% normal goat serum and 0.5% Triton X-100) for 1 hour at room temperature. After blocking, the primary antibody Chicken anti-NeuN (Sigma, ABN91, 1:1000) was added to the blocking solution and incubated with antibodies at 4°C overnight. The next day, the sections were washed three times with PBS containing 0.3% Triton X-100 for 20 minutes each, and then the secondary antibody goat anti-chicken-Alexa Fluor 488 (Life Technologies, A11039) was added and incubated at room temperature for 1 hour. The sections were washed again with PBS containing 0.3% Triton X-100 for three times, each time for 20 minutes, and then mounted with VECTASHIELD antifade mounting medium containing DAPI (Vector Laboratories, H1200). Finally, images were taken using a Dragonfly spinning disk confocal microscope (Andor).

[0253] 6.2 Results

[0254] Figure 6 shows immunofluorescence staining of neurons (green signal) in the ventral horn (A), dorsal horn (C), and central gray matter (E) of the spinal cord transverse sections taken 1 mm from the spinal cord injury site in mice treated with a control group (treated with PBS-containing matrix gel) and an experimental group (treated with IGFBP2 protein-containing matrix gel) 1, 3, and 7 days after spinal cord injury. Figures 6 (B, D, and F) show the statistical analysis of neuronal density in the control and experimental groups for the experiments shown in A, C, and E.

[0255] As shown in FIG6 , after spinal cord injury, there was obvious neuronal loss in various regions of the spinal cord at the injury site of the mouse (including the ventral horn, dorsal horn and intermediate gray matter of the spinal cord).

[0256] In the ventral horn of the spinal cord, the neuron density is about 30 / mm one day after injury. 2 , reduced to 20 / mm 7 days after injury 2 However, after treatment with IGFBP2-containing matrix gel, 30 cells / mm2 were still maintained 7 days after injury. 2 .

[0257] In the dorsal horn of the spinal cord, the neuron density was approximately 130 / mm 1 day after injury. 2 , 7 days after injury, it decreased to about 110 / mm 2, while the IGFBP2-treated group had 150 cells / mm 7 days after injury. 2 .

[0258] In the central gray matter of the spinal cord, the neuron density is about 60 / mm one day after injury. 2 , 7 days after injury, it decreased to 40 / mm 2 , while the neuronal density of the IGFBP2-treated group was 75 / mm 7 days after injury. 2 .

[0259] As shown in Figure 6, IGFBP2-treated mice showed more neuronal preservation compared to control mice at the same time point.

[0260] Treatment with matrix gel containing IGFBP2 protein can delay neuronal death in spinal cord injury.

[0261] Treatment of the injury site with IGFBP2 protein matrix gel can significantly reduce neurodegeneration and neuronal loss caused by spinal cord injury.

[0262] Example 7 IGFBP2 protein matrix gel treatment promotes sensory function recovery after spinal cord injury in mice

[0263] 7.1 Methods

[0264] 7.1.1 Spinal Cord Hemisection Injury and IGFBP2 Protein Matrix Gel Injection

[0265] The injection of IGFBP2 protein matrix gel was the same as in Example 6. The spinal cord was injured by right spinal cord hemisection.

[0266] 7.1.2 von Frey Hair Assessing Sensory Function After Spinal Cord Injury

[0267] Mice were placed in a plastic box with a metal grid bottom for 30 minutes. Once stable, they were stimulated vertically with either a 0.6g or 2.0g von Frey hair to the mid-plantar aspect of the foot until the hair bent and the hind limb lifted, a positive response. Each intensity of von Frey hair stimulation was applied 10 times to the left and right paws, and the proportion of paw-lifting responses was recorded. The assessment was blinded throughout, and the scorers were unaware of the specific treatment. The mice were scored before and weekly after injury.

[0268] 7.2 Results

[0269] Figure 7 shows the recovery of mechanical tactile function on the injured side of mice at specific time points after spinal cord injury, assessed using 0.6g (A) and 2.0g (B) filaments in the von Frey Hair test, respectively, in a control group (treated with PBS-containing matrix gel) and an experimental group (treated with IGFBP2 protein-containing matrix gel). Statistical significance was determined using the Student's t-test. "ns" indicates no significant difference between the two groups. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0270] As shown in Figure 7, the mechanical tactile function of the experimental group and the control group on the injured side was almost completely lost one day after injury. When stimulated by 0.6g fiber filaments 10 times, the mechanical tactile function of the mice in the experimental group recovered to a positive response of 1.8±0.3 times / 10 stimulations 2 weeks after injury, while the control group mice did not recover significantly at this time (0.6±0.3 times) and there was a significant difference between them and the IGFBP2-treated group.

[0271] The sensory function of animals in the IGFBP2-treated group showed sustained and rapid improvement, reaching a positive response of 6.5±0.4 times / 10 stimulations 4 weeks after injury and recovering to a maximum of 8.1±0.3 times / 10 stimulations 8 weeks after injury.

[0272] In contrast, mice in the control group recovered only 3.3±0.3 times at 8 weeks after injury. Even when given stronger pressure stimulation (e.g., using a 2.0g fiber), the sensory function of mice in the control group still showed poor recovery at 8 weeks after injury, with specific experimental data showing 2.7±0.3 positive responses per 10 stimulations. In contrast, the positive response of the IGFBP2-treated group was 8.7±0.2 times per 10 stimulations at 8 weeks after injury.

[0273] Regardless of whether weak or strong pressure stimulation was given, the recovery of sensory function in the IGFBP2-treated mice after spinal cord injury was significantly faster and higher than that in the control group mice.

[0274] Treatment with IGFBP2 protein matrix gel after spinal cord injury can significantly accelerate and improve the recovery of sensory function.

[0275] Example 8 IGFBP2 protein matrix gel treatment promotes motor function recovery in mice after spinal cord injury

[0276] 8.1 Methods

[0277] 8.1.1 Spinal Cord Hemisection Injury and IGFBP2 Protein Matrix Gel Injection

[0278] The spinal cord injury method and IGFBP2 protein matrix gel injection used were the same as those in Example 7.

[0279] 8.1.2 BMS Scoring

[0280] All mice to be measured were first stabilized in the testing room for 1 hour before behavioral assessment. Blind assessment was performed throughout the process, and the scorers were unaware of the specific treatment methods. The mice to be measured were scored before injury and weekly after injury. The specific operating steps are as follows: the mice were placed in an open field for 4 minutes. Two scorers blindly assessed the hind limb motor properties and evaluated the hind limb motor performance through the BMS score, including joint movement, weight support, plantar stepping, coordination, paw position, trunk and tail control. The BMS score ranges from 0 to 9 points (0 indicates complete paralysis and 9 indicates normal activity).

[0281] 8.2 Results

[0282] As shown in Figure 8 , the left and right hind feet of all mice could move normally before injury.

[0283] After right spinal cord hemisection, both the control group (treated with PBS-containing matrix gel) and the experimental group (treated with IGFBP2 protein-containing matrix gel) showed loss of motor function in the injured hindlimb immediately after injury, manifested by plantar inversion and a BMS score of 0; whereas the motor function of the uninjured hindlimb was only slightly affected (Table 2).

[0284] Two weeks after injury, the motor function of the injured hind limbs of the control group mice (PBS matrix gel treatment group) recovered spontaneously to a certain extent, but the motor function of the injured hind limbs of the mice in the IGFBP2 protein matrix gel treatment group recovered significantly faster than that of the control group.

[0285] The motor function of the hind limb of the healthy side recovered to a level close to that of the healthy side 3-4 hours after injury, while the control group needed more than 8 weeks to recover to a level close to that of the healthy side.

[0286] Table 2: BMS scores of mice in each group at different time points after injury (Mean ± SEM)

[0287] The results showed that IGFBP2 protein matrix gel treatment promoted motor function recovery after spinal cord injury in mice.

[0288] Example 9 IGFBP2 protein matrix gel treatment improves the accuracy of fine motor movements in mice after spinal cord injury

[0289] 9.1 Methods

[0290] 9.1.1 Spinal Cord Hemisection Injury and IGFBP2 Protein Matrix Gel Injection

[0291] The spinal cord injury method and IGFBP2 protein matrix gel injection used were the same as those in Example 7.

[0292] 9.1.2 Irregular ladder climbing test

[0293] All mice to be measured were first stabilized in the test room for 1 hour before behavioral assessment. Before injury, mice were trained on a regular ladder so that there was no significant difference in the error rate of mice. After injury, the movement of mice on an irregular ladder was video recorded weekly. The distance between the crossbars of the irregular ladder varied from 1 to 2 cm. The mice to be tested were placed on one side of the ladder and made to crawl from one side to the other, back and forth 3 times, and the process was recorded by video. Finally, the video recording was analyzed frame by frame. The number of errors (Error) and hits (Hit) were recorded according to the Metz and Whishaw evaluation criteria. Errors were divided into complete errors (Miss) and slips (Slip). Complete errors were defined as deep falls after the mouse's hind limbs missed the crossbar. Slips were defined as deep or slight falls after the mouse's hind limbs slipped from the ground. Correct assessments were defined as the mouse's hind limbs stepping on the crossbar and being able to proceed to the next step, including partial placement (only the toes or heels were placed on the crossbar) and correct placement (the middle of the sole of the foot was placed on the crossbar). Immediately after injury and 2 to 8 weeks after injury, the mice were tested once a week in the irregular ladder climbing test, and the error rate and hit rate changes were tracked and statistically analyzed.

[0294] 9.2 Results

[0295] As shown in FIG9 , immediately after injury, when the mouse climbed the irregular ladder, the plantar posterior foot on the injured side turned upward, and the plantar posterior foot could not step on the horizontal bar in all steps, with an error rate of 100% and a hit rate of 0%.

[0296] Two weeks after the injury, the error rate of the control group mice dropped to about 90%, and the hit rate was 10%; while the error rate of the mice in the IGFBP2 protein matrix gel treatment group dropped to about 70%, and the hit rate increased to 30%, which was three times the hit rate of the control group mice.

[0297] Compared with the control group mice, the mice in the IGFBP2 protein matrix gel-treated group had a consistently lower error rate and a consistently higher hit rate in this experimental test, indicating that IGFBP2 protein matrix gel treatment can significantly and continuously improve the accuracy of fine movements in mice after spinal cord injury.

[0298] Example 10 IGFBP2 protein matrix gel treatment improves overall motor coordination in mice after spinal cord injury

[0299] 10.1 Methods

[0300] 10.1.1 Spinal Cord Hemisection Injury and IGFBP2 Protein Matrix Gel Injection

[0301] The spinal cord injury method and IGFBP2 protein matrix gel injection used were the same as those in Example 7.

[0302] 10.1.2 Mouse Gait Analysis

[0303] Gait analysis of mice was performed using a gait meter (Shanghai Xinruan). The mice were first placed in a room to acclimate to the environment for 30 minutes, and then placed in a dark box on one side of the gait meter. They were made to walk from one side to the other along a glass track, and their gait was recorded. During the entire walking process, the mice could not turn around or stop, and they had to pass through the track at a relatively uniform speed. Finally, a series of gait parameters were visualized and evaluated, such as stride frequency and swing time, movement speed, and coordination. At each time point, the average value of each mouse in each behavioral test was calculated.

[0304] 10.2 Results

[0305] As shown in Figure 10, the motor coordination of mice was assessed using a gait analysis system 8 weeks after spinal cord injury. Green represents the gait parameters of mice without injury, black represents the gait parameters of mice in the control group (PBS matrix gel treatment), and red represents the gait parameters of mice in the experimental group (IGFBP2 protein matrix gel treatment).

[0306] Figure 10 A represents the stride frequency: before injury, the stride frequency of mice was about 15 steps / second; after injury, the stride frequency of the control group mice dropped to 10 steps / second, while the stride frequency of the mice in the IGFBP2 protein matrix gel treatment group was 14 steps / second, which was significantly higher than that of the control group.

[0307] Figure 10B shows the overall crawling speed: before injury, the overall crawling speed of the mice was about 13 cm / s; after injury, the movement speed of the control group mice dropped to 8 cm / s, while the movement speed of the IGFBP2 protein matrix gel treatment group was 11 cm / s, which was significantly higher than that of the control group.

[0308] Figure 10, C, shows the swing speed of the injured hind limbs: before injury, the swing speed of the mouse hind limbs was about 30 cm / s; after injury, the swing speed of the hind limbs of the control group mice dropped to about 10 cm / s, while that of the IGFBP2 protein matrix gel-treated group was about 20 cm / s, which was twice the swing speed of the injured hind limbs of the control group mice.

[0309] Figure 10, D, shows the proportion of support time of the injured hind limbs: when uninjured, the proportion of support time of the mouse's hind limbs was approximately 60%; after injury, the proportion of support time of the hind limbs of the control group mice dropped to approximately 25%, while the proportion of support time of the IGFBP2 protein matrix gel treatment group was approximately 45%, nearly twice that of the control group.

[0310] The above results indicate that various gait parameters of the group treated with IGFBP2 protein matrix gel were significantly improved, and IGFBP2 protein matrix gel treatment can significantly improve the coordination of the overall motor ability of mice after spinal cord injury.

[0311] Examples 8, 9 and 10 show that IGFBP2 protein matrix gel treatment can promote rapid recovery and significantly improve motor function, fine motor accuracy and overall motor coordination in mice after spinal cord injury.

[0312] As shown in FIG11 , IGFBP2 protein can promote the proliferation and / or activation of astrocytes and the growth of nerve axons, and has a significant promoting significance for the survival of neurons and the recovery of sensory and motor functions after spinal cord injury.

[0313] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of insulin-like growth factor binding protein 2, characterized in that, For preparing a preparation or pharmaceutical composition, the preparation or pharmaceutical composition is used (i) treatment of nerve damage, (ii) promoting the proliferation and / or activation of glial cells; (iii) inhibiting neuronal degeneration and / or death; (iv) promoting axonal growth; (v) promoting recovery of sensory function after nerve injury; and / or (vi) Treatment of neurodegenerative diseases.

2. The use according to claim 1, characterized in that The glial cells include astrocytes.

3. The use according to claim 1, characterized in that The nerve damage includes spinal cord injury.

4. The use according to claim 3, characterized in that The spinal cord injury includes complete spinal cord transection injury and / or hemi-spinal cord transection injury.

5. The use according to claim 1, characterized in that The neurons are derived from cortical neurons.

6. The use according to claim 1, wherein The promoting of motor recovery after nerve injury comprises: (u1) Improve fine motor accuracy after neurological injury; and / or (u2) Improve overall motor coordination after nerve injury.

7. A pharmaceutical composition, characterized in that Include: (i) an active ingredient comprising insulin-like growth factor binding protein 2; and (ii) a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein Used for (i) treatment of nerve damage, (ii) promoting the proliferation and / or activation of glial cells; (iii) inhibiting neuronal degeneration and / or death; (iv) promoting axonal growth; (v) promoting recovery of sensory function after nerve injury; and / or (vi) Treatment of neurodegenerative diseases.

9. A method for activating glial cells in vitro, characterized in that: Including steps: Insulin-like growth factor binding protein 2 is overexpressed in glial cells to activate glial cells.

10. A method for promoting neuron growth in vitro, characterized in that: Including steps: Bringing insulin-like growth factor binding protein 2 into contact with neurons, thereby promoting neuronal growth.

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