CN114181915A

CN114181915A - Application of bioactive polypeptide synthesized based on KDM2B sequence in mesenchymal stem cell neural differentiation and regeneration repair

Info

Publication number: CN114181915A
Application number: CN202111002469.5A
Authority: CN
Inventors: 范志朋; 曹杨杨; 张琛
Original assignee: Beijing Stomatological Hospital
Current assignee: Beijing Stomatological Hospital
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2022-03-15
Anticipated expiration: 2041-08-30
Also published as: CN114181915B

Abstract

The invention relates to the technical field of bioengineering, in particular to a bioactive polypeptide synthesized based on histone demethylase and application thereof in a mesenchymal stem cell neural differentiation process. Experiments show that the KDM2B bioactive polypeptide has a function in the differentiation process of mesenchymal stem cell nerves and a function in the regeneration of spinal cord nerve injured tissues. The invention relates to a possible protein interaction binding site of histone demethylase KDM2B and histone methylase EZH2, application of KDM2B and bioactive polypeptide synthesized based on the same in a mesenchymal stem cell neural differentiation process, application of KDM2B and bioactive polypeptide synthesized based on the same in spinal cord nerve injury tissue regeneration, and promotion of the obtained KDM2B and bioactive polypeptide synthesized based on the same in root tip papilla mammary stem cell neural differentiation and spinal cord nerve injury regeneration.

Description

Application of bioactive polypeptide synthesized based on KDM2B sequence in mesenchymal stem cell neural differentiation and regeneration repair

Technical Field

The invention relates to the technical field of bioengineering, in particular to a bioactive polypeptide synthesized based on histone demethylase KDM2B and application thereof in the differentiation process of mesenchymal stem cell nerves and the regeneration and repair of damaged nerve tissues.

Background

The cranial and maxillofacial nerve tissues are widely distributed and are easy to be damaged. Recent epidemiological investigation shows that 55.2% of craniomaxillofacial trauma patients have nerve injuries with different degrees, 2.2% of patients are additionally accompanied by spinal cord contusion, and 24.0% of patients in subsequent treatment complications have nerve dysfunction. The traditional treatment modes mainly use neuroprotective measures such as tepidity and the like, use neuroprotective medicines such as glucocorticoids, glutamic acid antagonists and the like, use electricity, heat, force and the like to stimulate nerve self-healing and the like, and the treatment is complex, long in period and low in nerve function recovery rate, so that the high disability rate is caused. The self/variant nerve transplantation treatment has the defects of difficult material taking of donor tissues, secondary damage of the material taking part and the like, and meanwhile, transplanted nerves cannot be well shaped and still cannot well recover the tissue structure appearance and the damaged function. At present, the clinical treatment effect of the injured nerve still needs to be improved.

Biological regenerative therapy based on mediation of Mesenchymal Stem Cells (MSCs) will be a new therapeutic approach for the future repair of impaired neurological function. In response to the tissue injury signal, the MSCs have good differentiation ability to promote the formation of nerve repair cells and axon regeneration, and finally promote the regeneration and repair of injured nerves. However, the source of the neurogenic stem cells is limited, the material is difficult to obtain, and more seed stem cells still need to be discovered. The odontogenic MSCs develop from the neural crest which originally originates from the ectoderm and is closely originated from the neural tissue, so the odontogenic MSCs have the advantages of transformation and application. Among them, the Stem Cells of the Papilla of the teeth from the Apical papillala (SCAP) exist in the Papilla tissue of the teeth at the Apical portion of the teeth, and the SCAP is found to form dental pulp nerve-like tissue in vivo, which suggests that the SCAP is the available seed Cells for nerve regeneration. The main problems that currently limit the potential applications of SCAPs are the low neural differentiation efficiency and the unclear regulatory mechanisms. Therefore, how to effectively discover key regulation targets and promote the restoration potential of the odontogenic mesenchymal stem cells is very important.

Recent studies have established that epigenetic regulation of chromatin is a key mechanism that determines the neural lineage differentiation of MSCs. Through the disclosure of a whole-genome epigenetic regulation map, the methylation modification scores of lysine 27 site (H3K27) and lysine 4 site (H3K4) on the differentiated MSCs histone H3 are obviously increased. Methylation modification of histones mostly occurs at lysine residues (K), which is the main form of covalent modification of histones in epigenetic mechanisms. Histone methylation modification groups existing in a promoter region of a gene block the transcriptional expression of the gene (such as H3K27me3) or promote the transcriptional expression of the gene (such as H3K4me3), and histone methylation/demethylase plays a key role in the methylation modification of histone respectively, and also has certain functional interaction (such as formation of a complex through functional domain combination).

A whole genome chromatin immunoprecipitation (ChIP) sequencing on embryonic stem cells showed that binding of histone methylase EZH2 was highly correlated with H3K27me3 modification of the promoter region of neural differentiation regulatory genes. EZH2 belongs to the core member of Polycomb groups (PcGs), and its modified H3K27me3 is a histone methylation modification that inhibits gene transcription, thought to hinder differentiation progression for specific cell fates. Research finds that EZH2 inhibits the differentiation of neural stem cells to form neurogenic cells, and the application of an H3K27me3 specific antagonist EPZ005687 can effectively improve the neuronal differentiation capacity of mesencephalon ventral-derived neural stem cells, and suggests that EZH2 has a possible inhibitory effect on neural differentiation. Therefore, how to effectively regulate the functional action of EZH2 is very critical.

Studies have shown that KDM2B is involved in recruiting the PRC2-EZH2 complex to anchor downstream target genes and subsequent covalent modification of histones, suggesting that there may be a cross-over in epigenetic regulation between KDM2B and EZH 2. KDM2B belongs to histone demethylase, and is mainly involved in the regulation and control of various cell processes by playing a role of removing trimethylation modification (H3K4me3) of histone H3K4 site. The researchers analyzed and found that the function of KDM2B depends on various functional domains such as JmjC, CxC, PHD and the like contained in the KDM. However, the relationship between the neural differentiation function of KDM2B and the odontogenic MSCs is not clear; the possible interaction between KDM2B and EZH2 proteins is unclear.

At present, a technical method for analyzing protein binding is being developed so that researchers can follow a wide view on information on specific binding sites at which two proteins interact, which is "polypeptide microarray analysis", a protein-based immunohybridization assay, using hybridization of a protein of interest with a microarray chip synthesized based on the full-length amino acid sequence of another protein, amplifying the signal of such hybridization binding by means of an enzyme-linked label, thereby recognizing possible binding fragment sites between two proteins and information on the specific amino acid sequence of such possible sites. Therefore, a possible binding functional domain segment between KDM2B and EZH2 can be researched by using a polypeptide microarray method, the key interaction site of a potential apparent modification enzyme for accurately regulating and controlling the neural differentiation of the odontogenic MSCs is understood, a new biological medicine for promoting the neural differentiation of the odontogenic MSCs is researched, and the effect of damaged neurobiological regeneration under clinical conditions is improved.

The purpose of the research of the invention is to clarify the effect of KDM2B on the neural differentiation and regeneration of odontogenic mesenchymal stem cells, to clarify the possible interaction of KDM2B and EZH2, and to research the possible binding site fragment information between KDM2B and EZH2 by using a polypeptide microarray method.

Disclosure of Invention

In view of the above, the invention provides a bioactive polypeptide synthesized based on histone demethylase KDM2B and a regulation and control method thereof in a mesenchymal stem cell neural differentiation process and damaged neural tissue regeneration and repair, and aims to solve the problem that the prior art does not relate to the regulation and control of a KDM2B gene and the bioactive polypeptide thereof in a odontogenic mesenchymal stem cell neural differentiation process and damaged neural tissue regeneration and repair.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of KDM2B overexpression in preparation of a preparation or a medicament for inducing in-vitro neural differentiation of deciduous papilla stem cells of apical teeth.

In some embodiments of the invention, the use of overexpression of KDM2B in the preparation of a formulation or medicament for promoting the formation of β iii-TUBULIN-positive and NESTIN-positive neurospheres in vitro in a deciduous stem cell of a root tip.

The invention also provides application of KDM2B in preparation of a preparation or a medicament for promoting regeneration and repair of spinal nerve injured tissue mediated by root apical papilla stem cells in vivo.

More importantly, the invention provides polypeptides having:

(I) and an amino acid sequence as shown in any of SEQ ID Nos. 1-266; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described in (I) and has the same function with the amino acid sequence described in (I); or

(III) an amino acid sequence having 90% or more identity to the amino acid sequence of (I) or (II);

and more of the substitution, deletion or addition of one or more amino acids is 2, 3, 4 or 5.

In addition, the invention also provides nucleic acid molecules encoding the polypeptides.

The invention also provides an expression vector comprising the nucleic acid molecule.

More importantly, the invention also provides the application of the polypeptide, the nucleic acid molecule and the expression vector in the preparation of a preparation or a medicament for inducing the in vitro neural differentiation of the deciduous papilla stem cells of the apical teeth.

The invention also provides the application of the polypeptide, the nucleic acid molecule and the expression vector in preparing a preparation or a medicament for promoting regeneration and repair of spinal nerve injured tissues mediated by in vivo apical deciduous papillary stem cells.

In some embodiments of the invention, the binding site of KDM2B to EZH2 comprises:

the positive binding site is selected from the group consisting of 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, and peptide 233;

the amino acid sequence of the positive binding site is shown 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;

the negative binding site is selected from peptide 83 and peptide 84;

the amino acid sequence of the negative binding site is shown as SEQ ID No. 267.

In addition, the polypeptide also comprises a polypeptide with an amino acid sequence shown in SEQ ID No. 267-270.

The invention also provides a medicament or a preparation, which comprises the polypeptide and pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides a preparation method of the polypeptide, which comprises the following steps:

step 1, culturing mesenchymal stem cells, constructing plasmids and transfecting viruses;

step 2, forming beta III-TUBULIN and NESTIN double-positive neurospheres, inducing and culturing mesenchymal stem cells by using a neural stem cell dominant culture medium, inducing the neurospheres formed in 9 days, fixing 4% paraformaldehyde, carrying out membrane permeation treatment on the Triton, then sealing the basic albumin liquid, then incubating overnight at 4 ℃ by using a specific primary antibody liquid, sequentially incubating a fluorescence calibration species specific secondary antibody, a cytoskeletal dye PDI and a cell nucleus dye DAPI, and then visualizing the cells by means of fluorescence excitation; mainly aiming at that the specific primary antibody is a polyclonal antibody for resisting a neuron specific expression gene microtubulin beta III-TUBULIN and a neural progenitor cell marker NESTIN NESTIN;

step 3, replanting the rat spinal cord nerve injury model and the root tip tooth papilla stem cells, and replanting about 1 × 10⁶Root tip tooth papilla stem cellTransplanting the rat into T10 spinal cord tissue total cutting sites of 10-week-old rats, and performing BBB hindlimb motor ability scoring on the rats at 0 week, 1 week, 2 weeks and 3 weeks after transplantation; histopathological analysis, obtaining spinal cord tissue at the injury part after three weeks, making paraffin sections, hematoxylin-eosin (HE) staining, and immunohistochemical staining of neuron specific expression TUBULIN beta III-TUBULIN and nerve fiber silk specific protein NEF-M;

further, transplanting rat T10 spinal cord tissue total cutting sites by root tip tooth papilla stem cells, specifically, respectively inserting needles towards the midline direction at the center, left side and right side of the total cutting sites by a micro injection system (total 30ul), and slowly injecting 10ul at each site;

step 4, performing co-immunoprecipitation reaction, dissolving cells by RIPA lysate to extract total protein, incubating a protein sample and specific primary immunoglobulin A/G beads overnight at 4 ℃, washing by Triethanolamine Buffered Saline (TBS), boiling and denaturing at 99 ℃, performing western blot analysis, separating the protein sample by using 10% SDS polyacrylamide gel, transferring the protein sample into polyvinylidene difluoride (PVDF) by using a semi-dry transfer membrane device, coating 5% skimmed milk on the membrane, standing for 2 hours, and incubating by using a primary antibody overnight; incubating the immune complex with rabbit or mouse immunoglobulin G antibody and visualizing it with a chemiluminescent substrate reagent; the specific primary antibody is an anti-KDM 2B polyclonal antibody and an EZH2 polyclonal antibody;

step 5, polypeptide microarray hybridization and data analysis, namely synthesizing a KDM2B polypeptide microarray chip by an overlaying design mode design and full-automatic polypeptide chip synthesizer according to the full-length amino acid sequence of the KDM 2B; performing immune hybridization reaction on the polypeptide microarray chip and recombinant protein, activating the polypeptide microarray chip, sealing, performing oscillation incubation overnight at 4 ℃ with a biotin-labeled EZH2 protein sample reaction solution, incubating with an HRP chromogenic substrate, and visualizing an ECL chemiluminescent reagent in a Chemmpchemi digital imager; scanning and data analysis are carried out on the color development points of the chip, TotalLab image analysis software is used for analyzing the optical density value of the color development points of the imaging picture, and a Spot Edge Average algorithm in the software is used for calculating the color development intensity percentage value of each color development point.

And 6, synthesizing and purifying bioactive polypeptides to obtain bioactive polypeptide sequences, marking the cell-penetrating peptides and FITC green fluorescent groups, sequentially synthesizing corresponding amino acids according to sequences under the swelling of resin-dichloromethane solution, detecting ninhydrin, then capping with pyridine and acetic anhydride, washing, precipitating crude products with diethyl ether, purifying by liquid chromatography after centrifugation, and freeze-drying by a freeze-dryer to obtain bioactive polypeptide powder.

The invention also provides application of co-immunoprecipitation analysis on the combination of the bioactive polypeptide provided by the invention to KDM2B/EZH2 complex; the application of beta III-TUBULIN and NESTIN double-positive neurosphere formation in the neurosphere differentiation of bioactive polypeptide on the deciduous head stem cells of the apical teeth; the rat spinal cord injury nerve tissue local site replanting is used for the application of bioactive polypeptide to spinal cord nerve injury tissue regeneration.

The invention provides a bioactive polypeptide synthesized based on histone demethylase KDM2B and application thereof in a mesenchymal stem cell neural differentiation process and spinal cord neural injury tissue regeneration, wherein the effect of the KDM2B bioactive polypeptide in the mesenchymal stem cell neural differentiation process and the effect in the spinal cord neural injury tissue regeneration are discovered through polypeptide microarray chip hybridization and data analysis, Western blot analysis, neurosphere-like induction formation, beta III-TUBULIN and NESTIN double-positive neurosphere-like immunofluorescence staining and rat spinal cord tissue full-cutting injury model local site replanting test. The invention relates to a possible protein interaction binding site of histone demethylase KDM2B and histone methylase EZH2, relates to the action of KDM2B and bioactive polypeptide in the differentiation process of mesenchymal stem cell nerves, and relates to the action of KDM2B and bioactive polypeptide in regeneration of spinal cord nerve injury tissues, and the obtained KDM2B and bioactive polypeptide synthesized based on the same can play a promoting role in neural differentiation of root tip dental papilla stem cells and regeneration of spinal cord nerve injury tissues.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for the description of the embodiments or the prior art will be briefly described below.

Fig. 1 illustrates the expression of KDM2B in mesenchymal stem cells and in spinal cord tissue provided by an embodiment of the invention; wherein, the real-time fluorescence quantitative reverse transcription PCR result of figure 1(A) shows that the expression of KDM2B is increased in the process of inducing the differentiation of the deciduous papilla stem cells of the apical teeth into nerves in vitro, and the peak value appears on the third day of induction; figure 1(B, C) immunohistochemical staining results show reduced expression of KDM2B in spinal nerve injured tissue compared to normal spinal nerve tissue; GAPDH as an internal control; data T test is used to determine statistical significance; -All error bars represent the s.d. (n-3); p is less than or equal to 0.01; red scale bar:100 μm;

FIG. 2 is a schematic diagram showing that overexpression of KDM2B promotes formation of β III-TUBULIN and NESTIN positive neurosphere-like cells in vitro, according to an embodiment of the present invention; wherein, the real-time fluorescence quantitative reverse transcription PCR result of the figure 2(A) and the protein immune blotting result of the figure 2(B) show that the over-expression root tip dental papilla stem cells of KDM2B are constructed; FIG. 2(C) over-expression of KDM2B promotes root apical papilla stem cell neurobulbar formation in vitro, when neuro-induced for 9 days, compared to the control Vector group; FIG. 2(D) immunofluorescence staining results and FIG. 2(E) quantitative results show that over-expression of KDM2B promotes formation of β III-TUBULIN positive neurobulboids in vitro of deciduous papilla stem cells of apical teeth; FIG. 2(F) immunofluorescence staining results and FIG. 2(G) quantitative results show that over-expression of KDM2B promotes formation of NESTIN-positive neurospheres in vitro of deciduous cells of the apical teeth; FIG. 2(H-K) real-time fluorescent quantitative reverse transcription PCR results show that overexpression of KDM2B up-regulates the expression of TH (H), NCAM (I), NEF (J), NEUROD (K) in apical papilla stem cells; GAPDH as an internal control; data T test is used to determine statistical significance; -All error bars represent the s.d. (n-3); p is less than or equal to 0.01; white scale bar:100 μm;

FIG. 3 is a schematic diagram showing that knockdown expression of KDM2B inhibits the formation of β III-TUBULIN and NESTIN positive neurospheres in vitro by using papillary stem cells of apical teeth provided in the examples of the present invention; wherein, the real-time fluorescence quantitative reverse transcription PCR result of FIG. 3(A) shows that the construction of the KDM2B knockdown expression root tip papilla stem cells is completed; FIG. 3(B) knockdown expression of KDM2B inhibited root tip papilla stem cell formation in vitro as compared to the control Scaramsh group at 9 days of adult neural induction; FIG. 3(C) immunofluorescence staining results and FIG. 3(D) quantitative results show that the knockdown expression of KDM2B inhibits beta III-TUBULIN positive neurosphere formation in vitro of root apical papilla stem cells; FIG. 3(E) immunofluorescence staining results and FIG. 3(F) quantitative results show that knockdown expression of KDM2B inhibits Nestin positive neurosphere formation in vitro of root-tip papilla stem cells; GAPDH as an internal control; data T test is used to determine statistical significance; -All error bars represent the s.d. (n-3); p is less than or equal to 0.01; white scale bar:100 μm;

fig. 4 is a schematic diagram showing that overexpression of KDM2B provided by the embodiment of the present invention promotes regeneration and repair of spinal nerve damaged tissue mediated by apical papilla stem cells in vivo; when the papillary stem cells of the apical teeth are transplanted for 3 weeks, the spinal cord nervous tissue in fig. 4(A) shows that the injury healing of the KDM2B overexpression group is obvious in general observation; FIG. 4(B) BBB behavioral score results show that KDM2B overexpression group significantly improves rat hindlimb motor ability; fig. 4(C) hematoxylin-eosin (HE) staining results show that KDM2B overexpression group significantly promotes repair and regeneration of spinal cord injury nerve fibers; FIG. 4(D) immunohistochemical staining results show that the KDM2B overexpression group significantly increased β III-TUBULIN and NEF-M expression in spinal cord injury neural tissue; GAPDH as an internal control; data T test is used to determine statistical significance; -All error bars represent the s.d. (n-3); p is less than or equal to 0.05 and P is less than or equal to 0.01; white scale bar:5mm, Red scale bar:100 μm;

FIG. 5 is a schematic diagram showing the CO-immunoprecipitation (CO-IP) method provided in the examples of the invention detecting the binding of KDM2B to EZH2 protein;

FIG. 6 is a schematic diagram of a microarray chip for the protein EZH2 and KDM2B according to an embodiment of the present invention; wherein the hybridization spot plot of FIG. 6(A) and the gray scale value results of the microarray polypeptide spots of FIG. 6(B) indicate positive binding peptide fragment sites; FIG. 6(C) a schematic diagram of the EZH2 and KDM2B protein binding domain fragment; FIG. 6(D) Co-immunoprecipitation (CO-IP) results show that 10ug/ml bioactive polypeptides peptide 46-47(PP1 group), peptide 122-123(PP2 group), and peptide 131-132(PP3 group) effectively block the binding of EZH2 and KDM 2B;

FIG. 7 is a schematic diagram showing that bioactive polypeptides 46-47(PP1 group), polypeptide 122-123(PP2 group) and polypeptide 131-132(PP3 group) provided by the embodiment of the present invention promote the formation of β III-TUBULIN and NESTIN positive neurosphere-like cells in vitro of the deciduous papillary stem cells of apical teeth; wherein, when figure 7(A) becomes neural induction for 9 days, peptide 46-47, peptide 122-123 and peptide 131-132 all significantly promote the formation of root tip papilla stem cell neurospheres compared with the control ConPP addition group; immunofluorescence staining and quantitative results show that the peptide 46-47, peptide 122-123 and peptide 131-132 addition groups all remarkably promote the formation of beta III-TUBULIN (B, C) positive and NESTIN (D, E) positive neurospheres in vitro of the root tip papilla stem cells; data T test is used to determine statistical significance; -All error bars represent the s.d. (n-3); p is less than or equal to 0.01; white scale bar:5 mm;

FIG. 8 is a schematic diagram showing that bioactive polypeptide peptides 46-47(PP1 group) provided by the embodiment of the invention promote the regeneration and repair of spinal cord injury nerve tissues; wherein, after the 10ug/ml peptide 46-47 pre-treatment of the deciduous head stem cells of the apical teeth is transplanted for 4 weeks, the general observation of the spinal cord tissue in FIG. 8(A) shows that the healing of the injured nerve tissue of the 10ug/ml peptide 46-47 pre-treatment group is obvious; FIG. 8(B) BBB behavioral scoring results show that 10ug/ml peptide 46-47 pretreatment group significantly improved hind limb locomotor ability in rats; meanwhile, after the spinal cord injury local site is independently and continuously injected with 10ug/ml peptide 46-47 for 4 weeks, the general observation of the spinal cord tissue in fig. 8(C) shows that the injured nerve tissue of the 10ug/ml peptide 46-47 injection group is obviously healed; FIG. 8(D) BBB ethological scoring result shows that 10ug/ml peptide 46-47 injection group significantly improves hindlimb mobility of rats; FIG. 8(E) Combined analysis BBB behavioural score results show that both the 10ug/ml peptide 46-47 pre-treated stem cell replanting group and the 10ug/ml peptide 46-47 injection group alone can significantly improve the hindlimb motor ability of rats at 4 weeks after intervention, and there is no significant difference between the two groups; data T test is used to determine statistical significance; -All error bars represent the s.d. (n-3); p is less than or equal to 0.05 and P is less than or equal to 0.01; white scale bar:5 mm;

FIG. 9 shows a schematic of the polypeptide spots on a KDM2B polypeptide microarray membrane and a Coomassie staining pattern; left side schematic representation of polypeptide spots on KDM2B polypeptide microarray membrane; the right panel shows a coomassie stained image after chip completion.

Detailed Description

The invention discloses a bioactive polypeptide synthesized based on histone demethylase and application thereof in a mesenchymal stem cell neural differentiation process. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein may be made and equivalents employed without departing from the spirit and scope of the invention.

The invention aims to provide a bioactive polypeptide synthesized based on histone demethylase KDM2B and a regulation and control method thereof in a mesenchymal stem cell neurodifferentiation process, and aims to solve the problem that the prior art does not relate to regulation and control of a KDM2B gene and the bioactive polypeptide thereof in a mesenchymal stem cell neurodifferentiation process.

The invention is realized in such a way, a bioactive polypeptide synthesized based on histone demethylase KDM2B and a regulation and control method thereof in the process of mesenchymal stem cell neurodifferentiation, and the method for designing and analyzing KDM2B polypeptide microarray and synthesizing the bioactive polypeptide based on KDM2B comprises the following steps:

firstly, polypeptide microarray chip design information of KDM2B protein is obtained by querying a Uniprot protein information website (https:// www.uniprot.org /) to obtain a full-length sequence of human KDM2B protein, synthesizing a polypeptide chip (detailed in Table 12) according to 1336 amino acid sequences of KDM2B protein, designing a first polypeptide of the array by using 15 amino acid sequences as an observation window from a first amino acid site through overlappinging design, then shifting 5 amino acid sites backwards, using 15 amino acid sequences as a second observation window, designing a second polypeptide of the array, and sequentially continuing by the method to finally obtain 266 polypeptides to form the polypeptide microarray chip based on the KDM2B protein.

Step two, polypeptide array synthesis, wherein the activated substrate chip membrane is placed on a full-automatic polypeptide chip synthesizer, and Fmoc (9-fluorenylmethoxycarbonyl) -amino acid raw material solution is automatically transferred to a specific position on the activated membrane according to a program to react with the membrane; then, the membrane is immersed into BSA protein blocking solutions I and II in sequence to carry out side chain blocking; then, washing the membrane with DMF (dimethylformamide) to remove the Fmoc protecting group at the amino terminal, and then drying with ethanol; repeating the steps until the polypeptide array is completely synthesized. Finally, the side chain protecting group is removed with a specific organic reagent, followed by CH₂Cl₂Washing membrane, washing with ethanol, drying, and storing at-20 deg.C.

Step three, performing immune hybridization binding reaction on the polypeptide array and the recombinant protein, activating the polypeptide microarray chip, adding a sealing liquid, sealing for 4 hours at room temperature by shaking, and washing the chip; taking EZH2 protein sample reaction solution (concentration is 1.5mg/ml), and carrying out protein labeling by EZ-link NHS-PEO4-Biotinylation kit (prod # 21455); mixing biotin-labeled EZH2 protein sample reaction solution (final concentration of 1ug/ml) diluted by using a sealing solution with a polypeptide microarray chip, performing shaking incubation at 4 ℃ overnight, and performing incubation on a control group by using the sealing solution; incubating a chromogenic substrate Streptavidin-HRP (High Sensitivity Streptavidin-HRP (prod #21133)), diluting a sealing solution (1:10000), incubating the polypeptide microarray chip with 5ml, shaking at room temperature for 2 hours, and washing the chip; ECL chemiluminescent reagents were visualized on a Chempchemi digital imager.

And step four, scanning the chip and analyzing the data of the color development chip, wherein the color development chip uses a Chempchemi chemiluminescence imaging system to scan and image at 425nm, and the color development time is 200 s. The imaging picture uses TotalLab image analysis software to analyze the optical density value of the color development points, and uses a 'Spot Edge Average' algorithm in the software to calculate the optical density value of each color development point by taking the peripheral background value of each color development point as reference.

Step five, synthesis of bioactive polypeptide, wherein in the project, 7 bioactive polypeptides are synthesized (the amino acid sequences are detailed in Table 17). Wherein, the left amino terminal of each bioactive polypeptide is added with a cell-penetrating peptide sequence: YGRKKRRQRRR to facilitate its passage through cell permeable membrane and into cell. Meanwhile, each bioactive polypeptide is calibrated by using a FITC green fluorescent group; the synthesis sequence is as follows: from the carboxy terminus to the amino terminus of each biologically active polypeptide sequence.

Specifically, the synthesis steps are as follows: weighing n equivalents of resin, placing into a reactor, adding DCM (dichloromethane) to swell for half an hour, then pumping off DCM, adding 2n equivalents of the first amino acid in the sequence, adding 2n equivalents of DIEA, appropriate amount of DMF and DCM (appropriate amount is that the resin can be fully stirred), DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, and nitrogen bubbling reaction for 60 min. Then adding about 5n equivalent of methanol, reacting for half an hour, pumping out reaction liquid, and washing with DMF and MEOH; the second amino acid in the sequence (also 2n equivalents), 2n equivalents HBTU (1-hydroxy, benzo, trichloroazol tetramethyl hexafluorophosphate) and DIEA were charged to the reactor, bubbled with nitrogen for 30min, the liquid was washed off, assayed for ninhydrin, and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin; sequentially adding amino acids in the subsequent polypeptide sequence according to the mode of the step 2) and carrying out reaction modification; blowing the resin to dry with nitrogen, taking the resin out of the reaction column, pouring the resin into a flask, adding a certain amount of cutting fluid (the composition is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water) (the ratio of the cutting fluid to the resin is about 10 ml/g) into the flask, shaking and filtering the resin; obtaining filtrate, then adding a large amount of ether into the filtrate to separate out a crude product, centrifuging, and cleaning to obtain the crude product of the bioactive polypeptide sequence;

and sixthly, purifying and freeze-drying the bioactive polypeptide, purifying the crude product to the required purity by means of a high performance liquid chromatography method, and concentrating and freeze-drying the purified liquid in a freeze dryer to finally obtain light yellow powder, namely the bioactive polypeptide.

As shown in fig. 6, the KDM2B polypeptide microarray design, analysis and KDM 2B-based synthesis of biologically active polypeptides according to the embodiments of the present invention comprises the following steps:

s601 polypeptide microarray chip design information of KDM2B protein

The full-length sequence of the human KDM2B protein is obtained through a Uniprot protein information website (https:// www.uniprot.org /) query, a polypeptide chip (detailed in Table 12) is synthesized according to 1336 amino acid sequences of the KDM2B protein, and an overlaying design is carried out, namely, a first polypeptide of the array is designed by taking the length of 15 amino acid sequences as an observation window from a first amino acid site, then the first polypeptide is shifted backwards by 5 amino acid sites, and then the length of the following 15 amino acid sequences is taken as a second observation window, a second polypeptide of the array is designed and sequentially carried out by the method, 266 polypeptides are finally obtained, and the polypeptide microarray chip based on the KDM2B protein is formed.

S602: polypeptide array synthesis

The activated substrate chip membrane is placed on a full-automatic polypeptide chip synthesizer, and Fmoc (9-fluorenylmethyloxycarbonyl) -amino acid raw material solution is automatically transferred to a specific position on the activated membrane according to a program to react with the membrane; then, the membrane is immersed into BSA protein blocking solutions I and II in sequence to carry out side chain blocking; then, washing the membrane with DMF (dimethylformamide) to remove the Fmoc protecting group at the amino end, and then drying with ethanol; repeating the steps until the polypeptide array is completely synthesized. Finally, the side chain protecting group is removed with a specific organic reagent, followed by CH₂Cl₂Washing membrane, washing with ethanol, drying, and storing at-20 deg.C.

S603: polypeptide array and recombinant protein immune hybridization combination reaction

Adding the sealing liquid into the polypeptide microarray chip, and oscillating for 4 hours at room temperature; taking EZH2 protein sample reaction solution (concentration is 1.5mg/ml), and carrying out protein labeling by EZ-link NHS-PEO4-Biotinylation kit (prod # 21455); using 5ml of biotin-labeled EZH2 protein sample reaction solution (final concentration is 1ug/ml) diluted by a blocking solution to shake with the polypeptide microarray chip at 4 ℃ overnight, and incubating a control group by the blocking solution; after diluting (1:10000) a chromogenic substrate Streptavidin-HRP (High Sensitivity Streptavidin-HRP (prod #21133)) with a blocking solution, 5ml was shaken with a polypeptide microarray chip at room temperature for 2 hours; ECL chemiluminescent reagents visualize it.

S604: chip scanning and color rendering point data analysis

The chip is scanned and imaged by using a chempchemic chemiluminescence imaging system at 425nm, and the development time is 200 s. And analyzing the optical density value of the color development point of the imaging picture by using TotalLab image analysis software, and calculating the color development percentage value of each point by using a 'Spot Edge Average' algorithm in the software.

S605: synthesis of biologically active polypeptides

In the present invention, 7 biologically active polypeptides were synthesized (amino acid sequences are shown in Table 17). Wherein, the left amino terminal of each bioactive polypeptide is added with a cell-penetrating peptide sequence: YGRKKRRQRRR to facilitate its passage through cell permeable membrane and into cell. Meanwhile, each bioactive polypeptide is calibrated by using a FITC green fluorescent group; the synthesis sequence is as follows: from the carboxy terminus to the amino terminus of each biologically active polypeptide sequence.

s606: purifying and freeze-drying the bioactive polypeptide:

purifying the crude product to required purity by high performance liquid chromatography, concentrating the purified liquid in a freeze dryer, and freeze-drying to obtain light yellow powder as bioactive polypeptide.

S607: methods of use of biologically active polypeptides:

the cells were dissolved in a dry cell culture medium (e.g., an α -MEM base solution containing 15% fetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100ug/ml streptomycin) or cell phosphate buffered saline at a storage concentration of 10ug/ul, and stored at-80 ℃ after split charging, avoiding repeated freeze-thawing. When in use, a corresponding culture solution system (such as the stem cell culture solution and the adult nerve differentiation inducing solution used in the project) is added according to the working concentration of 10ug/ml, and the preparation is prepared at present.

The specific embodiments are merely illustrative of the invention and do not limit the invention.

In conclusion, the invention researches the role of KDM2B and the bioactive polypeptide thereof in neural differentiation of deciduous cell stem cells of apical teeth and regeneration and repair of damaged neural tissues by using the bioactive polypeptide synthesized based on KDM 2B.

Data corresponding to the figures in the invention:

TABLE 1 data of FIG. 1(A)

TABLE 2 data of FIG. 1(C)

TABLE 3 data of FIG. 2(A)

TABLE 4 data of FIG. 2(E)

TABLE 5 data of FIG. 2(G)

TABLE 6 data of FIG. 2(H-K)

TABLE 7 data of FIG. 3(A)

TABLE 8 data of FIG. 3(E)

TABLE 9 data of FIG. 3(F)

TABLE 10 data of FIG. 4(B)

TABLE 11 data of FIG. 4(E)

TABLE 12 data of FIG. 6(B)

TABLE 13 data of FIG. 7(C)

TABLE 14 data of FIG. 7(E)

TABLE 15 raw data used in FIGS. 8(B), (D), (E)

The raw materials and reagents used in the synthesis and application of the bioactive polypeptide provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 cell culture and in vitro neural differentiation Induction

All stem cells related to the invention obey the behavioral guidelines of human embryonic stem cell research, the utilization of human tissues is approved by the ethical committee of the university of capital medical science, and volunteers give informed consent and sign informed consent before operation. Briefly, after disinfecting the teeth with 75% alcohol, the phosphate buffer solution was washed 10 times. Tooth root tip papilla tissue was carefully separated using a sterile scalpel blade, and after being minced with sterile scissors, 1ml each of a 3mg/ml collagenase I (Worthington Biochemical corp., Lakewood, NJ) and a 4mg/ml dispase (Roche Diagnostics corp., Indianapolis, IN) solution was added, and the mixture was subjected to shake digestion at 37 ℃ for 1 hour. Single cell suspensions were obtained through a 70-um filter (BD Biosciences, San Jose, Calif.). Next, these cells were inoculated into a low-limit Eagle medium (MEM) (Invitrogen, Carlsbad, Calif.), plus 15% fetal bovine serum, 2mmol/l glutamine, 100U/ml penicillin, and 100U/ml penicillinmg/ml streptomycin in 5% CO₂And the medium was changed every 3 days in a 37 ℃ humidified incubator.

All stem cells were identified as described previously for stem cell surface markers, and subsequent experiments used passage 3-5 stem cells. For in vitro neural differentiation induction, neural stem cell dominant culture medium (Neurobasal A solution added with 2% B27, 40ng/ml bFGF, 20ng/ml EGF, 2mM L-glutamine, 100U/ml penicillin and 100ug/ml streptomycin) is used for induction culture of stem cells, and the stem cells are replaced once every 3 days;

for the formation observation of the neurospheres, the neurospheres are observed under an inverted microscope when the neurospheres are induced for 9 days; for an immunofluorescence staining experiment, collecting neurospheres which induce 9 days, fixing with 4% paraformaldehyde, carrying out membrane permeation treatment on Triton, then carrying out basic albumin liquid sealing treatment, incubating the specific primary antibody liquid at 4 ℃ overnight, and sequentially incubating a fluorescence calibration species secondary antibody, a cytoskeletal dye PDI and a nuclear dye DAPI and then visualizing by means of fluorescence excitation; the primary specific antibody is anti-beta III-TUBULIN, NESTIN polyclonal antibody (Abcam, Cambridge, USA).

Example 2 plasmid construction and viral infection

Plasmid construction and virus transfection, the construction of the plasmid is carried out according to a standard method, a target complementary shRNA sequence designed by a target KDM2B gene is cloned into a virus vector pLKO.1 plasmid loop, sequencing identification is carried out, and the construction of a KDM2B shRNA plasmid is completed; designing a PCR primer of KDM2B gene full length, amplifying to obtain a full length gene sequence of KDM2B, adding an HA-Tag label, connecting the HA-Tag label to a retrovirus expression vector pQCXIN, sequencing and identifying to construct a plasmid containing the full length KDM2B gene sequence of the HA-Tag label; then, carrying out virus packaging, collecting, identifying virus titer, subpackaging and storing in a refrigerator at the temperature of-80 ℃; viral transfection, 10^-7The titer of retrovirus or lentivirus is cultured with the deciduous head stem cells of the apical teeth overnight under the participation of the coacervate amine, and the transfected cells are screened by antibiotics after 48 hours; the target complementary shRNA sequence designed by targeting KDM2B gene is KDM2 Bsh: 5'-ATTTGACGGGTGGATAATCTG-3' are provided.

Example 3 Total RNA isolation, Reverse Transcription (RT) PCR and real-time fluorescent quantitative reverse transcription PCR

Three groups of total RNA samples derived from the root tip papilla stem cells of different individuals were extracted and purified by using an extraction reagent and an RNA extraction kit (QIAGEN, GmBH, Germany), and then dissolved in RNase-Free Water (QIAGEN). Total RNA was quantified by spectrophotometer ND-2100 (seemer femtoler) and RNA integrity was assessed using agilent 2100 (agilent technologies). For mRNA detection, equal amounts of cDNA samples were synthesized by reverse transcription using the random primer kit (QIAGEN) according to the manufacturer's standard (Invitrogen). Real-time PCR reactions were performed using fluorescent PCR (qiagen) and icycleiq multicolor real-time fluorescent quantitative PCR technology detection systems. Primer design used in-line primer 3 program (primer sequences detailed in Table 18), GAPDH as internal control, relative mRNA levels by using 2^-ΔΔCtAnd (4) calculating by using the method.

Example 4 Co-immunoprecipitation and Western blot analysis

Three total protein samples from root tip papilla stem cells from different individuals were extracted using RIPA lysate lysis. For the co-immunoprecipitation, the total protein sample of equal mass was incubated with the specific primary immunoglobulin A/G beads overnight at 4 deg.C, washed with Triethanolamine Buffered Saline (TBS), denatured by boiling at 99 deg.C, and subjected to Western blot analysis or at-80 deg.C for future use;

for western blot analysis, equal mass total protein samples were separated with 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) using a semi-dry transfer membrane device, coated with 5% skim milk on the membrane and left for 2h, then incubated overnight with primary antibody; incubating the immunocomplex with a rabbit or mouse immunoglobulin G antibody and visualizing it with a chemiluminescent substrate reagent; the primary antibody directed primarily specifically against KDM2B, EZH2 polyclonal antibody (Abcam).

Example 5 rat spinal cord nerve injury model and root apical tooth papilla Stem cell transplantation

The invention is allowed by animal care and use committee of Beijing oral hospital affiliated to the university of capital medical science; will be about 1X 10⁶The root apical papilla stem cells were transplanted to the T10 spinal cord tissue total cutting sites of a 10-week-old rat spine. In particular, by micro-injectionThe injection system (30 ul in total) was inserted into the middle, left and right of the full-dissection site towards the midline, and 10ul was injected into each site. Mainly aiming at the fact that root apical papilla stem cells of a control Vector group and a KDM2B overexpression group are respectively replanted into 5 rats, and the procedures are carried out according to animal protocol approval specifications; BBB hindlimb locomotor ability scoring was performed on rats at 0, 1, 2 and 3 weeks post-transplantation, respectively;

for histopathological analysis, spinal cord tissue from the transplant was harvested three weeks later and fixed with 10% formalin, paraffin embedded, 5um sections, and stained with hematoxylin-eosin (HE); immunohistochemical staining, dewaxing and dehydrating a 5-micrometer section, removing endogenous peroxidase, sealing, incubating a specific primary antibody solution at 4 ℃ overnight, sequentially incubating biotin-horseradish peroxidase, and visualizing the section by means of 3, 3-Diaminobenzidine (DAB) substrate color development; the primary specific antibody is anti-beta III-TUBULIN, NEF-M polyclonal antibody (Abcam).

Example 6 polypeptide microarray chip design and analysis

The full-length sequence of the human KDM2B protein is obtained by the inquiry of a Uniprot protein information website (https:// www.uniprot.org /), and the full-length amino acid sequence of the KDM2B is designed and synthesized into a polypeptide chip by Overlapping; synthesizing a polypeptide array by using a full-automatic polypeptide chip synthesizer, carrying out incubation hybridization with biotin-labeled target protein after closing, visualizing a chemical chromogenic substrate, and imaging by using a Chempchemi digital imager; the chromogenic chip picture uses TotalLab image analysis software to analyze the optical density value of the chromogenic point, particularly, the positive chromogenic polypeptide locus is calculated by taking the background value around the chromogenic point as a reference, the highest value of the optical density value of the chromogenic point on the film is set as 100%, the optical density values of the rest points are percentage numerical values of the optical density value of the point, and the point on the polypeptide chip film with the optical density value exceeding 30% and the point on the negative reaction film with the optical density value lower than 30% is selected as the positive chromogenic point.

Further, a schematic of the polypeptide spots on the KDM2B polypeptide microarray membrane and coomassie staining pattern as shown in fig. 9:

as shown in the left diagram of FIG. 9, the polypeptides in the array have 18 rows and 15 columns, the numbering increases from left to right and from top to bottom, the array consists of 266 polypeptide spots, namely 266 peptides, and 1 copy of the polypeptide microarray chip is synthesized according to the schematic diagram of the chip, and each polypeptide sequence is detailed in Table 12. The right image of fig. 9 is a coomassie stained image after chip completion.

Further, a KDM2B polypeptide microarray membrane is subjected to hybridization reaction with EZH2 protein;

hybridization experiments polypeptide chip hybridization reactions were performed using NHS-PEO4-Biotinylation labeled EZH2 protein solution (1ug/ml), amplification reactions were performed using Streptavidin-HRP, and the reaction steps and conditions were as described above, followed by 5min of color development (results are shown in FIG. 6A), which shows that the labeled protein was clearly bound to and developed at some of the polypeptide spots on the chip.

Further, KDM2B polypeptide microarray film positive color reaction point analysis;

and (4) analyzing the optical density data of the color development points by using a TotalLab software picture (the original data of the optical density values of all the points are shown in an accessory Excel table), setting the highest value of the optical density values of the color development points on the film as 100 percent, and setting the optical density values of the rest points as percentage values of the optical density values of the points, and detecting the obtained gray value table of the positive color development sites (the result is shown in figure 6B). In this table, the abscissa is the polypeptide spot number, i.e., 266 polypeptides corresponding to the array, and the ordinate is the percentage of the optical density value of the spot. Empirically, positive chromogenic spots were selected with spot optical density values on the polypeptide chip membrane of more than 30% and spot optical density values on the negative reaction membrane of less than 30%, and the following sequences were clearly different (table 16):

TABLE 16 KDM2B polypeptide microarray chip and EZH2 protein immune hybridization coloration positive site polypeptide section

Chip peptide sequence number	Sequence numbering	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
			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

EXAMPLE 7 Synthesis and purification of biologically active Polypeptides

Obtaining a bioactive polypeptide sequence, calibrating a cell-penetrating peptide and a FITC green fluorescent group, sequentially synthesizing corresponding amino acids according to the sequence after resin-dichloromethane liquid swelling, detecting ninhydrin, then capping with pyridine and acetic anhydride, cleaning, separating out a crude product with diethyl ether, purifying by liquid chromatography after centrifugation, and freeze-drying by a freeze dryer to obtain bioactive polypeptide powder;

further, to test the protein binding site accuracy of KDM2B with EZH2, we synthesized 6 bioactive polypeptides based on the positive binding reporter sites in table 16 (table 17), while randomly selecting synthetic negative binding reporter sites peptide 83-84 as control polypeptides (ConPP group).

Table 17 amino acid sequences of biologically active polypeptides synthesized based on KDM2B

Grouping of synthetic polypeptides	Synthetic polypeptide numbering	Sequence numbering	Amino acid sequence
				ConPP group	peptide 83-84	SEQ ID No.267	EEEACDQQPQEEEEKDEEGE
PP1 group	peptide 46-47	SEQ ID No.268	VKKYCLMSVKGCFTDFHIDF
				PP2 group	peptide 122-123	SEQ ID No.269	ARRRRTRCRKCEACLRTECG
PP3 group	peptide 131-132	SEQ ID No.270	CIAPVLPHTAVCLVCGEAGK
				PP4 group	peptide 139-142	SEQ ID No.271	CNEIIHPGCLKIKESEGVVNDELPNCWECP
PP5 group	peptide 151-153	SEQ ID No.272	QKMNRDNKEGQEPAKRRSECEEAPR
				PP6 group	peptide 231	SEQ ID No.273	LRDLVLSGCSWIAVSALCSSSCPLL

Further, the results of co-immunoprecipitation analysis showed that the addition of 10ug/ml peptide 46-47, peptide 122-123, and peptide 131-132 to stimulate the root tip papilla stem cells for 24 hours could significantly block the binding of KDM2B to EZH2 (FIG. 6D).

EXAMPLE 8 use of bioactive Polypeptides for neuro-differentiation of root-tip dental papilla Stem cells

Furthermore, when the root apical papilla stem cells are induced and cultured for 9 days by adding 10ug/ml peptide into the neural stem cell dominant culture medium, the results show that the 10ug/ml peptide 46-47, peptide 122-123 and peptide 131-132 obviously improve the formation of beta III-TUBULIN positive (figure 7B) and NESTIN positive (figure 7C) neurospheres in vitro by the root apical papilla stem cells.

EXAMPLE 9 use of biologically active Polypeptides for regeneration repair of damaged neural tissue

Further, after pre-treating the root apex papilla stem cells with 10ug/ml peptide 46-47 for 24 hours, we immediately transplanted them to 10-week-old rats T10 spinal cord tissue total cutting sites (5 independent rats per group). 4 weeks after stem cell transplantation, spinal cord tissue was viewed overall with significant healing of injured nerve tissue from 10ug/ml peptide 46-47 pretreatment compared to control ConPP pretreatment (FIG. 8A); BBB ethological scoring results showed that hind limb locomotor ability of rats in the 10ug/ml peptide 46-47 pretreatment group was significantly improved (FIG. 8B).

Further, we injected 30ul of 10ug/ml peptide 46-47 alone to 10-week-old rats T10 spinal cord tissue total cutting site (5 independent rats per group) at a frequency of once weekly injection by means of microinjection system. After 4 weeks, spinal cord tissue was viewed overall with significant healing of injured nerve tissue from groups injected with 10ug/ml peptide 46-47 alone compared to control ConPP alone (fig. 8C); BBB ethological scoring results showed that the hindlimb motor ability of rats injected with peptide 46-47 at 10ug/ml alone was significantly improved (FIG. 8D).

Further, we analyzed BBB behavioral scores for rats in the control ConPP-pretreated stem cell replanting group, the 10ug/ml peptide 46-47-pretreated stem cell replanting group, and the 10ug/ml peptide 46-47-injected group alone in combination, and the results showed that hindlimb locomotor ability was significantly improved in both the 10ug/ml peptide 46-47-pretreated stem cell replanting group and the 10ug/ml peptide 46-47-injected group at week 4 of intervention, with no significant difference between the two groups (fig. 8E).

TABLE 18 primers for real-time quantitative PCR assay

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

Application of KDM2B overexpression in preparation of a preparation or a medicament for inducing differentiation of deciduous papilla stem cells of apical teeth into nerves in vitro.
2. Use according to claim 1, wherein the overexpression of KDM2B is used in the preparation of a formulation or medicament for promoting the formation of β iii-TUBULIN positive and NESTIN positive neurospheres in vitro on deciduous stem cells of the apical teeth.
The application of the over-expression of KDM2B in preparing a preparation or a medicament for promoting regeneration and repair of spinal nerve injured tissues mediated by root apical papilla stem cells in vivo.
4. A polypeptide, characterized in that it has:

(I) and an amino acid sequence as shown in any of SEQ ID Nos. 1-266; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described in (I) and has the same function with the amino acid sequence described in (I); or

(III) an amino acid sequence having 90% or more identity to the amino acid sequence of (I) or (II);

and 2 of the substitutions, deletions or additions of one or more amino acids.
5. A nucleic acid molecule encoding the polypeptide of claim 4.
6. An expression vector comprising the nucleic acid molecule of claim 5.
7. Use of the polypeptide of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or a pharmaceutically acceptable salt thereof, directly or indirectly in the preparation of a formulation or medicament for inducing the in vitro neural differentiation of deciduous papilla stem cells of apical teeth.
8. The use of the polypeptide of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or a pharmaceutically acceptable salt thereof, directly or indirectly in the preparation of a formulation or medicament for promoting regeneration and repair of spinal nerve injured tissue mediated by deciduous cells at the root tip in vivo.
9. The use as claimed in claim 7 or claim 8 wherein the binding site for KDM2B to EZH2 comprises:

the positive binding site is selected from the group consisting of 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, and peptide 233;

the amino acid sequence of the positive binding site is shown 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;

the negative binding site is selected from peptide 83 and peptide 84;

the amino acid sequence of the negative binding site is shown as SEQ ID No. 267.
10. A medicament or formulation comprising a polypeptide according to claim 4 and a pharmaceutically acceptable excipient.