Method for the preparation of adipose stem cells and their use in neural repair

Abstract

The application provides a preparation method of fat stem cells and the use thereof in nerve repair, and further, a specific differentiation-promoting peptide CF-8 for promoting differentiation of fat mesenchymal stem cells into nerve-like cells is screened from a deer antler polypeptide, wherein the nerve-like cells differentiated after the action of the differentiation-promoting peptide have a good therapeutic effect on treating traumatic brain injury, and have a good application prospect.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for preparing adipose stem cells and their use in nerve repair. Background Technology

[0002] Traumatic brain injury (TBI) refers to damage to the structure and function of the brain caused by external force applied to the head. This injury can be caused by a variety of factors, including traffic accidents, falls, and violent attacks. Based on the severity of the injury, TBI can be classified as mild, moderate, and severe.

[0003] In the pathophysiology of traumatic brain injury (TBI), neuroregulatory mechanisms play a crucial role. These mechanisms involve the interaction of neurotransmitters, neuromodulators, neural circuits, and the neuroendocrine system, collectively influencing key pathological processes such as post-injury inflammatory response, apoptosis, and neural remodeling. Neurotransmitters and neuromodulators are core mediators of neuroregulation; they act on specific receptors to trigger intracellular signal transduction, thereby affecting neuronal function and survival. Following TBI, the levels of various neurotransmitters and neuromodulators undergo significant changes. For example: Glutamate: As a major excitatory neurotransmitter, excessive release in the injury area can lead to excitotoxicity, exacerbating neuronal damage. Gamma-aminobutyric acid (GABA): As a major inhibitory neurotransmitter, dysfunction of GABA can lead to neuronal overexcitation. Nitric oxide (NO): Produced by neurons and glial cells, excessive amounts can trigger oxidative stress and neurotoxicity.

[0004] Neuronal death is a key event in the complex pathophysiology of traumatic brain injury (TBI), with programmed cell death (i.e., neuronal apoptosis) and necrosis being the two main modes of death. They play different roles in the development and progression of neurological deficits after TBI and are influenced by complex neuroregulatory mechanisms. Neuronal apoptosis is a highly regulated form of cell death that is ubiquitous in the body's development and homeostasis. After TBI, various factors, including direct damage caused by mechanical forces, oxidative stress, inflammatory responses, and excitotoxicity, can trigger neuronal apoptosis. This process typically involves the activation of a series of signaling pathways, such as death receptor pathways (e.g., Fas / CD95 and TNFR1) and non-death receptor pathways (e.g., intrinsic mitochondrial pathways). Death receptor pathways execute cell death by activating the caspase cascade, while non-death receptor pathways involve loss of mitochondrial membrane potential, cytochrome C release, apoptosis-inducing factor-1 (Apaf-1) accumulation, and caspase-9 activation. Ultimately, activated effector caspases (such as caspase-3, -6, and -7) cleave multiple substrates, leading to cellular structural damage, chromatin condensation, and apoptotic body formation, which are eventually cleared by phagocytes. Excessive neuronal apoptosis after TBI is closely associated with long-term sequelae such as cognitive and motor impairments. Studies have shown that inhibiting neuronal apoptosis after TBI may be a potential strategy for treating TBI. For example, blocking apoptosis signaling pathways by using death receptor inhibitors or mitochondrial protectants may reduce neuronal loss and improve neurological prognosis. Unlike apoptosis, necrosis is usually a non-regulated, passive form of cell death, mainly caused by severe cellular damage such as intense oxidative stress, energy depletion, and calcium overload. Neuronal necrosis is common in the acute phase of TBI, especially in areas directly compressed by hematoma or severely ischemic. Necrotic cells are characterized by cell swelling, mitochondrial dysfunction, cell membrane rupture, and the release of contents (such as organelles, proteins, and lipids) into the extracellular environment. The released necrotic cell contents (called "damage-associated molecular pattern markers," DAMPs) can further exacerbate the inflammatory response and tissue damage following TBI. For example, pyroptosis, a form of inflammatory cell necrosis, releases inflammatory cytokines such as IL-1β, which recruit more immune cells to the injury site, amplifying the inflammatory storm and further damaging surrounding neural tissue. Therefore, necrosis is not only a form of cell death but also a pathological process that can amplify damage. In the pathological process of TBI, neuronal apoptosis and necrosis do not exist in isolation; there are complex interactions and transformations between them. Certain initial injury stimuli may simultaneously trigger apoptosis and necrosis pathways, and there are also mutual regulatory mechanisms between apoptosis and necrosis signaling pathways.For example, severe oxidative stress may initially lead to mitochondrial damage and cell necrosis, but may subsequently activate apoptosis pathways by releasing cytochrome C. Conversely, some apoptosis inhibitors may also indirectly reduce necrosis by suppressing inflammatory responses. Understanding these complex interactions is crucial for a comprehensive understanding of the neuronal death mechanisms in TBI.

[0005] Drug intervention is one of the important means of treating traumatic brain injury. Currently, a variety of drugs are used to treat traumatic brain injury, including anti-inflammatory drugs, neuroprotective agents, and antioxidants. These drugs can improve patient prognosis by reducing the inflammatory response of brain tissue, promoting neuronal repair and regeneration, and inhibiting oxidative stress through different mechanisms. Anti-inflammatory drugs: Anti-inflammatory drugs can reduce the inflammatory response of brain tissue, reduce cell death and neurodegenerative changes. Commonly used anti-inflammatory drugs include nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and indomethacin, and glucocorticoids such as dexamethasone. Neuroprotective agents: Neuroprotective agents can protect neurons from damage and promote neuronal repair and regeneration. Commonly used neuroprotective agents include N-methyl-D-aspartate receptor antagonists (NMDA receptor antagonists) and calcium channel blockers (Ca2+ channel blockers). Antioxidants: Antioxidants can scavenge free radicals and reduce the damage of oxidative stress to neurons. Commonly used antioxidants include vitamin E, vitamin C, and selenium. Erythropoietin (EPO): EPO is a protein produced by the kidneys that stimulates the bone marrow to produce new red blood cells. Studies have shown that EPO can promote the repair and regeneration of neurons after traumatic brain injury. Neurotrophic Factors: Neurotrophic factors can promote the growth and differentiation of neurons and enhance their function. Research has found that certain neurotrophic factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can promote the repair and regeneration of neurons after traumatic brain injury. Other Medications: In addition to the above-mentioned medications, other drugs are also used to treat traumatic brain injury, such as antidepressants and anti-anxiety drugs. These drugs can improve the patient's mental state and cognitive function by regulating the balance of neurotransmitters.

[0006] Stem cells, as pluripotent primitive cells, can differentiate into various cell types in specific tissues and have shown positive effects in a variety of experimental nerve injuries. However, there is still no consensus on which type of stem cells to choose. Dadon et al. believe that bone marrow mesenchymal stem cells are very promising for the treatment of brain nerve injuries and other neurological diseases. Due to the inconvenience and high cost of obtaining bone marrow mesenchymal stem cells, their use has many limitations. In contrast, adipose-derived mesenchymal stem cells are readily available and inexpensive to prepare, and research on their use in nerve injury repair is still limited, warranting further investigation. Summary of the Invention

[0007] In one aspect, the present invention provides a polypeptide that effectively promotes the differentiation of adipose-derived mesenchymal stem cells into nerve cells, the polypeptide being isolated from deer antler.

[0008] The differentiation-promoting peptide is named CF-8, and its amino acid sequence is shown in SEQ ID NO: 1.

[0009] The present invention also provides the use of the differentiation-promoting peptide CF-8 in the preparation of a reagent for promoting the differentiation of adipose-derived mesenchymal stem cells into neural-like cells.

[0010] Furthermore, the present invention also provides the use of neural-like cells differentiated from adipose-derived mesenchymal stem cells in the preparation of medicaments for treating traumatic brain injury.

[0011] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0012] Furthermore, the present invention also provides the use of nerve-like cells differentiated from adipose-derived mesenchymal stem cells in the preparation of medicaments for treating nerve injuries. The aforementioned neurological damage can therefore include schizophrenia, obsessive-compulsive personality disorder, major depression, bipolar disorder, anxiety disorders, normal aging, epilepsy, retinal degeneration, traumatic brain injury, spinal cord injury, post-traumatic stress disorder, panic disorder, Parkinson's disease, dementia, Alzheimer's disease, mild cognitive impairment, chemotherapy-induced cognitive dysfunction ("chemobrain"), Down syndrome, and hearing loss. Loss, tinnitus, spinocerebellar ataxia, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, stroke, and disorders caused by the abuse of radiation therapy, chronic stress, or neuroactive drugs such as alcohol, opioids, methamphetamine, phencyclidine, and cocaine.

[0013] In this invention, the drug is further used to treat other neuropathies. These neuropathies include peripheral neuropathy and central neuropathy. Examples of diseases accompanied by peripheral neuropathy include: diabetic neuropathy, metabolic peripheral neuropathy associated with uremia, peripheral neuropathy associated with vitamin B deficiency, peripheral neuropathy associated with infectious diseases such as bifidobacteria, botulism, herpes virus (shingles), drug-induced peripheral neuropathy associated with anticonvulsants such as phenytoin sodium, antibacterial agents (chloramphenicol, nitrofurantoin, sulfonamides, etc.), chemotherapeutic agents (taxanes: paclitaxel, docetaxel, etc.), platinum preparations: oxaliplatin, cisplatin, carboplatin, nedaplatin, etc., vinca alkaloids: vincristine, vinblastine, vindesine, etc.), drug-induced peripheral neuropathy associated with the use of sedatives (barbiturates, cyclohexenebarbital, etc.), chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, and entrapment neuropathy. This invention relates to various peripheral neuropathy conditions, including: neuropathy (e.g., carpal tunnel syndrome, thoracic outlet syndrome, cubital tunnel syndrome, piriformis syndrome, meibomian tube syndrome, peroneal nerve restraint neuropathy, etc.); immune peripheral neuropathy such as multifocal motor neuropathy; peripheral neuropathy associated with allergic diseases such as nodular periarteritis, allergic vasculitis, and systemic lupus erythematosus; toxic peripheral neuropathy associated with the uptake of heavy metals such as lead, mercury, arsenic, and thallium; organic solvents such as diluents; organophosphate pesticides; toxic substances such as tri-o-toluyl phosphate (TOCP); alcohols; peripheral neuropathy caused by nerve compression due to cancer; and peripheral neuropathy associated with hereditary diseases (e.g., hypothyroidism, renal failure, Charcot-Marie-Tooth disease, Refsum disease, porphyria, Fabry disease, hereditary stress-sensitive peripheral neuropathy, etc.). Preferably, the differentiation-promoting polypeptide of the present invention is used in combination with a pharmaceutically acceptable carrier. The present invention therefore also provides pharmaceutical compositions suitable for application to the target. Such compositions comprise an effective amount of the compound of the present invention together with a pharmaceutically acceptable carrier. The carrier can be a liquid, in which case the composition is suitable for parenteral administration, or it can be a solid, i.e., formulated as a tablet or pill for oral administration. Alternatively, the carrier can be an aerosolizable liquid or solid form, in which case the composition is suitable for inhalation. If for parenteral administration, the composition should be pyrogen-free and present in an acceptable parenteral carrier. Alternatively, the active polypeptide can be encapsulated in liposomes using known methods.

[0014] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0015] The pharmaceutical compositions of the present invention may also contain isotonicity agents, such as sugars, polyols, such as mannitol, sorbitol, glycerol, or sodium chloride.

[0016] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for a chosen route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, or buffers, which may improve the shelf life or efficacy of the pharmaceutical composition. The compounds of the present invention can be prepared with a carrier that protects the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with waxes or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art.

[0017] In one embodiment, the compounds of the present invention may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is considered, except for any conventional media or agents currently incompatible with the active compounds. Complementary active compounds may also be included in the composition.

[0018] Pharmaceutical compositions intended for injection must generally be sterile and stable under manufacturing and storage conditions. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, by using a coating such as lecithin, in the case of dispersions, appropriate flowability can be maintained by maintaining the required particle size, and by using surfactants. In many cases, isotonic agents, such as sugars, polyols such as glycerol, mannitol, sorbitol, or sodium chloride, are preferably included in the composition. Extended absorption of the injectable composition can be achieved by including a delayed-absorption agent, such as monostearate or gelatin. Sterile injectable solutions can be prepared by incorporating a desired amount of an active compound with one of the ingredients or combinations thereof (if desired), such as those listed above, into a suitable solvent, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired components, such as those listed above. Examples of preparation methods for sterile injectable solutions using sterile powders are vacuum drying and freeze-drying (lyophilization), which produce a powder containing the active ingredient plus any other desired components from its preceding sterile-filtered solution.

[0019] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, the phrases “parenterally” and “via parenterally” refer to administration methods other than enteral and local application, typically by injection, including intradermal, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injections, subcutaneous, and infusions. Further, the method of administration of the present invention is intracerebral injection.

[0020] Beneficial effects

[0021] This invention provides a method for preparing adipose-derived stem cells and their use in nerve repair. Furthermore, the inventors screened specific differentiation-promoting peptides CF-8 from deer antler polypeptides to promote the differentiation of adipose-derived mesenchymal stem cells into neuron-like cells. The neuron-like cells differentiated after treatment with the differentiation-promoting peptide have good therapeutic effects on traumatic brain injury and have good application prospects. Attached Figure Description

[0022] Figure 1 Figure showing the impact of each group on the neurological damage severity score (NSS).

[0023] Figure 2 Results of bar rotation time for each group Detailed Implementation

[0024] Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0025] Example 1: Screening and Identification of Stem Cell Differentiation-Promoting Peptides

[0026] Fresh antlers from farmed sika deer were ground into powder, passed through a 100-mesh sieve, and mixed with double-distilled water at a ratio of 1g:30ml. The mixture was then boiled repeatedly six times. Neutral protease was added at an enzyme-to-substrate ratio of 1:120, and the mixture was hydrolyzed at 47℃ and pH 7.1 for 6 hours, followed by enzyme inactivation at high temperature. Subsequently, trypsin was added at an enzyme-to-substrate ratio of 1:80 for hydrolysis at 37℃ and pH 7.9 for 3 hours, after which the enzyme was inactivated. The components were obtained using ultrafiltration. The effects of each component on stem cell differentiation were examined according to the method described in "Experimental Study on the Induction of Rat Bone Marrow Mesenchymal Stem Cell Differentiation into Neuron-like Cells by Danshen Injection," Yu Qin et al., *Chinese Journal of Integrated Traditional and Western Medicine in Emergency and Critical Care*, Vol. 13, No. 4, July 2006. The results showed that the components with a molecular weight less than 3 kDa exhibited the best differentiation-promoting properties. Fractions with molecular weights less than 3 kDa were subjected to Sephadex-G25 gel column chromatography to obtain the CF fraction with the best differentiation-promoting properties. The CF fraction was further differentiated using DEAE-Sepharose FF ion-exchange chromatography to obtain the CF-8 fraction with the best differentiation-promoting properties. The CF fraction was further purified repeatedly using HPLC C18 reverse-phase column, and its amino acid sequence was determined by ESI-QqTOF mass spectrometry as shown in SEQ ID NO: 1. The CF-8 peptide was then synthesized artificially for later use.

[0027] Example 2: Preparation of adipose-derived mesenchymal stem cells

[0028] Adult male SD rats were anesthetized, and the epididymis was dissected under strict aseptic conditions. The fat pad of the epididymal tail was separated, suspended in 0.01 mol / L PBS, and minced. The cells were then digested with 0.15% collagenase at 37°C with shaking for 40 min. After digestion was terminated with FBS, the cells were centrifuged at 1000 r / min for 10 min, resuspended in DMEM / F12 containing 10% FBS, filtered through a 200-mesh sieve, and the cell density was adjusted to 1×10⁻⁶ cells / mL. 4 / ml of cells were seeded into culture dishes and incubated in a 37°C CO2 incubator. After 4 hours of inoculation, the cell culture medium was transferred to new culture dishes, and adherent cells were removed. The medium was changed for the first time after 48 hours, and then every 3 days thereafter. Cells were passaged when they reached confluence with the bottom of the dish. For passage, cells were digested with 0.25% trypsin and seeded into new culture dishes at a 1:3 ratio.

[0029] Third-generation cells were digested with 0.25% trypsin, centrifuged, washed three times with 0.01 mol / L PBS, and centrifuged again. The supernatant was discarded, and cold PBA (PBS + 2% BSA) was added. Antibodies (anti-CD90-FITC, anti-CD29APC, CD11b-APC, CD45-PE, CD49d-FITC, CD106-PE) targeting the cell surface antigens were added. The cells were incubated in the dark for 15 min, then PBS containing 5% FBS was added, and the cells were centrifuged twice. The supernatant was discarded, and PBS was added before flow cytometry analysis. Centrifugation was performed at 1000 rpm for 5 min each time. Flow cytometry analysis showed that ADSCs showed strong positive expression of CD90 and CD29, and negative expression of CD11b, CD49d, CD45, and CD106. Furthermore, the cells were predominantly long spindle-shaped, consistent with the characteristics of adipose-derived mesenchymal stem cells.

[0030] Example 3: Identification of the Differentiation-Promoting Properties of Differentiation-Promoting Peptide CF-8 in Adipose-Derived Stem Cells

[0031] Experimental group: Adipose-derived mesenchymal stem cells prepared in Example 2 were resuspended in DMEM / F12 containing 2% B27, 10 ng / ml bFGF, and 20 ng / ml EGF, and were cultured at a concentration of 1×10⁻⁶. 5The culture medium was seeded at 1 / ml into 24-well plates, with half the volume changed daily. Fresh medium was added every 3 days. The next step was performed after clonal cell spheroids formed. A blank control group was also included, cultured in DMEM / F12 medium containing 10% FBS. Step 2: The suspended clonal cell spheroids were seeded into 24-well plates pre-coated with poly-L-lysine coverslips. After cell attachment, the original medium was discarded and replaced with DMEM / F12 medium containing 10 ng / ml GDNF, 10 ng / ml BDNF, and 1 μmol / L RA. After 1 day, the medium was replaced with Neurobasal medium containing 10 ng / ml GDNF, 10 ng / ml BDNF, 1 μmol / L RA, and 2% B27. The medium was changed every 3 days. The control group was cultured in Neurobasal medium containing 2% B27 after cell attachment. Phenotypic identification was performed after 9 days.

[0032] Low concentration differentiation-promoting peptide CF-8 group: The steps and times were the same as the previous experimental groups, except that 50 μg / ml of differentiation-promoting peptide CF-8 was added to the culture medium in each step.

[0033] High concentration differentiation-promoting peptide CF-8 group: The steps and times were the same as the previous experimental groups, except that 200 μg / ml of differentiation-promoting peptide CF-8 was added to the culture medium in each step.

[0034] Phenotypic identification of neurons was performed on the induced cells: The cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, permeabilized with 0.1% Triton-100 for 30 min, blocked with 1% BSA for 1 h, and incubated overnight at 4℃ with mouse anti-rat NeuN antibody (1:100) and MAP2 antibody (1:100), respectively. The cells were then warmed to room temperature for 45 min the next day, incubated with fluorescent secondary antibody in the dark for 2 h, washed three times with PBS, and stained with DAPI for 10 min. The cells were then observed and photographed under a fluorescence microscope. The positive rates of NeuN and MAP2 in the cells were calculated. The results are shown in Table 1.

[0035] ,

[0036] MAP2 is a thermostable phosphoprotein belonging to the structural microtubule-associated protein family, primarily expressed in the cell bodies and dendrites of the central nervous system. NeuN is a neuronal nuclear protein; a positive result for both indicates that the cells possess neuronal characteristics. As shown in Table 1, the blank control group produced very few markers for naturally differentiated neuron-like cells, indicating a very low number of naturally differentiated neurons. In contrast, the experimental group, after induction with a specialized culture medium, produced a large number of neuron-like cells. The addition of the differentiation-promoting peptide CF-8 significantly increased the proportion of neuron-like cells in a concentration-dependent manner, demonstrating a significant synergistic effect. This indicates that the differentiation-promoting peptide CF-8 can significantly promote the differentiation of adipose-derived mesenchymal stem cells into neuron-like cells, with a remarkable effect, and can be used for the differentiation-promoting application of adipose-derived mesenchymal stem cells.

[0037] Example 3: Experimental use of adipose-derived mesenchymal stem cells to treat traumatic brain injury

[0038] Experimental Group 1: Adipose-derived mesenchymal stem cells prepared in Example 2 were resuspended in DMEM / F12 containing 2% B27, 10 ng / ml bFGF, 20 ng / ml EGF, and 200 μg / ml differentiation-promoting peptide CF-8, and were cultured at a concentration of 1×10⁻⁶. 5 The medium was seeded at a concentration of 1 / ml into 24-well plates, with half the medium changed daily. Fresh medium was added every 3 days. Once clonal cell spheroids formed, the suspended clonal cell spheroids were seeded into 24-well plates pre-coated with poly-L-lysine coverslips. After cell attachment, the original medium was discarded and replaced with DMEM / F12 medium containing 10 ng / ml GDNF, 10 ng / ml BDNF, 1 μmol / L RA, and 200 μg / ml differentiation-promoting peptide CF-8. After 1 day, the medium was replaced with Neurobasal medium containing 10 ng / ml GDNF, 10 ng / ml BDNF, 1 μmol / L RA, 2% B27, and 200 μg / ml differentiation-promoting peptide CF-8. The medium was changed every 3 days. After 9 days of culture, NeuN-positive cells were screened using flow cytometry to obtain NeuN-positive neuron-like cells.

[0039] Experimental Group 2: The method is the same as that of Experimental Group, except that differentiation-promoting peptides are not used.

[0040] Preparation of a traumatic brain injury (TBI) model: Mice were anesthetized with 3.5% chloral hydrate via intraperitoneal injection (1 ml / 100g body weight). After anesthesia, Kunming mice were fixed in a stereotaxic apparatus. The skull was exposed, the periosteum was dissected, and a bone window with a diameter of approximately 6 mm was drilled 1-2 mm posterior to the anterior fontanelle and 2 mm to the left of the sagittal suture to expose the dura mater. A 40g weight was dropped freely from a height of 25 cm along a vertical tube, impacting a ramus pin (with a cone-shaped tip and a diameter of 3 mm) on the dura mater, inducing TBI. One day after establishing the mouse TBI model, mice were anesthetized and fixed in a stereotaxic apparatus. 10 ml (approximately 1 x 10 ml) was aspirated using a glass microsyringe. 6 Neuromorphic cell suspensions prepared from the BrdU-labeled experimental group were injected at a rate of 2 μl / min. The needle was left in place for 5 minutes after injection, and then slowly withdrawn within 5 minutes. The needle insertion sites were: 3 mm posterior to the anterior fontanelle on the injured side and 2 mm to the left of the suture line. The insertion depth was 3 mm subcortical. The entire transplantation process was performed under aseptic conditions. Postoperatively, the scalp was sutured, and the patient received intraperitoneal gentamicin injections for one week, with adequate food and water.

[0041] The neurological damage severity score (NSS) was used to score the damage, with a maximum score of 18. Scores of 13-18 indicated severe damage, 7-12 indicated moderate damage, and 1-6 indicated mild damage. On the day of cell transplantation (day 0) and 21 days after transplantation, a double-blind method was used to randomly select 10 mice from each group to score the neurological damage severity. Results are as follows... Figure 1 As shown.

[0042] from Figure 1 The results showed that on day 0 after TBI, mice exhibited varying degrees of abnormalities in motor function, sensory function, balance function, and physiological reflexes, with all three groups showing high NSS scores. On day 21 post-transplantation, the NSS score in the control group showed slight improvement, while the NSS score in experimental group 2 was significantly lower than that in the control group. The NSS score in experimental group 1 (4.13±0.41) was significantly lower than that in the control group (8.46±0.32) and experimental group 2 (6.35±0.33). This fully demonstrates that the neuron-like cells obtained by treatment with the differentiation-promoting peptide CF-8 have stronger biological activity and can significantly improve the therapeutic effect of brain injury, with statistically significant differences compared to the control group (P<0.01).

[0043] In addition, each group of animals was placed on a stationary rotundus for 3 minutes, and then the rotation speed was increased from 5 rpm to 40 rpm within 5 minutes. Each animal was tested 3 times, with a 5-minute rest period after each test. If a mouse was passively spun around the rotundus without walking on it, it was considered to have fallen. The time from the start of each mouse's journey to the fall was recorded (in seconds), and the time was assessed on the same day as the neurological function impairment score. Results are as follows: Figure 2 As shown.

[0044] Fatigue transfer bars are used to evaluate the coordination and balance of movement. Figure 2 The results showed no significant difference in rotator times among the groups on the day of transplantation. However, at 21 days post-operation, the rotator times in experimental group 1 (332±13) s and experimental group 2 (298±10) s were significantly longer than those in the control group (259±25) s (P<0.01). This indicates that the treatment effectively improved motor coordination and balance, demonstrating its effectiveness.

[0045] All publications and patents mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated and incorporated herein by reference in its entirety. Although the invention has been described in conjunction with its specific embodiments, it should be understood that it can be further modified, and this application is intended to cover any variations, uses, or alterations of the invention that are generally based on the principles of the invention and include deviations from this disclosure, provided that they fall within the known or customary practice in the field to which the invention pertains and, if applicable, the essential features set forth above herein.

Claims

1. A differentiation-promoting peptide CF-8 isolated from deer antler, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1. The differentiation-promoting peptide CF-8 can effectively promote the differentiation of adipose-derived mesenchymal stem cells into neural-like cells and improve the activity of differentiated cells.

2. The use of a differentiation-promoting peptide CF-8 in the preparation of a culture medium for promoting the differentiation of adipose-derived mesenchymal stem cells into neural-like cells, characterized in that, The amino acid sequence of the differentiation-promoting peptide CF-8 is shown in SEQ ID NO:

1.

3. A method for promoting the differentiation of adipose-derived mesenchymal stem cells into neural-like cells in vitro, characterized in that... The method includes the step of promoting the differentiation of adipose-derived mesenchymal stem cells in a culture medium using the differentiation-promoting peptide CF-8 as described in claim 1.

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