WO2023028746A1 - 基于kdm2b序列合成的生物活性多肽在间充质干细胞神经分化和再生修复中的应用 - Google Patents

基于kdm2b序列合成的生物活性多肽在间充质干细胞神经分化和再生修复中的应用 Download PDF

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WO2023028746A1
WO2023028746A1 PCT/CN2021/115296 CN2021115296W WO2023028746A1 WO 2023028746 A1 WO2023028746 A1 WO 2023028746A1 CN 2021115296 W CN2021115296 W CN 2021115296W WO 2023028746 A1 WO2023028746 A1 WO 2023028746A1
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peptide
seq
kdm2b
stem cells
polypeptide
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French (fr)
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范志朋
曹杨杨
张琛
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首都医科大学附属北京口腔医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the invention relates to the technical field of bioengineering, in particular to a biologically active polypeptide synthesized based on histone demethylase KDM2B and its application in the process of mesenchymal stem cell neural differentiation and regeneration and repair of damaged nerve tissue.
  • Craniomaxillofacial nerve tissue is widely distributed and vulnerable to injury. Epidemiological surveys in recent years have shown that 55.2% of patients with craniofacial trauma have varying degrees of nerve damage, 2.2% of patients are also accompanied by spinal cord contusion, and 24.0% of patients with subsequent treatment complications have neurological dysfunction.
  • the treatment methods mainly used neuroprotective measures such as warming, neuroprotective drugs such as glucocorticoids, glutamate antagonists, etc., and used electricity, heat, force, etc. to stimulate nerve self-healing, etc. These treatments are complex and cyclical. Long, low recovery rate of neurological function, leading to high disability rate.
  • Autologous/allogeneic nerve transplantation has difficulties in obtaining donor tissue and secondary damage to the site where the material is obtained. At the same time, the transplanted nerve cannot be well shaped, and the shape of the tissue structure and damaged function cannot be well restored. At present, the clinical treatment effect of injured nerves still needs to be improved.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • the good differentiation ability of MSCs can promote the formation of nerve repair cells and axon regeneration, and ultimately promote the regeneration and repair of damaged nerves.
  • the sources of neurogenic stem cells are limited and difficult to obtain, and more seed stem cells still need to be discovered.
  • Odontogenic MSCs develop from the neural crest, which originally originated from the ectoderm and is closely related to neural tissue, so odontogenic MSCs have the advantage of transformation application.
  • SCAP Stem Cells from the Apical Papilla
  • the histone methylation modification group present in the gene promoter region hinders the transcriptional expression of the gene (such as H3K27me3) or promotes the transcriptional expression of the gene (such as H3K4me3), and histone methylation/demethylation enzymes in the group
  • the methylation modification of proteins plays a key regulatory role, and there are also certain functional interactions (such as the formation of complexes through the combination of functional domains).
  • EZH2 belongs to the core member of the Polycomb group (PcGs), and its modified H3K27me3 is a gene transcriptional repressive histone methylation modification, which is considered to hinder the differentiation process of specific cell fates.
  • KDM2B was involved in the recruitment of the PRC2-EZH2 complex to the anchoring of downstream target genes and the subsequent covalent modification of histones, suggesting that there may be a crossover in epigenetic regulation between KDM2B and EZH2.
  • KDM2B is a histone demethylase, which mainly participates in the regulation of various cellular processes by removing the trimethylation modification (H3K4me3) of the histone H3K4 site.
  • H3K4me3 trimethylation modification
  • this method is "peptide microarray analysis", protein-based immunoassay Hybridization test, using the protein of interest to hybridize with a microarray chip synthesized based on the full-length amino acid sequence of another protein, and using enzyme-linked labels to amplify the signal of this hybridization combination, thereby identifying possible binding fragment sites and Specific amino acid sequence information for this possible site. Therefore, the possible binding functional domain fragments between KDM2B and EZH2 can be studied by using the method of peptide microarray.
  • the purpose of this study is to clarify the effect of KDM2B on the neural differentiation and regeneration of odontogenic mesenchymal stem cells, to clarify the possible interaction between KDM2B and EZH2, and to study the possible binding site fragments between KDM2B and EZH2 by using the method of polypeptide microarray information.
  • the present invention provides a biologically active polypeptide based on the synthesis of histone demethylase KDM2B and its regulation method in the neural differentiation process of mesenchymal stem cells and the regeneration and repair of damaged nerve tissue, aiming to solve the existing problems
  • the technology does not involve the regulation of the KDM2B gene and its biologically active polypeptides in the neural differentiation process of dental-derived mesenchymal stem cells and the regeneration and repair of damaged nerve tissue.
  • the invention provides an application of KDM2B overexpression in preparing preparations or medicines for inducing neural differentiation of apical papilla stem cells in vitro.
  • the overexpression of KDM2B is used in the preparation of preparations or medicines for promoting the formation of ⁇ III-TUBULIN-positive and NESTIN-positive neurospheres from apical papilla stem cells in vitro.
  • the present invention also provides the application of the overexpression of KDM2B in the preparation of preparations or medicines for promoting the regeneration and repair of spinal cord nerve injury tissue mediated by apical papilla stem cells in vivo.
  • the number of substitutions, deletions or additions of one or more amino acids is 2, 3, 4 or 5.
  • the present invention also provides nucleic acid molecules encoding said polypeptides.
  • the present invention also provides expression vectors, including said nucleic acid molecules.
  • the present invention also provides the use of the polypeptide, the nucleic acid molecule, and the expression vector directly or indirectly in the preparation of preparations or drugs for inducing neurogenic differentiation of apical papilla stem cells in vitro.
  • the present invention also provides the polypeptide, the nucleic acid molecule, and the expression vector, which can be directly or indirectly used in the preparation of preparations for promoting the regeneration and repair of spinal cord nerve injury tissue mediated by apical papilla stem cells in vivo or pharmaceutical applications.
  • the binding sites of KDM2B and EZH2 include:
  • the positive binding site is selected from peptide 5, peptide 46, peptide 47, peptide 122, peptide 123, peptide 131, peptide 132, peptide 139, peptide 142, peptide 151, peptide 152, peptide 153, peptide 231, peptide 232, peptide 233 ;
  • amino acid sequence of the positive binding site is as SEQ ID No.5, SEQ ID No.46, SEQ ID No.47, SEQ ID No.122, SEQ ID No.123, SEQ ID No.131, SEQ ID No. 132. SEQ ID No.139, SEQ ID No.142, SEQ ID No.151, SEQ ID No.152, SEQ ID No.153, SEQ ID No.231, SEQ ID No.232 or SEQ ID No.233 Show;
  • the negative binding site is selected from peptide 83, peptide 84;
  • amino acid sequence of the negative binding site is shown in SEQ ID No.267.
  • polypeptides provided by the present invention also include polypeptides with amino acid sequences as shown in any of SEQ ID No. 267-270.
  • the present invention also provides medicine or preparation, including said polypeptide and pharmaceutically acceptable auxiliary materials.
  • the present invention also provides a preparation method of the polypeptide, comprising the following steps:
  • Step 1 mesenchymal stem cell culture, plasmid construction and virus transfection
  • Step 2 ⁇ III-TUBULIN, NESTIN double-positive neurospheres are formed, mesenchymal stem cells are induced and cultured with neural stem cell dominant medium, and the neurospheres formed after induction are 9 days, fixed with 4% paraformaldehyde, and treated with Triton for membrane permeabilization After blocking treatment with basic albumin solution, and then incubated overnight at 4°C with specific primary antibody solution, fluorescently labeled species-specific secondary antibody, cytoskeleton dye PDI, and nuclear dye DAPI were incubated in sequence, and then visualized with fluorescence excitation; mainly for specificity
  • the primary antibody is a polyclonal antibody against neuron-specific expression gene tubulin ⁇ III-TUBULIN and neural progenitor cell marker nestin NESTIN;
  • Step 3 Rat spinal cord injury model and apical papilla stem cell reimplantation. About 1 ⁇ 106 apical dental papilla stem cells were transplanted to the T10 total spinal cord tissue excised site of 10-week-old rats, respectively. At 0, 1, 2 and 3 weeks, the BBB hindlimb motor performance score was performed on the rats; for histopathological analysis, the injured spinal cord tissue was obtained after three weeks, and paraffin sections were made for hematoxylin-eosin (HE) staining , Immunohistochemical staining of neuron-specific expression of tubulin ⁇ III-TUBULIN and neurofilament-specific protein NEF-M;
  • HE hematoxylin-eosin
  • the apical papilla stem cells were transplanted to the total cut site of the spinal cord tissue in the T10 vertebrae of rats.
  • the microinjection system (30 ul in total) was inserted into the center, left side and right side of the total cut site respectively towards the midline. , slowly inject 10ul at each point;
  • Step 4 co-immunoprecipitation reaction, RIPA lysate to dissolve cells to extract total protein, incubate protein samples with specific primary anti-IgA/G beads at 4°C overnight, wash with triethanolamine buffered saline solution (TBS), and denature by boiling at 99°C , for Western blot analysis, protein samples were separated by 10% SDS polyacrylamide gel and transferred to polyvinyl difluoride (PVDF) using a semi-dry transfer membrane device, smeared with 5% skimmed milk on the membrane and left for 2h, then Incubate overnight with the primary antibody; the immune complex is incubated with rabbit or mouse immunoglobulin G antibody and visualized with chemiluminescence substrate reagent; the main specific primary antibody is anti-KDM2B, EZH2 polyclonal antibody;
  • PVDF polyvinyl difluoride
  • Step 5 Polypeptide microarray hybridization and data analysis.
  • the KDM2B polypeptide microarray chip is synthesized by Overlapping design method and fully automatic peptide chip synthesizer; the polypeptide microarray chip is immunohybridized with the recombinant protein, and the peptide After the microarray chip is activated, it is blocked and incubated overnight at 4°C with biotin-labeled EZH2 protein sample reaction solution, incubated with HRP chromogenic substrate, and visualized with ECL chemiluminescent reagent on Chempchemi digital imager; chip chromogenic spot scanning and data analysis , the imaged images were analyzed using TotalLab image analysis software to analyze the optical density value of the chromogenic point, and the "SpotEdgeAverage" algorithm in the software was used to calculate the percentage value of the color intensity of each chromogenic point.
  • Step 6 synthesis and purification of biologically active polypeptides, obtaining biologically active polypeptide sequences, penetrating peptides and FITC green fluorescent group calibration, sequentially synthesizing corresponding amino acids in sequence under resin-dichloromethane solution swelling, ninhydrin detection, and then It is capped with pyridine and acetic anhydride, washed, and the crude product is precipitated with ether, purified by liquid chromatography after centrifugation, and freeze-dried by a lyophilizer to obtain a biologically active polypeptide powder.
  • the present invention also provides co-immunoprecipitation analysis for the application of the biologically active polypeptide provided by the present invention in binding to the KDM2B/EZH2 complex; The application of differentiation into neurospheres; the application of biologically active polypeptides to the regeneration of spinal cord injury tissue by replanting the local site of spinal cord injury nerve tissue in rats.
  • the invention provides a biologically active polypeptide based on the synthesis of histone demethylase KDM2B and its application in the differentiation process of mesenchymal stem cells and the regeneration of spinal cord nerve injury tissue, through polypeptide microarray chip hybridization and data analysis , Western blot analysis, induced formation of neurospheres and immunofluorescent staining of ⁇ III-TUBULIN and NESTIN double-positive neurospheres, and replantation experiments at local sites in rat spinal cord tissue severed injury models found that KDM2B biologically active polypeptides in mesenchymal stem cells The role in the differentiation process, and the role in the regeneration of spinal cord nerve injury tissue.
  • the present invention relates to the possible protein interaction binding site between histone demethylase KDM2B and histone methylase EZH2, the role of KDM2B and bioactive polypeptides in the process of neural differentiation of mesenchymal stem cells, KDM2B and The role of bioactive peptides in tissue regeneration of spinal cord nerve injury.
  • the obtained KDM2B and the bioactive peptides synthesized based on it may play a role in promoting the neurogenic differentiation of apical papilla stem cells and tissue regeneration of spinal cord nerve injury.
  • Figure 2 shows a schematic diagram of the overexpression of KDM2B provided by the embodiment of the present invention to promote the formation of ⁇ III-TUBULIN and NESTIN positive neurospheres in apical dental papilla stem cells in vitro; wherein, Figure 2 (A) real-time fluorescent quantitative reverse transcription PCR results and Fig. 2 (B) Western blot results show that the overexpression of KDM2B apical papilla stem cells has been constructed; Fig.
  • Fig. 3 is a schematic diagram showing that knockdown expression of KDM2B provided by the embodiment of the present invention inhibits the formation of ⁇ III-TUBULIN and NESTIN positive neurospheres in apical dental papilla stem cells in vitro; among them, Fig. 3 (A) real-time fluorescent quantitative reverse transcription PCR results It shows that the knockdown expression of KDM2B in apical papilla stem cells has been constructed; Fig. 3 (B) At 9 days after neurogenesis induction, compared with the control Scramsh group, the knockdown expression of KDM2B inhibits the formation of neurospheres in apical papilla stem cells in vitro; Fig.
  • Fig. 4 shows the schematic diagram that the overexpression of KDM2B provided by the embodiment of the present invention promotes the regeneration and repair of spinal cord injury tissue mediated by apical papilla stem cells in vivo; when apical papilla stem cells are reimplanted for 3 weeks, Fig.
  • Figure 5 shows a schematic diagram of detecting the binding of KDM2B and EZH2 protein by the co-immunoprecipitation (CO-IP) method provided by the embodiment of the present invention
  • Figure 6 shows a schematic diagram of the EZH2 protein and KDM2B polypeptide microarray chip provided by the embodiment of the present invention; wherein, Figure 6 (A) hybridization dot diagram and Figure 6 (B) microarray polypeptide dot gray value results indicate positive binding peptide sites ; Figure 6 (C) Schematic diagram of EZH2 and KDM2B protein binding functional domain fragments; Figure 6 (D) Co-immunoprecipitation (CO-IP) results show that 10ug/ml biologically active peptide peptide 46-47 (PP1 group), peptide 122-123 (PP2 group), peptide 131-132 (PP3 group) effectively blocked the combination of EZH2 and KDM2B;
  • CO-IP Co-immunoprecipitation
  • Fig. 7 shows that biologically active polypeptide peptide 46-47 (PP1 group), peptide 122-123 (PP2 group), peptide 131-132 (PP3 group) provided by the embodiment of the present invention promotes the in vitro ⁇ III-TUBULIN and NESTIN of apical tooth papilla stem cells Schematic diagram of the formation of positive neurospheres; among them, Fig. 7 (A) 9 days after the induction of neurogenesis, compared with the control group added with ConPP, peptide 46-47, peptide 122-123, and peptide 131-132 added groups all significantly promoted root growth.
  • FIG. 8 shows the schematic diagram of the biologically active polypeptide peptide 46-47 (PP1 group) provided by the embodiment of the present invention to promote the regeneration and repair of spinal cord injury nerve tissue; wherein 10ug/ml peptide 46-47 pretreated apical papilla stem cells after 4 weeks of replantation ,
  • Figure 8 (A) The general view of the spinal cord tissue showed that the 10ug/ml peptide 46-47 pretreatment group healed the injured nerve tissue significantly;
  • Figure 8 (B) BBB behavioral score showed that the 10ug/ml peptide 46-47 pretreatment group significantly improved Rat hindlimb motor ability; at the same time, after 4 weeks of continuous injection of 10ug/ml peptide 46-47 alone at the local site of spinal cord injury,
  • Figure 8 (C) shows the general view of the spinal cord tissue in the 10ug/ml peptide 46-47 injection group to damage the nerve tissue healing is obvious;
  • Figure 8 (D) BBB behavioral score results show that 10ug/
  • Figure 9 shows a schematic diagram of polypeptide spots on the KDM2B polypeptide microarray membrane and a Coomassie staining image; the left side shows a schematic diagram of polypeptide spots on the KDM2B polypeptide microarray membrane; the right side shows a Coomassie staining image after the chip is completed.
  • the invention discloses a biologically active polypeptide synthesized based on histone demethylase and its application in the process of neural differentiation of mesenchymal stem cells.
  • the purpose of the present invention is to provide a biologically active polypeptide based on the synthesis of histone demethylase KDM2B and its regulation method in the process of mesenchymal stem cell neuroblast differentiation, aiming to solve the problem that the prior art does not involve the KDM2B gene and its Issues in the regulation of bioactive peptides during the neurogenic differentiation of mesenchymal stem cells.
  • the present invention is achieved in this way, a biologically active polypeptide based on histone demethylase KDM2B synthesis and its regulation method in the process of mesenchymal stem cell neural differentiation, the KDM2B polypeptide microarray design, analysis and KDM2B-based
  • the synthetic biologically active polypeptide method comprises the following steps:
  • Step 1 the peptide microarray chip design information of KDM2B protein, query the full-length sequence of human KDM2B protein through the Uniprot protein information website (https://www.uniprot.org/), and synthesize peptides according to the 1336 amino acid sequence of KDM2B protein
  • the chip (listed in Table 12) was designed by Overlapping, that is, starting from the first amino acid position, using the sequence length of 15 amino acids as an observation window, designing the first polypeptide of the array, and then shifting backward by 5 Amino acid sites, with the length of the next 15 amino acid sequences as the second observation window, the second polypeptide of the array is designed, and in this way, 266 polypeptides are finally obtained to form a KDM2B protein-based Peptide microarray chip.
  • Step 2 Polypeptide array synthesis.
  • the activated matrix chip membrane is placed on a fully automatic peptide chip synthesizer, and the Fmoc (9-fluorenylmethoxycarbonyl)-amino acid raw material solution is automatically transferred to a specific position on the activated membrane according to the program and then the membrane is synthesized.
  • the membrane was sequentially immersed in BSA protein blocking solution I and II for side chain blocking; then, the membrane was washed with DMF (dimethylformamide) to remove the Fmoc protecting group at the amino terminal, and then dried with ethanol; repeat
  • DMF dimethylformamide
  • Step 3 Polypeptide array and recombinant protein immunohybridization reaction, activate the peptide microarray chip, add blocking solution, shake and seal at room temperature for 4 hours, wash the chip; take EZH2 protein sample reaction solution (concentration 1.5mg/ml), use EZ -link NHS-PEO4-Biotinylation kit (prod#21455) for protein labeling; the biotin-labeled EZH2 protein sample reaction solution diluted with blocking solution (final concentration 1ug/ml) was mixed with the peptide microarray chip, and incubated overnight at 4°C with shaking , the control group was incubated with blocking solution; the chromogenic substrate Streptavidin-HRP was incubated (High Sensitivity Streptavidin-HRP (prod#21133)), after the blocking solution was diluted (1:10000), the polypeptide microarray chip was incubated with 5ml and shaken at room temperature for 2 Hours, the chip was washed; ECL chemil
  • Step 4 chip scanning and color point data analysis, the color chip was scanned and imaged using Chempchemi chemiluminescence imaging system at 425nm, and the color development time was 200s.
  • the imaged images were analyzed with the TotalLab image analysis software for the optical density value of the chromogenic point, and the “Spot Edge Average” algorithm in the software was used to calculate the optical density value of each chromogenic point with reference to the surrounding background value of each chromogenic point.
  • Step 5 synthesis of biologically active polypeptides.
  • a total of 7 biologically active polypeptides were synthesized (the amino acid sequences are listed in Table 17).
  • the membrane-penetrating peptide sequence: YGRKKRRQRRR is added to the left amino terminal of each biologically active polypeptide, so as to facilitate its passage through the cell permeable membrane and enter the cell to play a role.
  • each biologically active polypeptide is calibrated with FITC green fluorescent group; synthesis sequence: from the carboxy-terminus to the amino-terminus of each biologically active polypeptide sequence.
  • the synthesis steps are as follows: Weigh n equivalents of resin into the reactor, add DCM (dichloromethane) to swell for half an hour, then remove the DCM, add 2n equivalents of the first amino acid in the sequence, add 2n equivalents of DIEA, an appropriate amount DMF, DCM (appropriate amount means that the resin can be fully agitated), DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, and nitrogen gas bubbling for 60 minutes.
  • DCM dichloromethane
  • DIEA diisopropylethylamine
  • DMF dimethylformamide
  • DCM dimethylformamide
  • wash add an appropriate amount of decapping solution to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash, and detect ninhydrin; add amino acids in the subsequent polypeptide sequence in turn according to the method of step 2) above and perform reaction modification;
  • the resin is blown dry with nitrogen, it is removed from the reaction column, poured into a flask, and then a certain amount of cutting solution (the composition is 95% TFA, 2 % ethanedithiol, 2% triisopropylsilane, 1% water), shake, and filter off the resin; obtain the filtrate, then add a large amount of ether to the filtrate, separate out the crude product, centrifuge, and wash to obtain the biologically active polypeptide sequence the crude product;
  • Step 6 Purify and freeze-dry the biologically active polypeptide. Purify the crude product to the required purity by means of high performance liquid chromatography. Put the purified liquid into a freeze dryer for concentration and freeze-drying, and finally obtain a light yellow powder that is the biologically active polypeptide. .
  • the KDM2B polypeptide microarray design and analysis of the embodiment of the present invention and the bioactive polypeptide method based on KDM2B synthesis include the following steps:
  • the activated matrix chip membrane is placed on the fully automatic peptide chip synthesizer, and the Fmoc(9-fluorenylmethoxycarbonyl)-amino acid raw material solution is automatically transferred to the specific position on the activated membrane to react with the membrane according to the program; then, the membrane is sequentially Immerse in BSA protein blocking solution I and II for side chain blocking; then, wash the membrane with DMF (dimethylformamide) to remove the Fmoc protecting group at the amino terminal, and then dry with ethanol; repeat the above steps until all peptide arrays The synthesis is complete. Finally, use a specific organic reagent to remove the side chain protecting group, then wash the membrane with CH 2 Cl 2 , wash with ethanol and dry, then use immediately or store at -20°C.
  • BSA protein blocking solution I and II for side chain blocking
  • DMF dimethylformamide
  • the chip was scanned and imaged using Chempchemi chemiluminescence imaging system at 425nm, and the color development time was 200s.
  • the imaging pictures were analyzed with TotalLab image analysis software to analyze the optical density value of the color point, and the "SpotEdgeAverage" algorithm in the software was used to calculate the color percentage value of each point.
  • each biologically active polypeptide is calibrated with FITC green fluorescent group; synthesis sequence: from the carboxy-terminus to the amino-terminus of each biologically active polypeptide sequence.
  • the synthesis steps are as follows: Weigh n equivalents of resin into the reactor, add DCM (dichloromethane) to swell for half an hour, then remove the DCM, add 2n equivalents of the first amino acid in the sequence, add 2n equivalents of DIEA, an appropriate amount DMF, DCM (appropriate amount means that the resin can be fully agitated), DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, and nitrogen gas bubbling for 60 minutes.
  • DCM dichloromethane
  • DIEA diisopropylethylamine
  • DMF dimethylformamide
  • DCM dimethylformamide
  • wash add an appropriate amount of decapping solution to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash, and detect ninhydrin; add amino acids in the subsequent polypeptide sequence in turn according to the method of step 2) above and perform reaction modification;
  • the resin is blown dry with nitrogen, it is removed from the reaction column, poured into a flask, and then a certain amount of cutting solution (the composition is 95% TFA, 2 % ethanedithiol, 2% triisopropylsilane, 1% water), shake, and filter off the resin; obtain the filtrate, then add a large amount of ether to the filtrate, separate out the crude product, centrifuge, and wash to obtain the biologically active polypeptide sequence the crude product;
  • the crude product is purified to the required purity by means of high performance liquid chromatography, and the purified liquid is concentrated and freeze-dried in a lyophilizer, and finally a pale yellow powder is obtained, which is a biologically active polypeptide.
  • Use stem cell culture medium such as the ⁇ -MEM base solution containing 15% fetal bovine serum, 2mmol/L glutamine, 100U/ml penicillin and 100ug/ml streptomycin used in this project
  • cell phosphate buffered saline It can be dissolved according to the storage concentration of 10ug/ul, and stored at -80°C after aliquoting to avoid repeated freezing and thawing.
  • the corresponding culture solution system such as the stem cell culture solution and neurogenesis differentiation induction solution used in this project
  • the present invention studies the role of KDM2B and its biologically active polypeptides in the neural differentiation of apical papilla stem cells and in the regeneration and repair of damaged nerve tissue based on the biologically active polypeptides synthesized by KDM2B.
  • the raw materials and reagents used in the synthesis and application of the biologically active polypeptide provided by the present invention can be purchased from the market.
  • Single cell suspensions were obtained through 70-um filters (BD Biosciences, San Jose, CA,). Next, these cells were inoculated in a minimum amount of Eagle medium (Minimum Eagle medium, MEM) (Invitrogen, Carlsbad, CA), plus 15% fetal bovine serum, 2mmol/l glutamine, 100U/ml penicillin and 100mg/ml chain Mycin, placed in a humidified incubator with 5% CO2 and 37 °C, and the medium was replaced every 3 days.
  • MEM Minimum Eagle medium
  • neural stem cell dominant medium Neuroblastasal A solution supplemented with 2% B27, 40ng/ml bFGF, 20ng/ml EGF, 2mM L-glutamine, 100U/ml penicillin and 100ug/ml streptomycin ) to induce cultured stem cells and replace them every 3 days;
  • Plasmid construction and virus transfection the construction of the plasmid was carried out according to the standard method, the target complementary shRNA sequence targeting the KDM2B gene design was cloned into the viral vector pLKO.1 plasmid ring, sequenced and identified, and the KDM2B shRNA plasmid was constructed; the KDM2B gene was designed The full-length PCR primers were used to amplify the full-length gene sequence of KDM2B, add the HA-Tag tag, connect it to the retroviral expression vector pQCXIN, sequence and identify it, and construct the full-length KDM2B gene containing the HA-Tag tag Sequence the plasmid; then carry out virus packaging, collection, virus titer identification, and store in -80 degree refrigerator after subpackaging; virus transfection, retrovirus or lentivirus with a titer of 10 -7 is mixed with the root tip under the participation of polybrene Dental papilla stem cells were cultured overnight, and the transfected cells
  • Example 3 total RNA isolation, reverse transcription (RT) PCR and real-time fluorescent quantitative reverse transcription PCR
  • RNA samples from apical papilla stem cells from different individuals were extracted and purified using extraction reagents and RNA extraction kits (QIAGEN, GmBH, Germany), and dissolved in RNase-Free Water (QIAGEN).
  • Total RNA was quantified by a spectrophotometer ND-2100 (Thermo Fisher), and RNA integrity was assessed using an Agilent 2100 (Agilent Technologies).
  • ND-2100 Thermo Fisher
  • Agilent 2100 Agilent 2100
  • an equal amount of cDNA samples were synthesized by reverse transcription using a random primer kit (QIAGEN) according to the manufacturer's standard (Invitrogen).
  • the present invention is approved by the Animal Care and Use Committee of Beijing Stomatological Hospital affiliated to Capital Medical University; about 1 ⁇ 10 6 apical papilla stem cells were transplanted to the site of complete amputation of spinal cord tissue in the T10 part of the spine of a 10-week-old rat.
  • the needles were respectively inserted in the center, left side and right side of the total cut site towards the midline, and 10ul was injected slowly at each site.
  • the apical papilla stem cells in the control Vector group and the KDM2B overexpression group were respectively replanted in 5 rats. These procedures were carried out in accordance with the approved specifications of the animal protocol; After 3 weeks, the BBB hindlimb motor ability score was carried out on the rats;
  • the spinal cord tissue of the transplanted part was fixed with 10% formalin, embedded in paraffin, sectioned with 5um, and stained with hematoxylin-eosin (HE); immunohistochemical staining, sectioned with 5um Wax dehydration, removal of endogenous peroxidase after blocking, specific primary antibody solution was incubated overnight at 4°C, biotin-horseradish peroxidase was incubated sequentially, with the help of 3,3-diaminobenzidine (DAB) substrate Color development makes it visualized; the main specific primary antibody is anti- ⁇ III-TUBULIN, NEF-M polyclonal antibody (Abcam).
  • HE hematoxylin-eosin
  • Embodiment 6 Polypeptide microarray chip design and analysis
  • the full-length sequence of human KDM2B protein was obtained through the Uniprot protein information website (https://www.uniprot.org/).
  • the full-length amino acid sequence of KDM2B was designed and synthesized by Overlapping peptide chips; peptide arrays were synthesized with the help of a fully automatic peptide chip synthesizer.
  • KDM2B polypeptide microarray membrane and EZH2 protein hybridization reaction KDM2B polypeptide microarray membrane and EZH2 protein hybridization reaction
  • Chip peptide number serial number amino acid sequence 5 SEQ ID No.5 AEKQKKKTVIYTKCF 46 SEQ ID No.46 VKKYCLMSVKGCFTD 47 SEQ ID No.47 LMSVKGCFTDFHIDF 122 SEQ ID No.122 ARRRRTRCRKCEACL 123 SEQ ID No.123 TRCRKCEACLRTECG 131 SEQ ID No.131 CIAPVLPHTAVCLVC 132 SEQ ID No.132 LPHTAVCLVCGEAGK 139 SEQ ID No.139 CNEIIHPGCLKIKES
  • apical papilla stem cells with 10ug/ml peptide 46-47 for 24 hours, and immediately transplanted them into the site of total spinal cord tissue excisement in the T10 vertebrae of 10-week-old rats (5 independent rats in each group).
  • the spinal cord tissue showed significant healing of injured nerve tissue in the 10ug/ml peptide 46-47 pretreatment group (Fig. 8A); BBB behavioral scores showed that 10ug/ml peptide 46-47 The motor ability of the hindlimbs of rats in the 46-47 pretreatment group was significantly improved (Fig. 8B).
  • bioactive polypeptides synthesized based on the histone demethylase KDM2B provided by the present invention and their application in the neural differentiation process of mesenchymal stem cells and the regeneration and repair of damaged nerve tissues have been introduced above in detail.
  • This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above embodiments is only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

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Abstract

本发明涉及基于组蛋白去甲基化酶合成的生物活性多肽及其在间充质干细胞神经向分化过程中的应用。本发明公开了组蛋白去甲基化酶KDM2B与组蛋白甲基化酶EZH2可能的蛋白互作结合位点，公开了 KDM2B及基于其合成的生物活性多肽在间充质干细胞神经向分化过程以及脊髓神经损伤组织再生中的作用，并基于此得到KDM2B及基于其合成的生物活性多肽可能在根尖牙乳头干细胞神经分化及脊髓神经损伤再生中起促进作用。

Description

基于KDM2B序列合成的生物活性多肽在间充质干细胞神经分化和再生修复中的应用 技术领域
本发明涉及生物工程技术领域，特别涉及基于组蛋白去甲基化酶KDM2B合成的生物活性多肽及其在间充质干细胞神经向分化过程和损伤神经组织再生修复中的应用。
背景技术
颅颌面的神经组织分布广泛，易受损伤。近年流行病学调查显示55.2％的颅颌面外伤患者存在不同程度神经损伤，2.2％的患者另伴有脊髓挫伤，后续治疗并发症中24.0％的患者出现神经功能障碍。以往治疗方式主要是使用神经保护措施如温热等，使用神经保护药物如糖皮质激素类、谷氨酸拮抗剂类等，使用电、热、力等刺激神经自我愈合等，这些治疗复杂、周期长，神经功能恢复率低，导致高致残率。自/异体神经移植治疗存在供体组织取材困难、取材处二次损伤等，同时移植神经无法进行良好塑型，仍不能很好恢复组织结构外形和受损功能。目前损伤神经的临床治疗效果仍待提升。
以间充质干细胞(Mesenchymal stem cells，MSCs)介导为基础的生物性再生治疗将是未来修复损伤神经功能的治疗新方式。作为对组织损伤信号的响应，MSCs良好的分化能力可以促进神经修复细胞形成和轴突再生等，最终促进损伤神经的再生修复。但是，神经源性干细胞来源有限、取材困难，仍需发掘更多种子干细胞。牙源性MSCs发育自神经嵴，神经嵴最初起源于外胚层，与神经组织发源密切，因此牙源性MSCs更具有转化应用的优势。其中，根尖牙乳头干细胞(Stem Cells from the Apical Papilla,SCAP)存在于牙齿根尖部牙乳头组织中，研究发现SCAP在体内可形成牙髓神经样组织，提示SCAP是神经再生的可用种子细胞。目前限制SCAP潜在应用的主要问题是神经分化效率低和调节机制不明确。因此，如何有效发掘关键调控靶点，促进牙源性间充质干细胞修复潜能显得尤为重要。
最近的研究确定了染色质的表观遗传调控是决定MSCs神经谱系分化的关键机制。通过全基因组表观遗传调控图谱的揭示，发现分化后MSCs组蛋白H3上赖氨酸27位点(H3K27)和赖氨酸4位点(H3K4)的甲基化修饰分值明显升高。组蛋白的甲基化修饰多发生在赖氨酸残基(K)上，是表观遗传机制中组蛋白共价修饰的主要形式。存在于基因启动子区的组蛋白甲基化修饰基团阻碍该基因的转录表达(如H3K27me3)或促进该基因的转录表达(如H3K4me3)，组蛋白甲基化/去甲基化酶在组蛋白的甲基化修饰中分别起到关键调控作用，同时也存在一定的功能相互作用(如通过功能域结合形成复合体)。
一项关于胚胎干细胞的全基因组染色质免疫沉淀(ChIP)测序表明，组蛋白甲基化酶EZH2的结合和神经分化调控基因的启动子区的H3K27me3修饰高度相关。EZH2属于多梳蛋白组(Polycomb group，PcGs)的核心一员，其修饰的H3K27me3是一种基因转录抑制的组蛋白甲基化修饰，被认为阻碍特定细胞命运的分化进程。研究发现EZH2抑制神经干细胞分化形成神经源性细胞，使用H3K27me3特异拮抗剂EPZ005687可有效提高中脑腹侧来源神经干细胞的神经元性分化能力，提示EZH2对神经分化可能的抑制作用。因此，如何有效调控EZH2的功能作用十分 关键。
研究发现KDM2B参与招募PRC2-EZH2复合体对下游靶基因的锚定和后续组蛋白的共价修饰，提示KDM2B与EZH2之间可能存在表观遗传调控作用上的交叉。KDM2B属于组蛋白去甲基化酶，主要通过发挥去除组蛋白H3K4位点的三甲基化修饰(H3K4me3)的作用，参与多种细胞进程的调控。研究者分析发现KDM2B功能的发挥依赖其含有的JmjC、CxxC、PHD等多种功能结构域。然而，KDM2B与牙源性MSCs神经分化功能的关系尚不清楚；KDM2B与EZH2蛋白之间可能的相互作用不清楚。
目前，一种分析蛋白质结合的技术方法正发展起来，使得研究者关于两个蛋白质相互作用的具体结合位点信息可以遵循一个广泛观点，这种方法是“多肽微阵列分析”，基于蛋白质的免疫杂交试验，利用感兴趣的蛋白与基于另一个蛋白全长氨基酸序列合成的微阵列芯片杂交，借助酶联标记放大这种杂交结合的信号，从而识别两个蛋白质之间可能的结合片段位点和这一可能位点具体的氨基酸序列信息。由此，通过使用多肽微阵列的方法可以研究KDM2B与EZH2间可能的结合功能域片段，理解这一过程有望明确调控牙源性MSCs神经分化的潜在表观修饰酶的关键相互作用位点，研发促进牙源性MSCs神经分化的生物性新药，提升临床条件下损伤神经生物性再生效果。
本发明研究的目的是阐明KDM2B对牙源性间充质干细胞神经分化与再生的作用，阐明KDM2B与EZH2的可能互作，通过使用多肽微阵列的方法研究KDM2B与EZH2间可能的结合位点片段信息。
发明内容
有鉴于此，本发明提供一种基于组蛋白去甲基化酶KDM2B合成的生物活性多肽及其在间充质干细胞神经向分化过程和损伤神经组织再生修复中的调控方法，旨在解决现有技术没有涉及KDM2B基因及其生物活性多肽在牙源性间充质干细胞神经分化过程和损伤神经组织再生修复中调控的问题。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了KDM2B过表达在制备诱导根尖牙乳头干细胞体外神经分化的制剂或药物中的应用。
在本发明一些具体实施方案中，KDM2B的过表达在制备促进根尖牙乳头干细胞体外βⅢ-TUBULIN阳性及NESTIN阳性类神经球的形成的制剂或药物中的应用。
本发明还提供了KDM2B的过表达在制备促进体内根尖牙乳头干细胞介导的脊髓神经损伤组织再生修复的制剂或药物中的应用。
更重要的是，本发明提供了多肽，其具有：
(I)、如SEQ ID No.1～266任意所示的氨基酸序列；或
(II)、如(I)所述的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(I)所述的氨基酸序列功能相同的氨基酸序列；或
(III)、与如(I)或(II)所述的氨基酸序列具有90％以上同一性的氨基酸序列；
所述取代、缺失或添加一个或多个氨基酸中的多个为2个、3个、4个或5个。
此外，本发明还提供了编码所述多肽的核酸分子。
本发明还提供了表达载体，包括所述的核酸分子。
更重要的是，本发明还提供了所述的多肽，所述的核酸分子，所述的表达载体，直接或间接在制备诱导根尖牙乳头干细胞体外成神经分化的制剂或药物中的应用。
同样重要的是，本发明还提供了所述的多肽，所述的核酸分子，所述的表达载体，直接或间接在制备促进体内根尖牙乳头干细胞介导的脊髓神经损伤组织再生修复的制剂或药物中的应用。
在本发明的一些具体实施方案中，KDM2B与EZH2的结合位点包括：
阳性结合位点选自peptide 5、peptide 46、peptide 47、peptide 122、peptide 123、peptide 131、peptide 132、peptide 139、peptide 142、peptide 151、peptide 152、peptide 153、peptide 231、peptide 232、peptide 233；
所述阳性结合位点的氨基酸序列如SEQ ID No.5、SEQ ID No.46、SEQ ID No.47、SEQ ID No.122、SEQ ID No.123、SEQ ID No.131、SEQ ID No.132、SEQ ID No.139、SEQ ID No.142、SEQ ID No.151、SEQ ID No.152、SEQ ID No.153、SEQ ID No.231、SEQ ID No.232或SEQ ID No.233所示；
所述阴性结合位点选自peptide 83、peptide 84；
所述阴性结合位点的氨基酸序列如SEQ ID No.267所示。
此外，本发明还提供的多肽还包括氨基酸序列如SEQ ID No.267～270任意所示的多肽。
本发明还提供了药物或制剂，包括所述的多肽以及药学上可接受的辅料。
此外，本发明还提供了所述多肽的制备方法，包括如下步骤：
步骤1，间充质干细胞培养，质粒构建与病毒转染；
步骤2，βⅢ-TUBULIN、NESTIN双阳性类神经球形成，间充质干细胞使用神经干细胞优势培养基诱导培养，诱导9天时形成的类神经球，4％多聚甲醛固定，Triton进行膜通透处理后基础白蛋白液封闭处理，然后用特异性一抗液4℃孵育过夜，荧光标定种属特异二抗、细胞骨架染料PDI、细胞核染料DAPI依次孵育后借助荧光激发使其可视化；主要针对特异性一抗是抗神经元特异表达基因微管蛋白βⅢ-TUBULIN、神经祖细胞标记物巢蛋白NESTIN多克隆抗体；
步骤3、大鼠脊髓神经损伤模型及根尖牙乳头干细胞回植，将约1×10 6个根尖牙乳头干细胞移植到10周龄大鼠T10部脊髓组织全切断位点，分别于移植后0周、1周、2周和3周对大鼠进行BBB后肢运动能力评分；组织病理学分析，三周后获取损伤处脊髓组织，制作石蜡切片，苏木精-伊红染色(HE)染色，神经元特异表达微管蛋白βⅢ-TUBULIN、神经纤维丝特异蛋白NEF-M的免疫组织化学染色；
进一步，根尖牙乳头干细胞移植大鼠T10脊椎部脊髓组织全切断位点，特别的，通过微注射体系(共30ul)于全切断位点的正中、左侧和右侧分别向中线方向进针，每位点分别缓慢注射10ul；
步骤4，免疫共沉淀反应，RIPA裂解液溶解细胞提取总蛋白，蛋白样本与特异一抗免疫球蛋白A/G珠子4℃孵育过夜，三乙醇胺缓冲盐水溶液(TBS)清洗后，99℃煮沸变性，进行蛋白质印迹分析，蛋白样本用10％SDS聚丙烯酰胺凝胶分离并利用半干转移膜装置转移到聚乙二烯二氟化物(PVDF)中，膜上涂抹5％脱脂牛奶放置2h，然后用一抗孵育一夜；免疫复合物与兔或小鼠 免疫球蛋白G抗体一同孵育并用化学发光底物试剂使其可视化；主要针对特异一抗是抗KDM2B、EZH2多克隆抗体；
步骤5，多肽微阵列杂交及数据分析，根据KDM2B全长氨基酸序列，通过Overlapping设计方式设计、全自动多肽芯片合成仪合成KDM2B多肽微阵列芯片；多肽微阵列芯片与重组蛋白免疫杂交反应，将多肽微阵列芯片活化后封闭处理，与生物素标记的EZH2蛋白样品反应液4℃震荡孵育过夜，HRP显色底物孵育，ECL化学发光试剂于Chempchemi数字成像仪可视化；芯片显色点扫描及数据分析，成像图片使用TotalLab图像分析软件分析显色点光密度值，使用软件中“SpotEdgeAverage”算法，计算每个显色点的显色强度百分比值。
步骤6，生物活性多肽的合成和纯化，获得生物活性多肽序列，穿膜肽及FITC绿色荧光基团标定，树脂-二氯甲烷液溶胀下按序列顺次合成相应氨基酸，茚三酮检测，然后用吡啶和乙酸酐封端，洗净、乙醚析出粗产物，离心后液相色谱法提纯，冻干机冻干获得生物活性多肽粉末。
本发明还提供了免疫共沉淀分析用于本发明提供的生物活性多肽对KDM2B/EZH2复合体结合的应用；βⅢ-TUBULIN、NESTIN双阳性类神经球形成用于生物活性多肽对根尖牙乳头干细胞成神经球分化的应用；大鼠脊髓损伤神经组织局部位点回植用于生物活性多肽对脊髓神经损伤组织再生的应用。
本发明提供了一种基于组蛋白去甲基化酶KDM2B合成的生物活性多肽及其在间充质干细胞神经向分化过程和脊髓神经损伤组织再生中的应用，通过多肽微阵列芯片杂交及数据分析、蛋白质印迹分析、类神经球诱导形成和βⅢ-TUBULIN、NESTIN双阳性类神经球免疫荧光染色、大鼠脊髓组织全切断损伤模型局部位点回植试验发现KDM2B生物活性多肽在间充质干细胞神经向分化过程中的作用，以及在脊髓神经损伤组织再生中的作用。本发明涉及组蛋白去甲基化酶KDM2B与组蛋白甲基化酶EZH2可能的蛋白互作结合位点、涉及KDM2B及生物活性多肽在间充质干细胞神经向分化过程中的作用、涉及KDM2B及生物活性多肽在脊髓神经损伤组织再生中的作用，得到KDM2B及基于其合成的生物活性多肽可能在根尖牙乳头干细胞成神经分化及脊髓神经损伤组织再生中起促进作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示本发明实施例提供的间充质干细胞中及脊髓组织中KDM2B的表达；其中，图1(A)实时荧光定量反转录PCR结果表明KDM2B在根尖牙乳头干细胞体外成神经分化诱导过程中表达升高，峰值出现在诱导的第三天；图1(B，C)免疫组织化学染色结果表明相较于正常脊髓神经组织，KDM2B在脊髓神经损伤组织中表达降低；GAPDH作为内部控制；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；**P≤0.01；Red scale bar:100μm；
图2示本发明实施例提供的KDM2B的过表达促进根尖牙乳头干细胞体外βⅢ-TUBULIN及NESTIN阳性类神经球形成量的示意图；其中，图2(A)实时荧光定量反转录PCR结果和图2(B)蛋白质免疫印迹结果表明构建完成KDM2B的过表达根尖牙乳头干细胞；图2(C)成神经诱导9天时，相较于对照Vector组，KDM2B的过表达促进根尖牙乳头干细胞体外类神经球形成； 图2(D)免疫荧光染色结果及图2(E)定量结果表明KDM2B的过表达促进根尖牙乳头干细胞体外βⅢ-TUBULIN阳性类神经球形成；图2(F)免疫荧光染色结果及图2(G)定量结果表明KDM2B的过表达促进根尖牙乳头干细胞体外NESTIN阳性类神经球形成；图2(H-K)实时荧光定量反转录PCR结果表明KDM2B的过表达上调了根尖牙乳头干细胞中TH(H)、NCAM(I)、NEF(J)、NEUROD(K)的表达；GAPDH作为内部控制；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；**P≤0.01；White scale bar:100μm；
图3示本发明实施例提供的KDM2B的敲低表达抑制根尖牙乳头干细胞体外βⅢ-TUBULIN及NESTIN阳性类神经球形成量的示意图；其中，图3(A)实时荧光定量反转录PCR结果表明构建完成KDM2B的敲低表达根尖牙乳头干细胞；图3(B)成神经诱导9天时，相较于对照Scramsh组，KDM2B的敲低表达抑制根尖牙乳头干细胞体外类神经球形成；图3(C)免疫荧光染色结果及图3(D)定量结果表明KDM2B的敲低表达抑制根尖牙乳头干细胞体外βⅢ-TUBULIN阳性类神经球形成；图3(E)免疫荧光染色结果及图3(F)定量结果表明KDM2B的敲低表达抑制根尖牙乳头干细胞体外NESTIN阳性类神经球形成；GAPDH作为内部控制；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；**P≤0.01；White scale bar:100μm；
图4示本发明实施例提供的KDM2B的过表达促进体内根尖牙乳头干细胞介导的脊髓神经损伤组织再生修复的示意图；其中根尖牙乳头干细胞回植3周时，图4(A)脊髓神经组织大体观显示KDM2B过表达组损伤愈合明显；图4(B)BBB行为学评分结果表明KDM2B过表达组显著提高大鼠后肢运动能力；图4(C)苏木精-伊红染色(HE)染色结果表明KDM2B过表达组显著促进脊髓损伤神经纤维修复再生；图4(D)免疫组织化学染色结果表明KDM2B过表达组显著调高了脊髓损伤神经组织中βⅢ-TUBULIN和NEF-M的表达；GAPDH作为内部控制；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；*P≤0.05，**P≤0.01；White scale bar:5mm，Red scale bar:100μm；
图5示本发明实施例提供的免疫共沉淀(CO-IP)方法检测KDM2B与EZH2蛋白结合的示意图；
图6示本发明实施例提供的EZH2蛋白与KDM2B多肽微阵列芯片的示意图；其中，图6(A)杂交斑点图及图6(B)微阵列多肽斑点灰度值结果表示阳性结合肽段位点；图6(C)EZH2与KDM2B蛋白结合功能域片段示意图；图6(D)免疫共沉淀(CO-IP)结果表明10ug/ml生物活性多肽peptide 46-47(PP1组)、peptide 122-123(PP2组)、peptide 131-132(PP3组)有效阻断EZH2与KDM2B的结合；
图7示本发明实施例提供的生物活性多肽peptide 46-47(PP1组)、peptide 122-123(PP2组)、peptide 131-132(PP3组)促进根尖牙乳头干细胞体外βⅢ-TUBULIN及NESTIN阳性类神经球形成量的示意图；其中，图7(A)成神经诱导9天时，相较于对照ConPP添加组，peptide 46-47、peptide 122-123、peptide 131-132添加组均显著促进根尖牙乳头干细胞类神经球形成；免疫荧光染色及定量结果表明peptide 46-47、peptide 122-123、peptide 131-132添加组均显著促进根尖牙乳头干细胞体外βⅢ-TUBULIN(B,C)阳性和NESTIN(D,E)阳性类神经球形成；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；**P≤0.01；White scale bar:5mm；
图8示本发明实施例提供的生物活性多肽peptide 46-47(PP1组)促进脊髓损伤神经组织再 生修复的示意图；其中10ug/ml peptide 46-47预处理根尖牙乳头干细胞回植4周后，图8(A)脊髓组织大体观显示10ug/ml peptide 46-47预处理组损伤神经组织愈合明显；图8(B)BBB行为学评分结果表明10ug/ml peptide 46-47预处理组显著提高大鼠后肢运动能力；同时，脊髓损伤局部位点单独应用10ug/ml peptide 46-47连续注射4周后，图8(C)脊髓组织大体观显示10ug/ml peptide 46-47注射组损伤神经组织愈合明显；图8(D)BBB行为学评分结果表明10ug/ml peptide 46-47注射组显著提高大鼠后肢运动能力；图8(E)联合分析BBB行为学评分结果表明干预施加4周时，10ug/ml peptide 46-47预处理干细胞回植组和单独10ug/ml peptide 46-47注射组均能显著提高大鼠后肢运动能力，且两组间无明显差别；数据T检验用来确定统计学意义；All error bars represent the s.d.(n＝3)；*P≤0.05，**P≤0.01；White scale bar:5mm；
图9示KDM2B多肽微阵列膜上多肽点示意图和考马斯染色图；左侧图示KDM2B多肽微阵列膜上多肽点示意图；右侧图示芯片完成后考马斯染色图像。
具体实施方式
本发明公开了基于组蛋白去甲基化酶合成的生物活性多肽及其在间充质干细胞神经向分化过程中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明的目的在于提供一种基于组蛋白去甲基化酶KDM2B合成的生物活性多肽及其在间充质干细胞成神经分化过程中的调控方法，旨在解决现有技术没有涉及KDM2B基因及其生物活性多肽在间充质干细胞成神经分化过程中调控的问题。
本发明是这样实现的，一种基于组蛋白去甲基化酶KDM2B合成的生物活性多肽及其在间充质干细胞成神经分化过程中的调控方法，该KDM2B多肽微阵列设计、分析及基于KDM2B合成的生物活性多肽方法包括以下步骤：
步骤一，KDM2B蛋白的多肽微阵列芯片设计信息，通过Uniprot蛋白信息网站(https://www.uniprot.org/)查询获得人源KDM2B蛋白全长序列，根据KDM2B蛋白的1336个氨基酸序列合成多肽芯片(详列于表12)，通过Overlapping设计，即从第一个氨基酸位点开始，以15个氨基酸序列长度做为一个观察窗，设计该阵列的第一条多肽，然后向后移位5个氨基酸位点，以后面15个氨基酸序列长度作为为第二个观察窗，设计该阵列的第二条多肽，并以此方法依次顺延，最终获得266条多肽，组成一个基于KDM2B蛋白为基础的多肽微阵列芯片。
步骤二，多肽阵列合成，活化过的基质芯片膜放置于全自动多肽芯片合成仪上，根据程序自动转移Fmoc(9-芴甲氧羰基)-氨基酸原料溶液到活化膜上的特定位置与膜进行反应；接着，膜按序浸入BSA蛋白封闭液I和II中，进行侧链封闭；然后，DMF(二甲基甲酰胺)洗膜移除氨基端的Fmoc保护基团，而后再用乙醇干燥；重复以上步骤，直至多肽阵列全部合成完毕。最后，用特定的有机试剂去除侧链保护基团，再用CH 2Cl 2洗膜，乙醇洗涤后干燥，立即使用或-20℃保存。
步骤三，多肽阵列与重组蛋白免疫杂交结合反应，将多肽微阵列芯片活化后加入封闭液，室 温下震荡封闭4小时，洗涤芯片；取EZH2蛋白样品反应液(浓度1.5mg/ml)，用EZ-link NHS-PEO4-Biotinylation kit(prod#21455)进行蛋白标记；用封闭液稀释的生物素标记的EZH2蛋白样品反应液(终浓度1ug/ml)与多肽微阵列芯片混合，4℃震荡孵育过夜，对照组用封闭液孵育；显色底物Streptavidin-HRP孵育(High Sensitivity Streptavidin-HRP(prod#21133))，封闭液稀释(1:10000)后，用5ml孵育多肽微阵列芯片，室温震荡2小时，洗涤芯片；ECL化学发光试剂于Chempchemi数字成像仪可视化。
步骤四，芯片扫描及显色点数据分析，显色芯片使用Chempchemi化学发光成像系统425nm扫描成像，显色时长200s。成像图片使用TotalLab图像分析软件分析显色点光密度值，使用软件中“Spot Edge Average”算法，以每个显色点周边背景值为参照计算每个显色点的光密度值。
步骤五，生物活性多肽的合成，本项目内，合成的生物活性多肽共7条(氨基酸序列详列于表17)。其中，每条生物活性多肽的左侧氨基端均添加了穿膜肽序列：YGRKKRRQRRR，以利于其通过细胞通透膜，进入细胞内发挥作用。同时，每条生物活性多肽均使用FITC绿色荧光基团进行标定；合成顺序：从每条生物活性多肽序列的羧基端到氨基端。
具体的，合成步骤如下：称取n当量树脂放入反应器，加入DCM(二氯甲烷)溶胀半小时，然后抽掉DCM，加入序列中第一个氨基酸2n当量，加2n当量的DIEA，适量的DMF、DCM(适量是指以可使树脂充分鼓动起来为宜)、DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5n当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净；往反应器中加入序列中第二个氨基酸(也为2n当量)，2n当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA，氮气鼓泡反应30min，洗掉液体，茚三酮检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基，洗净，茚三酮检测；依上步骤2)的方式依次加入后续多肽序列中的氨基酸并进行反应修饰；将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA，2％乙二硫醇，2％三异丙基硅烷，1％水)，震荡，滤掉树脂；得到滤液，然后向滤液中加入大量乙醚，析出粗产物，离心，清洗即可得到生物活性多肽序列的粗产物；
步骤六，生物活性多肽纯化、冻干，借助高效液相色谱方法将粗品提纯至要求纯度，纯化好的液体放入冻干机中进行浓缩、冻干，最终获得淡黄色粉末即为生物活性多肽。
如图6所示，本发明实施例的KDM2B多肽微阵列设计、分析及基于KDM2B合成的生物活性多肽方法包括以下步骤：
S601:KDM2B蛋白的多肽微阵列芯片设计信息
通过Uniprot蛋白信息网站(https://www.uniprot.org/)查询获得人源KDM2B蛋白全长序列，根据KDM2B蛋白的1336个氨基酸序列合成多肽芯片(详列于表12)，通过Overlapping设计，即从第一个氨基酸位点开始，以15个氨基酸序列长度做为一个观察窗，设计该阵列的第一条多肽，然后向后移位5个氨基酸位点，以后面15个氨基酸序列长度作为为第二个观察窗，设计该阵列的第二条多肽，并以此方法依次顺延，最终获得266条多肽，组成一个基于KDM2B蛋白为基础的多肽微阵列芯片。
S602：多肽阵列合成
活化过的基质芯片膜放置于全自动多肽芯片合成仪上，根据程序自动转移Fmoc(9-芴甲氧羰 基)-氨基酸原料溶液到活化膜上的特定位置与膜进行反应；接着，膜按序浸入BSA蛋白封闭液I和II中，进行侧链封闭；然后，DMF(二甲基甲酰胺)洗膜移除氨基端的Fmoc保护基团，而后再用乙醇干燥；重复以上步骤，直至多肽阵列全部合成完毕。最后，用特定的有机试剂去除侧链保护基团，再用CH 2Cl 2洗膜，乙醇洗涤后干燥，立即使用或-20℃保存。
S603：多肽阵列与重组蛋白免疫杂交结合反应
多肽微阵列芯片加入封闭液，室温震荡4小时；取EZH2蛋白样品反应液(浓度1.5mg/ml)，用EZ-link NHS-PEO4-Biotinylation kit(prod#21455)进行蛋白标记；使用5ml封闭液稀释的生物素标记的EZH2蛋白样品反应液(终浓度1ug/ml)与多肽微阵列芯片4℃震荡过夜，对照组用封闭液孵育；使用封闭液稀释(1:10000)显色底物Streptavidin-HRP(High Sensitivity Streptavidin-HRP(prod#21133))后，使用5ml与多肽微阵列芯片室温震荡2小时；ECL化学发光试剂使其可视化。
S604：芯片扫描及显色点数据分析
芯片使用Chempchemi化学发光成像系统425nm扫描成像，显色时长200s。成像图片使用TotalLab图像分析软件分析显色点光密度值，使用软件中“SpotEdgeAverage”算法，计算各点显色百分值。
S605：生物活性多肽的合成
本发明中，合成的生物活性多肽共7条(氨基酸序列详列于表17)。其中，每条生物活性多肽的左侧氨基端均添加了穿膜肽序列：YGRKKRRQRRR，以利于其通过细胞通透膜，进入细胞内发挥作用。同时，每条生物活性多肽均使用FITC绿色荧光基团进行标定；合成顺序：从每条生物活性多肽序列的羧基端到氨基端。
具体的，合成步骤如下：称取n当量树脂放入反应器，加入DCM(二氯甲烷)溶胀半小时，然后抽掉DCM，加入序列中第一个氨基酸2n当量，加2n当量的DIEA，适量的DMF、DCM(适量是指以可使树脂充分鼓动起来为宜)、DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5n当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净；往反应器中加入序列中第二个氨基酸(也为2n当量)，2n当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA，氮气鼓泡反应30min，洗掉液体，茚三酮检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基，洗净，茚三酮检测；依上步骤2)的方式依次加入后续多肽序列中的氨基酸并进行反应修饰；将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA，2％乙二硫醇，2％三异丙基硅烷，1％水)，震荡，滤掉树脂；得到滤液，然后向滤液中加入大量乙醚，析出粗产物，离心，清洗即可得到生物活性多肽序列的粗产物；
S606：生物活性多肽纯化、冻干：
借助高效液相色谱方法将粗品提纯至要求纯度，纯化好的液体放入冻干机中进行浓缩、冻干，最终获得淡黄色粉末即为生物活性多肽。
S607：生物活性多肽的使用方法：
使用干细胞培养液(如本项目中使用的为含有15％胎牛血清、2mmol/L谷氨酰胺、100U/ml青霉素和100ug/ml链霉素的α-MEM基液)或细胞磷酸盐缓冲盐水按照10ug/ul的储存浓度溶解即可，分装后于-80℃保存，避免反复冻融。使用时按10ug/ml的工作浓度加入相应培养液体系(如 本项目中使用的干细胞培养液及成神经分化诱导液)，现用现配。
所述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
综上，本发明通过基于KDM2B合成的生物活性多肽来研究KDM2B及其生物活性多肽在根尖牙乳头干细胞神经分化中的作用，以及在损伤神经组织再生修复中的作用。
本发明中附图对应的数据：
表1图1(A)的数据
Figure PCTCN2021115296-appb-000001
表2图1(C)的数据
Figure PCTCN2021115296-appb-000002
表3图2(A)的数据
Figure PCTCN2021115296-appb-000003
Figure PCTCN2021115296-appb-000004
表4图2(E)的数据
Figure PCTCN2021115296-appb-000005
表5图2(G)的数据
Figure PCTCN2021115296-appb-000006
表6图2(H-K)的数据
Figure PCTCN2021115296-appb-000007
Figure PCTCN2021115296-appb-000008
表7图3(A)的数据
Figure PCTCN2021115296-appb-000009
表8图3(E)的数据
Figure PCTCN2021115296-appb-000010
表9图3(G)的数据
Figure PCTCN2021115296-appb-000011
Figure PCTCN2021115296-appb-000012
表10图4(B)的数据
Figure PCTCN2021115296-appb-000013
表11图4(E)的数据
Figure PCTCN2021115296-appb-000014
Figure PCTCN2021115296-appb-000015
表12图6(B)的数据
Figure PCTCN2021115296-appb-000016
Figure PCTCN2021115296-appb-000017
Figure PCTCN2021115296-appb-000018
Figure PCTCN2021115296-appb-000019
Figure PCTCN2021115296-appb-000020
Figure PCTCN2021115296-appb-000021
Figure PCTCN2021115296-appb-000022
Figure PCTCN2021115296-appb-000023
表13图7(C)的数据
Figure PCTCN2021115296-appb-000024
Figure PCTCN2021115296-appb-000025
表14图7(E)的数据
Figure PCTCN2021115296-appb-000026
表15图8(B)、(D)、(E)使用的原始数据
Figure PCTCN2021115296-appb-000027
Figure PCTCN2021115296-appb-000028
本发明提供的生物活性多肽的合成及应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1细胞培养及体外神经分化诱导
本发明所涉及的所有干细胞均遵守人类胚胎干细胞研究的行为指南，人体组织的利用得到首都医科大学伦理委员会的批准，志愿者均知情同意，并术前签订知情同意书。简要说，75％酒精消毒牙齿后，磷酸缓冲液继续清洗10次。使用无菌手术刀片小心分离牙齿根尖部牙乳头组织，无菌剪刀剪碎后，加入3mg/ml的胶原蛋白酶I(Worthington Biochemical Corp.,Lakewood,NJ)和4mg/ml的分散酶(Roche Diagnostics Corp.,Indianapolis,IN)溶液各1ml，放入37℃震荡消化1小时。通过70-um的过滤器(BD Biosciences,San Jose,CA,)得到单一细胞的悬浮液。接着，将这些细胞接种于低限量Eagle培养基(Minimum Eagle medium，MEM)(Invitrogen,Carlsbad,CA)，加15％胎牛血清、2mmol/l谷氨酰胺、100U/ml青霉素和100mg/ml链霉素，置于5％CO 2和37℃的加湿保温箱中，每3天更换一次培养基。
所有干细胞按照之前描述进行干细胞表面标志物鉴定，后续实验使用第3-5代干细胞。对于体外神经分化诱导，使用神经干细胞优势培养基(Neurobasal A液中添加2％B27、40ng/ml bFGF、20ng/ml EGF、2mM L-谷氨酰胺、100U/ml青霉素和100ug/ml链霉素)诱导培养干细胞，每3天更换一次；
对于类神经球形成观察，分化诱导9天时于倒置显微镜下观察；对于免疫荧光染色实验，收集诱导9天时的类神经球，4％多聚甲醛固定，Triton进行膜通透处理后基础白蛋白液封闭处理，特异一抗液4℃孵育过夜，荧光标定种属二抗、细胞骨架染料PDI和细胞核染料DAPI依次孵育后借助荧光激发使可视化；主要针对特异一抗是抗βⅢ-TUBULIN、NESTIN多克隆抗体(Abcam, Cambridge,USA)。
实施例2质粒构建和病毒感染
质粒构建与病毒转染，按照标准方法进行质粒的构建，靶向KDM2B基因设计的目的互补shRNA序列被克隆进入到病毒载体pLKO.1质粒环中，测序鉴定，构建完成KDM2B shRNA质粒；设计KDM2B基因全长的PCR引物，扩增得到KDM2B的全长基因序列，添加HA-Tag标签，将其连接到逆转录病毒的表达载体pQCXIN上，测序鉴定，构建得到含有HA-Tag标签的全长KDM2B基因序列的质粒；然后进行病毒包装、收集，病毒滴度鉴定，分装后保存在-80度冰箱；病毒转染，10 -7滴度的逆转录病毒或慢病毒在凝聚胺参与下与根尖牙乳头干细胞培养过夜，48小时后抗生素筛选被转染的细胞；靶向KDM2B基因设计的目的互补shRNA序列是，KDM2Bsh：5’-ATTTGACGGGTGGATAATCTG-3’。
实施例3总RNA分离、逆转录(RT)PCR和实时荧光定量逆转录PCR
来源于不同个体的根尖乳头干细胞的三组总RNA样本通过使用抽提试剂和RNA抽提试剂盒(QIAGEN，GmBH，Germany)提取、纯化后，溶解于RNase-Free Water(QIAGEN)。总RNA通过分光光度仪ND-2100(赛默飞世尔)定量，使用安捷伦2100(安捷伦科技)评估RNA完整性。对于mRNA检测，依据生产厂家的标准(英杰)，使用随机引物试剂盒(QIAGEN)逆转录合成等量cDNA样本。使用荧光PCR(QIAGEN)和IcycleriQ多色实时荧光定量PCR技术检测系统进行实时PCR反应。引物设计使用在线引物3程序(引物序列详列于表18)，GAPDH作为内部控制，相对mRNA水平通过使用2 -ΔΔCt方法计算。
实施例4免疫共沉淀反应及蛋白质印迹分析
来源于不同个体的根尖乳头干细胞的三组总蛋白样本使用RIPA裂解液溶解提取。对于免疫共沉淀反应，等质量总蛋白样本与特异一抗免疫球蛋白A/G珠子4℃孵育过夜，三乙醇胺缓冲盐水溶液(TBS)清洗后，99℃煮沸变性，进行蛋白质印迹分析或-80℃备用；
对于蛋白质印迹分析，等质量总蛋白样本用10％SDS聚丙烯酰胺凝胶分离并利用半干转移膜装置转移到聚乙二烯二氟化物(PVDF)中，膜上涂抹5％脱脂牛奶放置2h，然后用一抗孵育一夜；免疫复合物与兔或小鼠免疫球蛋白G抗体一同孵育并用化学发光底物试剂使其可视化；主要针对特异性一抗是抗KDM2B、EZH2多克隆抗体(Abcam)。
实施例5大鼠脊髓神经损伤模型及根尖牙乳头干细胞回植
本发明通过首都医科大学附属北京口腔医院动物关怀和使用委员会允许；将约1×10 6个根尖牙乳头干细胞移植到一只10周龄大鼠脊椎T10部脊髓组织全切断位点。特别的，通过微注射体系(共30ul)于全切断位点的正中、左侧和右侧分别向中线方向进针，每位点分别缓慢注射10ul。主要针对的，对照Vector组和KDM2B过表达组根尖牙乳头干细胞是分别进行5只大鼠的回植，这些程序依照动物协议批准规范进行；分别于移植后0周、1周、2周和3周对大鼠进行BBB后肢运动能力评分；
对于组织病理学分析，三周后获取移植部脊髓组织用10％福尔马林固定，石蜡包埋，5um切片，苏木精-伊红染色(HE)染色；免疫组织化学染色，5um切片脱蜡脱水，去除内源过氧化物酶封闭后，特异性一抗液4℃孵育过夜，生物素-辣根过氧化物酶依次孵育后，借助3，3-二氨基联苯胺(DAB)底物显色使其可视化；主要针对特异性一抗是抗βⅢ-TUBULIN、NEF-M多克隆抗体(Abcam)。
实施例6多肽微阵列芯片设计及分析
通过Uniprot蛋白信息网站(https://www.uniprot.org/)查询获得人源KDM2B蛋白全长序列，KDM2B全长氨基酸序列通过Overlapping设计合成多肽芯片；借助全自动多肽芯片合成仪合成多肽阵列，封闭后与生物素标记目标蛋白孵育杂交，化学显色底物可视化，Chempchemi数字成像仪成像；显色芯片图片使用TotalLab图像分析软件分析显色点光密度值，特别的，阳性显色多肽位点以该显色点周边背景值为参照计算，设置膜上显色点光密度值最高的值为100％，其余各点光密度值为该点光密度值的百分比数值，选择多肽芯片膜上点光密度值超过30％且阴性反应膜上点光密度值低于30％的为阳性显色点。
进一步，KDM2B多肽微阵列膜上多肽点示意图和考马斯染色图，如图9所示：
如图9左侧图所示，阵列中多肽共18列15行，编号由左至右，由上至下依次增大，共由266个多肽点即266条肽组成，按照此芯片示意图合成1个拷贝的多肽微阵列芯片，每条多肽序列详列于表12。图9右侧图像为芯片完成后考马斯染色图像。
进一步，KDM2B多肽微阵列膜与EZH2蛋白杂交反应；
杂交实验使用NHS-PEO4-Biotinylation标记的EZH2蛋白液(1ug/ml)进行多肽芯片杂交反应，使用Streptavidin-HRP进行放大反应，反应步骤及条件如前所述，显色5min(结果如图6A所示)，由图可以看出，标记蛋白和芯片上某些多肽点有明显的结合并显色。
进一步，KDM2B多肽微阵列膜阳性显色反应点分析；
使用TotalLab软件图片显色点光密度数据分析(各点光密度值原始数据见附件Excel表格)，设置膜上显色点光密度值最高的值为100％，其余各点光密度值为该点光密度值的百分比数值，检测获得的阳性显色位点的灰度值表(结果如图6B显示)。该表中，横坐标为多肽点编号亦即对应阵列的266条多肽，纵坐标为个点光密度值百分比。根据经验，选择多肽芯片膜上点光密度值超过30％且阴性反应膜上点光密度值低于30％的为阳性显色点，则以下序列有明显差异(表16)：
表16 KDM2B多肽微阵列芯片与EZH2蛋白免疫杂交显色阳性位点的多肽段
芯片肽段序号 序列编号 氨基酸序列
5 SEQ ID No.5 AEKQKKKTVIYTKCF
46 SEQ ID No.46 VKKYCLMSVKGCFTD
47 SEQ ID No.47 LMSVKGCFTDFHIDF
122 SEQ ID No.122 ARRRRTRCRKCEACL
123 SEQ ID No.123 TRCRKCEACLRTECG
131 SEQ ID No.131 CIAPVLPHTAVCLVC
132 SEQ ID No.132 LPHTAVCLVCGEAGK
139 SEQ ID No.139 CNEIIHPGCLKIKES
142 SEQ ID No.142 EGVVNDELPNCWECP
151 SEQ ID No.151 QKMNRDNKEGQEPAK
152 SEQ ID No.152 DNKEGQEPAKRRSEC
153 SEQ ID No.153 QEPAKRRSECEEAPR
231 SEQ ID No.231 LRDLVLSGCSWIAVS
232 SEQ ID No.232 LSGCSWIAVSALCSS
233 SEQ ID No.233 WIAVSALCSSSCPLL
实施例7生物活性多肽的合成与纯化
获得生物活性多肽序列，穿膜肽及FITC绿色荧光基团标定，树脂-二氯甲烷液溶胀下按序列顺次合成相应氨基酸，茚三酮检测，然后用吡啶和乙酸酐封端，洗净、乙醚析出粗产物，离心后液相色谱法提纯，冻干机冻干获得生物活性多肽粉末；
进一步，为了检测KDM2B与EZH2的蛋白结合位点准确性，我们根据表16内的阳性结合报告位点合成了6条生物活性多肽(表17)，同时随机选择合成了阴性结合报告位点peptide 83-84作为对照多肽(ConPP组)。
表17基于KDM2B合成的生物活性多肽的氨基酸序列
Figure PCTCN2021115296-appb-000029
进一步，通过免疫共沉淀分析结果表明添加10ug/ml peptide 46-47、peptide 122-123、peptide 131-132刺激根尖牙乳头干细胞24小时后可显著阻断KDM2B与EZH2的结合(图6D)。
实施例8生物活性多肽对根尖牙乳头干细胞成神经分化的应用
进一步，根尖牙乳头干细胞使用神经干细胞优势培养基添加10ug/ml peptide诱导培养9天时，结果表明10ug/ml peptide 46-47、peptide 122-123、peptide 131-132明显提高了根尖牙乳头干细胞体外形成βⅢ-TUBULIN阳性(图7B)及NESTIN阳性(图7C)类神经球形成。
实施例9生物活性多肽对损伤神经组织再生修复的应用
进一步，我们选取并使用10ug/ml peptide 46-47预处理根尖牙乳头干细胞24小时后，即刻移植到10周龄大鼠T10脊椎部脊髓组织全切断位点(每组5只独立大鼠个体)。移植干细胞4周后，相较于对照ConPP预处理组，脊髓组织大体观显示10ug/ml peptide 46-47预处理组损伤神经组织愈合显著(图8A)；BBB行为学评分结果表明10ug/ml peptide 46-47预处理组大鼠后肢运动能力显著提高(图8B)。
进一步，我们借助微注射体系以每周注射一次的频率将30ul的10ug/ml peptide 46-47单独注射到10周龄大鼠T10脊椎部脊髓组织全切断位点(每组5只独立大鼠个体)。4周后，相较于单独注射对照ConPP组，脊髓组织大体观显示单独注射10ug/ml peptide 46-47组损伤神经组织愈合显著(图8C)；BBB行为学评分结果表明单独注射10ug/ml peptide 46-47组大鼠后肢运动能力显著提高(图8D)。
进一步，我们联合分析了对照ConPP预处理干细胞回植组、10ug/ml peptide 46-47预处理干细胞回植组和单独注射10ug/ml peptide 46-47组的大鼠BBB行为学评分，结果显示干预第4周时10ug/ml peptide 46-47预处理干细胞回植组和单独注射10ug/ml peptide 46-47组大鼠后肢运动能力均显著提高，两组间无明显差别(图8E)。
表18、进行实时定量PCR试验的引物
Figure PCTCN2021115296-appb-000030
以上对本发明所提供的基于组蛋白去甲基化酶KDM2B合成的生物活性多肽及其在间充质干细胞神经向分化过程和损伤神经组织再生修复中的应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本 发明权利要求的保护范围内。

Claims (10)

  1. KDM2B过表达在制备诱导根尖牙乳头干细胞体外成神经分化的制剂或药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，KDM2B的过表达在制备促进根尖牙乳头干细胞体外βⅢ-TUBULIN阳性及NESTIN阳性类神经球的形成的制剂或药物中的应用。
  3. KDM2B的过表达在制备促进体内根尖牙乳头干细胞介导的脊髓神经损伤组织再生修复的制剂或药物中的应用。
  4. 多肽，其特征在于，其具有：
    (I)、如SEQ ID No.1～266任意所示的氨基酸序列；或
    (II)、如(I)所述的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(I)所述的氨基酸序列功能相同的氨基酸序列；或
    (III)、与如(I)或(II)所述的氨基酸序列具有90％以上同一性的氨基酸序列；
    所述取代、缺失或添加一个或多个氨基酸中的多个为2个。
  5. 编码如权利要求4所述多肽的核酸分子。
  6. 表达载体，其特征在于，包括如权利要求5所述的核酸分子。
  7. 如权利要求4所述的多肽，如权利要求5所述的核酸分子，如权利要求6所述的表达载体，直接或间接在制备诱导根尖牙乳头干细胞体外成神经分化的制剂或药物中的应用。
  8. 如权利要求4所述的多肽，如权利要求5所述的核酸分子，如权利要求6所述的表达载体，直接或间接在制备促进体内根尖牙乳头干细胞介导的脊髓神经损伤组织再生修复的制剂或药物中的应用。
  9. 如权利要求7或8所述的应用，其特征在于，KDM2B与EZH2的结合位点包括：
    阳性结合位点选自peptide 5、peptide 46、peptide 47、peptide 122、peptide 123、peptide 131、peptide 132、peptide 139、peptide 142、peptide 151、peptide 152、peptide 153、peptide 231、peptide 232、peptide 233；
    所述阳性结合位点的氨基酸序列如SEQ ID No.5、SEQ ID No.46、SEQ ID No.47、SEQ ID No.122、SEQ ID No.123、SEQ ID No.131、SEQ ID No.132、SEQ ID No.139、SEQ ID No.142、SEQ ID No.151、SEQ ID No.152、SEQ ID No.153、SEQ ID No.231、SEQ ID No.232或SEQ ID No.233所示；
    所述阴性结合位点选自peptide 83、peptide 84；
    所述阴性结合位点的氨基酸序列如SEQ ID No.267所示。
  10. 药物或制剂，其特征在于，包括如权利要求4所述的多肽以及药学上可接受的辅料。
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