Polypeptide and application thereof

Abstract

The invention relates to a polypeptide and application thereof, and belongs to the technical field of biomedical treatment. The amino acid sequence of the polypeptide comprises a sequence as shown in SEQ ID NO. 1. The invention designs a novel polypeptide, verifies that the novel polypeptide can relieve the inhibition effect of myelin protein molecules and promote the neural stem cells to differentiate into neurons, discovers that the novel polypeptide can be combined with a microtubule stabilizer TPI-287 to promote the neural stem cells to differentiate into neurons, and further develops a product and a method for promoting the neural stem cells to differentiate into neurons. The method is of great significance in promoting nerve regeneration medicine to span from basic research to clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a polypeptide and its applications, particularly its application in promoting the differentiation of neural stem cells into neurons. Background Technology

[0002] Neural stem cells (NSCs) are recognized in the field of neuroregenerative medicine as a strategic resource for treating neurological injuries and degenerative diseases such as spinal cord injury, stroke, and Alzheimer's disease due to their unique self-renewal capacity and multi-directional differentiation potential. Their core value lies in rebuilding damaged neural circuits through cell replacement therapy.

[0003] Under ideal in vitro culture conditions, non-neuronal cells (NSCs) can differentiate into neurons, astrocytes, and other cells according to the laws of neural development. The effective generation of functional neurons is the core biological basis for restoring synaptic transmission, reconstructing neural pathways, and achieving therapeutic functions, directly determining the clinical outcome of improved motor, sensory, or cognitive functions after transplantation. However, when NSCs are transplanted into actual nerve injury lesions, their differentiation fate is drastically reprogrammed by the pathological microenvironment: the massive disintegration of myelin at the injury site releases high concentrations of inhibitory molecules. These myelin-derived inhibitory factors constitute a strong microenvironmental barrier, inhibiting neurogenesis pathways and driving gliosis, ultimately hijacking the differentiation direction of NSCs.

[0004] Therefore, developing directed differentiation technology that can mimic the characteristics of the neural injury microenvironment and specifically reverse its inhibitory effects, and forcibly guiding NSCs to efficiently generate functional neurons in adverse environments, has become a key innovative path to overcome the bottleneck of clinical translation in transplantation therapy. This is of great significance for promoting the transition of neuroregenerative medicine from basic research to clinical practice. For example, CN109481673A discloses a composition that promotes neural repair after brain injury, which alleviates the inhibitory effect of myelin protein molecules after brain injury and promotes the differentiation of neural stem cells into neurons. The composition includes an EGFR antibody drug and neural stem cells.

[0005] In conclusion, developing novel formulations that can alleviate the inhibitory effects of myelin molecules and promote the differentiation of neural stem cells into neurons is of great significance to the biomedical field. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a polypeptide and its application, particularly in promoting the differentiation of neural stem cells into neurons, with the aim of achieving efficient promotion of neural stem cell differentiation into neurons.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a polypeptide whose amino acid sequence includes the sequence shown in SEQ ID NO.1.

[0009] This invention designs a novel polypeptide and verifies that it can alleviate the inhibitory effect of myelin molecules and promote the differentiation of neural stem cells into neurons. It also finds that it can promote the differentiation of neural stem cells into neurons in combination with the microtubule stabilizer TPI-287.

[0010] SEQ ID NO. 1: PDSGRRYVVLPRHAVDIARRLRKFQREKKGKCRKA.

[0011] It is understood that, based on the peptide designed in this invention, functionally similar peptides obtained by using genetic modification methods in the art to perform amino acid substitution, deletion, or addition should all be within the scope of protection of this invention. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence identity between the changed amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In the art, conservative substitution with amino acids of similar or comparable properties usually does not change the function of the peptide; therefore, peptides obtained by conservative substitution with amino acids of similar or comparable properties are also within the scope of protection of this invention.

[0012] In a second aspect, the present invention provides a nucleic acid molecule that encodes the polypeptide described in the first aspect.

[0013] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0014] Fourthly, the present invention provides the application of the polypeptide described in the first aspect in promoting the differentiation of neural stem cells into neurons.

[0015] This invention further develops products and methods to promote the differentiation of neural stem cells into neurons, which is of great significance for advancing the transition of neuroregenerative medicine from basic research to clinical practice.

[0016] Fifthly, the present invention provides a complex for promoting the differentiation of neural stem cells into neurons, the complex comprising a microtubule stabilizer and the polypeptide described in the first aspect.

[0017] Preferably, the concentration ratio of the microtubule stabilizer to the polypeptide is 1 nM:(20~40)μg / mL.

[0018] In a sixth aspect, the present invention provides a culture medium for promoting the differentiation of neural stem cells into neurons, the culture medium comprising a basal culture medium containing the polypeptide described in the first aspect and / or the complex for promoting the differentiation of neural stem cells into neurons described in the fifth aspect.

[0019] Preferably, the basal culture medium includes Neurobasal basal culture medium.

[0020] Preferably, the culture medium for promoting the differentiation of neural stem cells into neurons further contains B27 additive, GlutaMAX, penicillin, and streptomycin.

[0021] In a seventh aspect, the present invention provides a method for promoting the differentiation of neural stem cells into neurons, the method comprising:

[0022] The polypeptide described in the first aspect and / or the complex described in the fifth aspect that promotes the differentiation of neural stem cells into neurons are applied to cultured neural stem cells to induce their proliferation and differentiation into neurons.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] This invention designs a novel polypeptide and verifies that it can alleviate the inhibitory effect of myelin molecules and promote the differentiation of neural stem cells into neurons. It also discovers that it can promote the differentiation of neural stem cells into neurons in combination with the microtubule stabilizer TPI-287. Furthermore, the invention aims to further develop products and methods to promote the differentiation of neural stem cells into neurons, which is of great significance for promoting the transition of neural regenerative medicine from basic research to clinical practice. Attached Figure Description

[0025] Figure 1 This image shows the differentiation of neural stem cells into astrocytes under different conditions using immunofluorescence staining to detect GFAP protein. Green represents GFAP-positive cells, and blue represents Hoechst 33342 staining of all cell nuclei. The scale bar is 100 μm. Image A shows neural stem cells cultured for 7 days with myelin added; Image B shows neural stem cells cultured for 7 days with both myelin and TPI-287 added; Image C shows neural stem cells cultured for 7 days with both myelin and NDP peptide added; Image D shows neural stem cells cultured for 7 days with both myelin and TPI-287 and NDP peptide added; Image E shows the statistical results of the proportion of GFAP-positive cells in the total cell count. * indicates p < 0.05.

[0026] Figure 2This image shows the differentiation of neural stem cells into neurons under different conditions using immunofluorescence staining of NeuN protein. Green represents NeuN-positive cells, and blue represents Hoechst 33342 staining of all cell nuclei. The scale bar is 100 μm. Image A shows neural stem cells cultured for 7 days with myelin added; Image B shows neural stem cells cultured for 7 days with both myelin and TPI-287 added; Image C shows neural stem cells cultured for 7 days with both myelin and NDP peptide added; Image D shows neural stem cells cultured for 7 days with both myelin and TPI-287 and NDP peptide added; Image E shows the statistical results of the proportion of NeuN-positive cells to total cells. * indicates p < 0.05.

[0027] Figure 3 This image shows the differentiation of neural stem cells into neurons under different conditions using immunofluorescence staining to detect MAP2 protein. Green represents MAP2-positive cells, and blue represents Hoechst 33342 staining of all cell nuclei. The scale bar is 100 μm. Image A shows neural stem cells cultured for 7 days with myelin added; Image B shows neural stem cells cultured for 7 days with both myelin and TPI-287 added; Image C shows neural stem cells cultured for 7 days with both myelin and NDP peptide added; Image D shows neural stem cells cultured for 7 days with both myelin and TPI-287 and NDP peptide added; Image E shows the statistical results of the proportion of MAP2-positive cells in the total cells. * indicates p < 0.05. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0030] Example 1

[0031] This embodiment involves polypeptide synthesis.

[0032] A polypeptide (named NDP) with the amino acid sequence: PDSGRRYVVLPRHAVDIARRLRKFQREKKGKCRKA (SEQ ID NO.1) was synthesized using a solid-phase peptide synthesis method with 2-chlorotriphenylmethyl chloride resin as the solid-phase support. Four amino acids with protecting groups are used: N-(9-fluorenylmethoxycarbonyl)-L-alanine (Fmoc-Ala-OH) (catalog number 531480, Sigma-Aldrich), N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-Phe-OH) (catalog number 338338, Sigma-Aldrich), N-(9-fluorenylmethoxycarbonyl)-N1-(tert-butyloxycarbonyl)-L-tryptophan (Fmoc-Trp(Boc)-OH) (catalog number 47561, Sigma-Aldrich), and N-(9-fluorenylmethoxycarbonyl)-O4-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH) (47623, Sigma-Aldrich). First, the carboxyl group of the first amino acid is coupled to the resin. Before each amidation reaction, the Fmoc protecting group is removed using a 20% piperidine / N,N-dimethylformamide (DMF) (catalog number PHR1553, Sigma-Aldrich) solution. Then, the carboxyl group of the other amino acid is activated with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) (catalog number 12804, Sigma-Aldrich) and N,N-diisopropylethylamine (DIPEA) (catalog number 03439, Sigma-Aldrich) to couple it to the free amino group. After the final coupling step, the N-terminal Fmoc protecting group is removed. After rinsing with DMF to remove excess reagent, the mixture is washed with dichloromethane (DCM) (catalog number 34856, Sigma-Aldrich). The peptide chains were dissociated by treating the resin with trifluoroacetic acid (TFA) (catalog number 8.08260, Sigma-Aldrich) for 1 hour, simultaneously removing the protecting groups of the amino acid side chains. After concentration using a rotary evaporator, the peptides and impurities were separated by precipitation with diethyl ether (catalog number 1.00923, Sigma-Aldrich). The crude peptide product was finally purified by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II preparative chromatograph with a C18 reversed-phase column.

[0033] Example 2

[0034] This embodiment describes the isolation and culture of neural stem cells.

[0035] Spinal cord-derived neural stem cells were isolated from the spinal cord tissue of 2-day-old SD rats. The spinal cord tissue was isolated under a dissecting microscope and immersed in HBSS buffer (Catalog No. #C14175500BT, Gibco). After discarding the buffer, the spinal cord tissue was minced with scissors, and 0.25% trypsin / EDTA (Catalog No. #40127ES60, Yisheng) was added for digestion at 37°C for 15 minutes. After digestion, an equal volume of 10% fetal bovine serum (Catalog No. #16000044, Invitrogen) was added to terminate the digestion, and the cells were repeatedly pipetted. After centrifugation at 500 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in culture medium. Cell suspensions were seeded into culture flasks and cultured statically in a 37°C, 5% CO2 incubator using neural stem cell proliferation medium [DMEM / F12 basal medium (catalog number #c11330, Invitrogen) supplemented with 2% B27 additive (catalog number #17504044, Invitrogen), 1% non-essential amino acids (catalog number #11140050, Invitrogen), 1% sodium pyruvate (catalog number #11360070, Invitrogen), 1% penicillin-streptomycin (catalog number #15140122, Invitrogen), 20 ng / mL epidermal growth factor (catalog number #AF-100-15, Proteintech), 20 ng / mL basic fibroblast growth factor (catalog number #AF-100-18B, Merck Millipore), and 6 mg / mL glucose (catalog number #G7021, Sigma-Aldrich)]. After 24 hours, nerve balls were formed and then transferred to suspension culture flasks for continued suspension culture at 37°C and 100 rpm.

[0036] Example 3

[0037] This embodiment describes the isolation of myelin proteins.

[0038] Spinal cord tissue from adult SD rats was homogenized in a solution containing 10 mM HEPES (catalog number H23830, Sigma-Aldrich), 5 mM EDTA (catalog number 03699, Sigma-Aldrich), and 0.32 M sucrose (s5016, Sigma-Aldrich). The homogenate was then plated onto an equal volume of 0.85 M sucrose solution (containing 10 mM HEPES, 5 mM EDTA, pH 7.4) and centrifuged at 24,600 rpm for 30 minutes. The crude myelin sheath fraction was collected from the liquid-liquid interface, resuspended in ice-cold distilled water, and centrifuged at 9,500 rpm for 15 minutes. The precipitate was subjected to two more hypotonic shaking treatments. The final precipitate was dissolved in a solution containing 10 mM HEPES, 5 mM EDTA, and 0.32 M sucrose, and then plated again onto a 0.85 M sucrose solution. The centrifugation and hypotonic shaking steps were repeated. Finally, the purified myelin was resuspended in PBS buffer and stored at -20°C. The concentration of myelin protein was determined using the BCA protein quantification kit (catalog number BCA01, Beijing Dingguo Changsheng Biotechnology Co., Ltd.) according to the manufacturer's instructions.

[0039] Example 4

[0040] This procedure involves the induction and differentiation of neural stem cells.

[0041] Cell culture plates were coated with laminin (1:100 diluted in PBS) (11243217001, Sigma-Aldrich) for 2 hours (37℃). To assess the differentiation capacity of neural stem cells under different culture conditions, suspension-cultured neural stem cell spheres were dissociated into single-cell suspensions using 0.25% trypsin / EDTA (catalog number #40127ES60, Yisheng) and seeded into the aforementioned laminin-coated cell culture plates. Cells were cultured for 7 days under different conditions, and differentiation was assessed. The differentiation medium consisted of Neurobasal medium (catalog number #21103049, Invitrogen) supplemented with 2% B27 additive (catalog number #17504044, Invitrogen), 1% GlutaMAX (catalog number #35050061, Invitrogen), and 1% penicillin-streptomycin (catalog number #15140122, Invitrogen). To simulate the inhibitory microenvironment of myelin after nerve injury, 100 μg / mL of myelin was added to the above differentiation medium as a baseline control group for subsequent comparative evaluation of cell differentiation status under different conditions. Simultaneously, under these baseline culture conditions, different reagents were added to detect their effects on neural stem cells. The first group was cultured with 1 nM of the microtubule stabilizer TPI-287 (catalog number HY-126899, MedChemExpress); the second group was cultured with 30 μg / mL of NDP peptide; and the third group was cultured with both TPI-287 (1 nM) and NDP peptide (30 μg / mL).

[0042] Immunofluorescence staining

[0043] Cell differentiation status was identified by immunofluorescence staining after 7 days of cell culture. For immunocytochemical analysis, cells cultured in culture plates were fixed with 4% paraformaldehyde (PFA) at room temperature for 10-15 minutes. After washing three times with PBS, the fixed samples were permeabilized with PBS containing 0.4% Triton X-100 and 10% bovine serum albumin (BSA) for 60 minutes. Primary antibody incubation was performed overnight at 4°C or 3 hours at room temperature. After washing three times with PBS, the samples were incubated with secondary antibody at room temperature for 45 minutes. Cell nuclei were stained with Hoechst 33342 (catalog number 94403, Sigma-Aldrich).

[0044] Images were acquired using a laser confocal microscope (Leica, Germany) at 20× or 40× magnification. Primary antibodies used included GFAP antibody (catalog number ab4674, abcam), NeuN antibody (catalog number ab177487, abcam), and MAP2 antibody (catalog number ab32454, abcam). Cell differentiation was analyzed by statistically analyzing the percentage of positive cells.

[0045] Research Results

[0046] Immunofluorescence staining of cultured neural stem cells can analyze cell differentiation, and the results are as follows: Figure 1 As shown, neural stem cells differentiated extensively into astrocytes in the presence of myelin, with 88.62% expressing the astrocyte marker GFAP. However, with the addition of the microtubule stabilizer TPI-287 in the presence of myelin, the proportion of GFAP-expressing cells decreased to 85.64%. With the addition of NDP peptide in the presence of myelin, the proportion of GFAP-positive cells was 60.03%. Furthermore, with the simultaneous addition of TPI-287 and NDP peptide in the presence of myelin, the proportion of GFAP-positive astrocytes decreased significantly to 5.89%. These results suggest that TPI-287 and NDP peptide can antagonize the differentiation of neural stem cells into astrocytes in the presence of myelin.

[0047] To further examine the differentiation of neural stem cells into neurons, immunofluorescence staining was performed using neuronal markers NeuN antibody and MAP2 antibody. The results are as follows: Figure 2 As shown, when myelin was added, the percentage of NeuN-positive neural stem cells was 7.08%; when microtubule stabilizer TPI-287 was added in the presence of myelin, the percentage of NeuN-expressing cells was 7.14%; when NDP peptide was added in the presence of myelin, the percentage of NeuN-positive cells was 20.6%; and when both TPI-287 and NDP peptide were added in the presence of myelin, the percentage of NeuN-positive neurons reached 71.89%.

[0048] like Figure 3As shown, in the presence of myelin, the percentage of neural stem cells positive for the neuronal marker MAP2 was 5.16%; while with the addition of the microtubule stabilizer TPI-287 in the presence of myelin, the percentage of MAP2-expressing cells was 16.48%; with the addition of NDP peptide in the presence of myelin, the percentage of MAP2-positive cells was 30.35%; and with the simultaneous addition of TPI-287 and NDP peptide in the presence of myelin, the percentage of MAP2-positive neurons reached 76.69%. These results suggest that TPI-287 and NDP peptide can promote the differentiation of neural stem cells into neurons in the presence of myelin.

[0049] In summary, this invention designs a novel polypeptide and verifies that it can alleviate the inhibitory effect of myelin molecules and promote the differentiation of neural stem cells into neurons. It also discovers that it can promote the differentiation of neural stem cells into neurons in combination with the microtubule stabilizer TPI-287. Furthermore, the development of products and methods to promote the differentiation of neural stem cells into neurons is of great significance for advancing the transition of neuroregenerative medicine from basic research to clinical practice.

[0050] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule of claim 2.

4. Use of the polypeptide of claim 1 in promoting differentiation of neural stem cells into neurons.

5. A complex for promoting differentiation of neural stem cells into neurons, characterized by, The complex comprises a microtubule stabilizer and the polypeptide of claim 1.

6. The complex for facilitating differentiation of neural stem cells into neurons according to claim 5, wherein The concentration ratio of the microtubule stabilizer and the polypeptide is 1 nM:(20-40) μg / mL.

7. A medium for promoting differentiation of neural stem cells into neurons, characterized by, The medium for promoting differentiation of neural stem cells into neurons comprises a basal medium containing the polypeptide of claim 1 and / or the complex of claim 5 or 6 for promoting differentiation of neural stem cells into neurons.

8. The medium for facilitating differentiation of neural stem cells into neurons according to claim 7, wherein, The basal medium comprises Neurobasal basal medium.

9. The medium for promoting differentiation of neural stem cells into neurons according to claim 7 or 8, wherein The medium for promoting differentiation of neural stem cells into neurons further comprises B27 supplement, GlutaMAX, penicillin and streptomycin.

10. A method of promoting differentiation of neural stem cells into neurons, the method comprising, The method comprises: The polypeptide of claim 1 and / or the complex of claim 5 or 6 for promoting differentiation of neural stem cells into neurons is used to act on cultured neural stem cells to induce proliferation and differentiation of the neural stem cells into neurons.

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