CN114522233A

CN114522233A - Polypeptide sequence of KDM6B and regulation and control application to mesenchymal stem cell function

Info

Publication number: CN114522233A
Application number: CN202111440575.1A
Authority: CN
Inventors: 范志朋; 张琛; 姜钰; 龙璐珏; 曹杨杨
Original assignee: Beijing Stomatological Hospital
Current assignee: Beijing Stomatological Hospital
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-05-24
Anticipated expiration: 2041-11-30
Also published as: CN114522233B

Abstract

The invention relates to the field of biomedicine, in particular to regulation and control application of KDM6B to mesenchymal stem cell functions. WDR5 is a co-binding protein that negatively regulates histone demethylases KDM6B and MLL1 function. WDR5 can form a protein complex with histone demethylase KDM6B to inhibit the function of KDM6B, and further regulate the expression and function of genes by regulating downstream senescence and osteogenesis related genes and the methylation state of histone in a gene promoter region, so as to finally achieve the effects of regulating the senescence and differentiation functions of stem cells and the repair and regeneration functions of bone/dental tissues. Aiming at the sequence of a KDM6B and WDR5 binding region, small molecular polypeptides are developed and utilized, and the function of mesenchymal stem cells is regulated by regulating and controlling the binding of a KDM6B/WDR5 complex. Finally, the function of the mesenchymal stem cells is restored under the conditions of aging and osteoporosis, thereby promoting the repair and regeneration of bone/tooth tissues.

Description

Polypeptide sequence of KDM6B and regulation and control application to mesenchymal stem cell function

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide sequence of KDM6B and application thereof in regulation and control of mesenchymal stem cell functions.

Background

With the extension of human life and the coming of aging society, the aging of population becomes a more and more serious social problem facing all countries today. Aging, a physiological phenomenon in life, causes neurodegenerative diseases, osteoporosis, tooth loss, cardiovascular diseases, tumors, metabolic diseases, and the like, which have become important threats to human health. The skeleton of human body is gradually degenerated, and the prevalence rate of osteoporosis is obviously increased. Osteoporosis affects not only the vertebrae, hip, phalanges, but also the jaw bone. The more severe the osteoporosis of the skeletal system of the whole body, the more evident is the loss of bone and mineralized tissue of the jaw bone. Unbalance is rebuilt to the jaw, remaining alveolar bone absorbs, jaw atrophy, can all accelerate the not hard up of tooth and drop, seriously influences jaw and keeps important functions such as face appearance, supplementary pronunciation and chew. Therefore, prevention and treatment of age-related osteoporosis and tooth loss in the elderly population will present a significant challenge. At present, the defects of the repair and treatment of senile diseases, osteoporosis and tooth loss exist. Therefore, the research on the regulation and control mechanism of the differentiation and regeneration functions of the mesenchymal stem cells under the aging condition has important significance for the repair and treatment of bone tissues and tooth tissues of the elderly patients and the improvement of the life quality of the elderly patients.

The current treatment method for osteoporosis is mainly to prevent fracture, and the most commonly used medicine in clinic is diphosphate. Although bisphosphonates reduce the incidence of fractures by 40% to 70%, there are also a number of side effects, including causing acute renal failure, esophageal cancer, and musculoskeletal pain. In addition, the long-term use of bisphosphonates increases the risk of fractures in patients, especially typical femoral fractures and osteonecrosis of the jaw bone. Other drugs utilize anabolic drugs to stimulate bone formation and reduce the risk of patient fractures. Parathyroid hormone (PTH) is the only drug approved by the united states food and drug administration to stimulate bone formation, but it is associated with the onset of osteosarcoma and can only be used for 2 years. Postmenopausal women are considered a risk group for accelerated bone loss [29], and hormone replacement therapy (estrogen and progesterone therapy, estrogen therapy alone, or selective estrogen receptor modulators) remains the first line of choice for clinical treatment. However, hormone therapy increases the risk of breast cancer in patients. The intake of calcium and vitamin D is only suitable for the prevention of osteoporosis and it is not completely effective in avoiding the development of osteoporosis. Although a lot of osteoporosis treatment medicines exist at the present stage, the side effects are obvious, the bone recovery capability is limited, and different patients have differences, and the clinical effect has a certain space for improving by only applying the medicine treatment. Therefore, understanding the etiology and molecular mechanisms of osteoporosis helps to find more effective treatments that prevent the deterioration of bone tissue microstructure and maintain bone homeostasis.

The tooth tissue defect and the tooth loss are common diseases and frequently-occurring diseases of the old people, the chewing, speaking, beauty and psychological health of patients are seriously influenced, the existing tooth loss repairing method belongs to non-biological prosthesis repairing, the price is high, the adjacent healthy teeth are often damaged, and the gap exists between the adjacent healthy teeth and the natural teeth. Therefore, dental tissue regeneration has become a hotspot of international oral medical research. Mesenchymal stem cells have the capacity of tissue damage repair and multidirectional differentiation, and can be differentiated into all kinds of cells of mesoderm, so that a stem cell-mediated tissue engineering technology becomes an important means for repairing various tissue damages. However, like other somatic cells, the functions of repair, renewal and differentiation of damage are diminished with the aging of cells, and even dysfunction occurs, thereby affecting the therapeutic effect of stem cells. The function of the mesenchymal stem cells of the aged patient is recovered from the aging state, the repairing and regenerating potential of the aging tissue is enhanced, and the immunological rejection reaction of the allogeneic mesenchymal stem cells is avoided. Therefore, the elucidation of the directional differentiation mechanism of the aged mesenchymal stem cells is the key of tissue repair and regeneration, and has important significance for the repair and treatment of bone tissues and tooth tissues of the old.

Disclosure of Invention

In view of this, the present inventors investigated the relationship of KDM6B, WDR5 and MLL1, and their roles and mechanisms in the regulation of senescence and osteogenic/dental differentiation in mesenchymal stem cells.

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

the invention provides application of a protein complex as a target in preparation of a reagent or a medicament for inhibiting mesenchymal stem cell aging and promoting mesenchymal stem cell osteogenic differentiation or odontoblastic differentiation;

the protein complex comprises a first protein complex and a second protein complex;

the first protein complex comprises WDR5 and KDM 6B;

the second protein complex comprises KDM6B, WDR5 and MLL 1.

Most importantly, the present invention also provides a biologically active peptide having:

(I) the amino acid sequence shown as SEQ ID No.1 or 2; 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).

The invention also provides nucleic acids encoding the above bioactive peptides.

In addition, the invention also provides a biological material capable of expressing the bioactive peptide, and the biological material comprises one or more of an expression vector, a plasmid, an expression cassette, a recombinant bacterium or a host cell.

Based on the research, the invention also provides application of the bioactive peptide in preparing a reagent or a medicament for inhibiting the aging of mesenchymal stem cells, promoting osteogenic differentiation of bone marrow mesenchymal stem cells and/or promoting odontogenic differentiation of deciduous head stem cells at root tips.

The invention also provides application of the bioactive polypeptide in preparation of a reagent or a medicament for preventing and treating osteoporosis and periodontitis and/or repairing mucous membrane and skin defects.

In addition, the invention also provides a reagent or a medicament, which comprises the bioactive peptide and pharmaceutically acceptable auxiliary materials.

The research starts from clinical transformation application, and on the basis of the early-stage research, the research explains how to regulate the action of KDM6B/WDR5 under specific clinical conditions to reverse stem cell senescence, promote stem cell bone/tooth differentiation and bone/tooth tissue repair and regeneration. The research is also helpful to clarify the molecular mechanism of function regulation of the MSCs in the aging and osteoporosis microenvironment, and provides target genes and theoretical basis for function reconstruction of the MSCs and tissue regeneration promotion. The research develops a new small molecular preparation which is used as a treatment medicine for aging and aging-related diseases to promote bone/tooth tissue regeneration and provide a basis for clinical transformation and application of the preparation. Through the research on the regulation and control functions and the molecular mechanism of KDM6B, WDR5 and MLL1 signal molecules on the aging and differentiation functions of mesenchymal stem cells, the following results are found:

WDR5 is a co-binding protein that negatively regulates the function of histone demethylases KDM6B and MLL 1. WDR5 can form a protein complex with histone demethylase KDM6B, and inhibit the function of KDM6B to finally achieve the functions of regulating the aging and differentiation functions of stem cells and the repairing and regenerating functions of bone/dental tissues. Based on mechanism research, small molecule polypeptides are developed and utilized aiming at the sequence of a KDM6B and WDR5 binding region, and the function of mesenchymal stem cells is regulated by regulating the binding of a KDM6B/WDR5 complex. Finally, the function of the mesenchymal stem cells is restored under the conditions of aging and osteoporosis, thereby promoting the repair and regeneration of bone/tooth tissues.

Including but not limited to:

(I) effects on bone marrow mesenchymal stem cells:

1. WDR5 and KDM6B form a protein complex in bone marrow mesenchymal stem cells.

2. Formation of complex of WDR5 and KDM6B proteins is increased in senescent bone marrow mesenchymal stem cells.

3. The

bioactive polypeptides

102 and 114 inhibit the aging of the mesenchymal stem cells and promote the osteogenic differentiation function of the mesenchymal stem cells.

4. The

bioactive polypeptides

102 and 114 inhibit the aging of the bone marrow mesenchymal stem cells of the aged mice and promote the osteogenic differentiation function of the bone marrow mesenchymal stem cells of the aged mice.

(II) effect on papilla deciduous stem cells of root tips:

1. KDM6B, WDR5 and MLL1 in the root apical papilla stem cells form a protein complex.

2. The KDM6B/WDR5 protein binding site is mutated, so that KDM6B is enhanced to inhibit the senescence of the deciduous head stem cells of the apical teeth, and the odontogenic differentiation function of the deciduous head stem cells of the apical teeth is promoted.

3. The

bioactive polypeptides

102 and 114 inhibit the aging of the deciduous cells of the apical teeth and promote the odontoblast differentiation function of the deciduous cells of the apical teeth.

And (III) application in the prevention and treatment of osteoporosis.

The bioactive polypeptide 114 has the function of enhancing the bone density of an osteoporosis mouse, and can effectively prevent the bone loss of the osteoporosis mouse.

And (IV) application in periodontitis treatment.

1. Bioactive polypeptide 114 has the function of reducing periodontal pocket depth, attachment loss, and gingival recession in a small pig periodontitis model.

2. The bioactive polypeptide 114 has the function of promoting the formation of alveolar bone new bone in a small pig periodontitis model.

3. Bioactive polypeptide 114 has the effect of promoting gingival soft tissue repair in a small pig periodontitis model.

Drawings

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

FIG. 1 shows that WDR5 forms protein complexes with KDM6B, MLL1 in mesenchymal stem cells; after WDR5 is knocked out in mesenchymal stem cells, Co-IP results show that the protein complexes of WDR5, KDM6B and MLL1 in a WDR5 knock-out group are reduced in formation, and beta-actin is used as an internal reference;

FIG. 2 shows that WDR5 binding to KDM6B, MLL1 was increased in mesenchymal stem cells under senescent conditions; Co-IP experiments are carried out in mesenchymal stem cells of 2-month-old C57BL/6 mice and 18-month-old C57BL/6 mice to detect the combination of KDM6B, WDR5 and MLL 1. Co-IP results show that compared with young mice, WDR5, KDM6B and MLL1 protein complexes in bone marrow mesenchymal stem cells of aged mice are increased, and beta-actin is used as an internal reference;

FIG. 3 shows the discovery of the KDM6B-WDR5 protein binding site using polypeptide microarray technology; wherein, A: a polypeptide microarray chip made of KDM6B is incubated with WDR5, and 7 positive polypeptide binding sites are found; b: the optical density value of the spot on the positive membrane of the array corresponding to KDM 6B; c: Co-IP result detection shows that only 102 and 114 of three bioactive polypeptides, namely peptide102, peptide114 and peptide152, can effectively open the combination of KDM6B/WDR5 protein complexes;

FIG. 4 shows that

bioactive polypeptides

102, 114 inhibit senescence and promote osteogenic differentiation of mesenchymal stem cells; wherein, A: the results of telomerase reverse transcriptase ELISA experiments show that peptide114 promotes the expression of telomerase reverse transcriptase in mesenchymal stem cells of bone marrow compared with a control group; B-C: the results of beta-gal staining and quantitative analysis show that peptide102 and peptide114 lead to an obvious reduction in the number of beta-gal positive cells of mesenchymal stem cells in bone marrow compared with a control group; d: peptide102 and peptide114 promoted ALP activity of mesenchymal stem cells in bone marrow as compared with the control group; E-F: alizarin red staining and calcium ion quantitative analysis results show that compared with a control group, the mineralization capacity of mesenchymal stem cells in peptide102 and peptide114 groups is obviously improved; p is less than or equal to 0.05 and P is less than or equal to 0.01;

FIG. 5 shows that

bioactive polypeptides

102, 114 inhibit senescence in senescent mesenchymal stem cells and promote osteogenic differentiation in senescent mesenchymal stem cells; wherein, A: the results of beta-gal staining and quantitative analysis show that peptide102 and peptide114 cause a significant decrease in the number of beta-gal positive cells of senescent bone marrow mesenchymal stem cells compared to the control group; b: peptide102 and peptide114 promoted ALP activity of senescent bone marrow mesenchymal stem cells compared to the control group; C-D: alizarin red staining and calcium ion quantitative analysis results show that compared with a control group, the mineralization capacity of the aged mesenchymal stem cells of peptide102 and peptide114 groups is obviously improved; p is less than or equal to 0.05 and P is less than or equal to 0.01;

FIG. 6 shows protein complexes of KDM6B with WDR5 in apical papilla stem cells; wherein, A: after KDM6B is knocked out from deciduous stem cells of the apical teeth, Co-IP results show that KDM6B and WDR5 protein complexes in a KDM6B knock-out group form less, and histone H3 serves as an internal reference; b: after WDR5 is knocked out from deciduous cells of the apical teeth, Co-IP results show that protein complexes of WDR5 and KDM6B in a WDR5 knock-out group are reduced in formation, and beta-actin is used as an internal reference;

FIG. 7 shows that the mutant KDM6B sequences 102 and 114 inhibit the senescence of the deciduous cells of the apical teeth and promote the deciduous cells of the apical teeth to differentiate into teeth; wherein, A: Co-IP results showed that KDM6B and WDR5 binding was reduced in the root apical papilla stem cells of HA-KDM6B-mut102 group and HA-KDM6B-mut114 group compared to the control group; b: the results of telomerase reverse transcriptase ELISA experiments show that compared with a control group, HA-KDM6B-mut114 promotes the expression of telomerase reverse transcriptase in the papilla of the apical teeth; C-D: the results of beta-gal staining and quantitative analysis show that compared with the control group, the HA-KDM6B-mut102 group and the HA-KDM6B-mut114 group cause the number of beta-gal positive cells of the papilla stem cells of the apical teeth to be obviously reduced; e: compared with the control group, the HA-KDM6B-mut102 group and the HA-KDM6B-mut114 group promoted ALP activity of the root apical papilla stem cells; F-G: alizarin red staining and calcium ion quantitative analysis results show that compared with a control group, the mineralization capacity of the papilla of the root tips of the HA-KDM6B-mut102 group and the HA-KDM6B-mut114 group is obviously improved; p is less than or equal to 0.05 and P is less than or equal to 0.01;

FIG. 8 shows that

bioactive polypeptides

102, 114 inhibit senescence of root apical papilla stem cells, promoting odontogenic differentiation of root apical papilla stem cells; wherein, A: Co-IP results showed that KDM6B and WDR5 binding was reduced in root tip papilla stem cells of peptide102 and peptide114 groups compared to the control group; b: the results of telomerase reverse transcriptase ELISA experiments show that peptide114 promotes expression of telomerase reverse transcriptase in root apical papilla stem cells compared with a control group; C-D: the results of beta-gal staining and quantitative analysis show that peptide102 and peptide114 lead to a significant reduction in the number of beta-gal positive cells in mammary stem cells from apical teeth compared to the control group; e: peptide102 and peptide114 promoted ALP activity of deciduous stem cells at the root tip compared to the control group; F-G: alizarin red staining and calcium ion quantitative analysis results show that compared with a control group, the mineralization capacity of root apex dental papilla stem cells of peptide102 and peptide114 groups is obviously improved; p is less than or equal to 0.05 and P is less than or equal to 0.01;

FIG. 9 shows that after 12 weeks of bioactive polypeptide injection, CBCT three-dimensional image was reconstructed and new bone formation volume was measured, showing that the new bone formation in periodontitis defect region of 114peptide group was higher than that of Control peptide group and PBS group; the difference between the formation amount of new bones of each group has significant meaning; p is less than or equal to 0.05;

FIG. 10 shows that after 12 weeks of bioactive polypeptide injection, the periodontal probing depth of the 114peptide group was significantly lower than that of the PBS group and the Control peptide group, as measured by periodontal probe; 114peptide has significant significance with the difference between the rest groups; p is less than or equal to 0.05;

FIG. 11 shows that the loss of attachment of the 114peptide group was significantly lower than those of the PBS group and the Control peptide group, as measured by periodontal probe, at 12 weeks after injection of the biologically active polypeptide; the difference between the 114peptide group and the rest groups has significant significance; p is less than or equal to 0.05;

FIG. 12 shows that after 12 weeks of bioactive polypeptide injection, the 114peptide group showed significantly lower gingival recession than the PBS group and the Control peptide group as measured by periodontal probe; the difference between the 114peptide group and the rest groups has significance; p is less than or equal to 0.05;

FIG. 13 shows the examined miniature pig lying on the CBCT scanner bed from a natural bite

And fixing the head, continuously scanning to obtain CBCT sectional images and performing three-dimensional reconstruction on the DICOM images by using Mimics17.0 image processing software. CBCT data are acquired before, after and after modeling respectively for post-treatment assessment; the postoperative three-dimensional reconstruction model shows that new bones are formed in the PBS group/Control peptide group/114 peptide; the forming effect of the new bone of the 114peptide group is better than that of the Control peptide group and the PBS group;

FIG. 14 shows that after 12 weeks of bioactive polypeptide injection, the wounds of PBS group/Control peptide group/114 peptide group in the bone defect area of the periodontal tissue of the minipig were healed, and no infection or necrotic tissue was observed; the soft tissue repair effect of the 114peptide group is better than that of the Control peptide group and the PBS blank Control group;

FIG. 15 shows that biologically active polypeptide 114 promotes healing of a defect in the mucosa of the palate in a mouse; injecting pbs, control peptide and 114peptide with equal amount around the palatine mucosa defect of the mouse, and observing the mucosa healing change condition of the mouse; a, b and c represent a mouse palate defect healing model; d, e and f are pbs group, control peptide group and 114peptide group at 14 days respectively; g, h and i are respectively pbs group, control peptide group and 114peptide group at 21 days, and the palatal mucosa is defective; the results show that the 114peptide group had significant healing of palatal mucosal defects compared to the pbs and control peptide groups;

FIG. 16 shows the areas of non-healing of palatal mucosal defects in mice on days 14 and 21 in the pbs, control peptide and 114peptide groups, the 114peptide group being statistically significant compared to the pbs and control peptide groups; p is less than or equal to 0.05;

FIG. 17 shows that 114peptide promotes healing of skin defects in the back of mice; injecting pbs, control peptide and 114peptide with equal amount around the whole skin defect with the diameter of 6mm on the back of the mouse, wherein a and b are skin defects of the mice of 0 day and 14 days of the pbs group respectively; c, d is the skin defect of the mice of the control peptide group at 0 day and 14 days respectively; e, f are skin defects of mice of the 114peptide group at 0 days and 14 days respectively; compared with the pbs injection group and the control peptide injection group, the 114peptide group has obvious healing of skin defects;

FIG. 18 shows that the 114peptide group was found to be statistically significant compared to the pbs group, the control peptide group by calculating the healing rate of skin defects on the back of mice at days 10 and 14 in the pbs group, the control peptide group and the 114peptide group; p is less than or equal to 0.05, P is less than or equal to 0.01, P is less than or equal to 0.001;

FIG. 19 shows that biologically active polypeptide 114 prevents femur bone loss in osteoporotic mice; a: carrying out Micro-CT on the dirty end of the femoral shaft of the mouse, and observing the change condition of the bone trabecula of the mouse; b, through micro-CT analysis of the femoral shaft dirty end of the mouse, the mice injected with PBS and Control peptide groups are found to have no obvious difference between the bone density of the femoral bone trabecula and the bone density of OVX groups; when the OVX group, the PBS group, the Control peptide group and the Sham group are compared, the trabecular bone density is obviously reduced; bone trabecular bone density of mice in the 114peptide group was significantly increased compared with those in the OVX, PBS and Control peptide groups.

Detailed Description

The invention discloses a polypeptide sequence of KDM6B and the application thereof in the regulation and control of mesenchymal stem cell function, and those skilled in the art can appropriately improve process parameters for reference of the content. 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 methods and applications of this 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 methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

KDM6B, a demethylase of H3K27me2/3, may play an important role in the development of bone and tooth tissues by influencing mesenchymal stem cell-specific lineage differentiation. KDM6B also participates in the process of osteoclast, researches show that after bone injury, osteoclast differentiation is increased to cause osteoporosis, and in the process, KDM6B reduces the methylation level of H3K27me3 on Nfatc1 gene, activates Nfatc1 gene expression to maintain bone mass and limits the occurrence of osteoporosis. In addition, down-regulation of histone demethylase KDM6B expression can lead to apoptosis and increased senescence in adult stem cells, which leads to loss of self-renewal capacity. We therefore hypothesized that KDM6B plays an important functional role in mesenchymal stem cell senescence and dysfunction. In this study, we investigated the effect of KDM6B on mesenchymal stem cell senescence. Experimental results show that KDM6B can inhibit the beta-gal and p16 of bone marrow mesenchymal stem cells and root apical tooth papilla stem cells^INK4AAnd up-regulates telomerase reverse transcriptase activity. Senescence-associated beta-galactosidase (SA-beta-gal) activity is one of the earliest biomarkers for identifying senescent cells in cultured cells and fresh tissue samplesIt is effectively demonstrated that senescent cells will gradually accumulate in the foci of senescence-associated diseases and senescent tissues in a variety of mammals. Another significant feature of senescent cells is the increased expression of cytostatic proteins, which results in the maintenance of the arrest state of senescent cells, and in turn in the continued accumulation of senescent cells, where p16^INK4AIs the most prominent cyclin. Currently, quercetin and fisetin have been demonstrated to stimulate tissues and cells against aging in various situations in vitro and in vivo. The most obvious result is that both can effectively reduce p16 and SA-beta-gal expression. In addition, inhibition of telomere shortening as a means of preventing and reducing cellular senescence in the context of senescence and telomere dysfunction is considered to be a reliable treatment. Systemic delivery of Tert reduces some of the senescence markers and senescence-associated conditions and extends the lifespan of wild-type mice, thus demonstrating that maintenance of telomere function plays a role in natural senescence.

The combination of literature and previous experimental data suggests that KDM6B has the functions of promoting mesenchymal stem cell osteogenesis/odontoblastic directional differentiation and inhibiting mesenchymal stem cell aging. Therefore, the KDM6B becomes a candidate target for preventing and treating senile diseases due to the effects of the KDM6B in aging and bone/tooth tissue regeneration, but how to effectively regulate the function of the KDM6B is not clear, and the regulation mechanism of the KDM6B needs to be deeply researched. The literature refers to the discovery that WDR5 can form protein complexes with KDM6B in HEK293 cells. To verify this result, we performed Co-IP experiments in both bone marrow mesenchymal stem cells and root apical dental papilla stem cells, and found that KDM6B and WDR5 could form protein complexes in both cells. In the aging animal model, the C57 mice of 18-24 months of age are highly matched with the C57 mice of 56-69 years of age of human, so that the aging state of the mesenchymal stem cells can be simulated more accurately. We therefore performed Co-IP experiments on mesenchymal stem cells from 2-and 18-month old mice. The results showed that KDM6B/WDR5 protein complex formation was increased in bone marrow mesenchymal stem cells of aged mice compared to young mice. WDR5 is involved in regulating numerous cell physiological activities such as epithelial mesenchymal transition, leukemia development, differentiation of chondrocytes and osteocytes, maintenance of the pluripotency of embryonic stem cells, and the like. Furthermore, methylation of WDR5 and histone plays an important role in growth and development of vertebrates. However, there is currently a lack of research on the effects of WDR5 on mesenchymal stem cell osteogenic/odontoblastic differentiation and aging. Based on the above results, we speculate that WDR5 can negatively regulate the effect of KDM6B on mesenchymal stem cells. In addition, the specific protein structure of WDR5 makes it play a central scaffold role, and can form a complex with various proteins to regulate the differentiation, proliferation and antiviral effects of stem cells. WDR5 was found to be a key co-binding protein catalyzing the activity of H3K4me3 transferase, MLL1 complex. Therefore, when the WDR5 was studied for its effect on stem cell regulation, the effect of MLL1 on it was also of great concern. Based on the above results, we suggest that WDR5 may be a negative regulator of KDM6B and MLL1 mesenchymal stem cell function regulation. To confirm this hypothesis, we found 7 binding sites for the KDM6B/WDR5 protein complex by protein microarray technology:

37R E S R V Q R S R M D S S V S (shown in SEQ ID No. 3);

93C E T L V E R V G R S A T D P (shown in SEQ ID No. 4);

102FITC- (Acp) -KEKSRRVLGNLDLQSYGRKKRRQRRR (shown as SEQ ID No. 1);

114FITC- (Acp) -ADLTISHCAADVVRAYGRKKRRQRRR (shown as SEQ ID No. 2);

128S R S H T T I A K Y A Q Y Q A (shown in SEQ ID No. 5);

152FITC- (Acp) -IVPMIHVSWNVARTVYGRKKRRQRRR (shown as SEQ ID No. 6);

153V A R T V K I S D P D L F K M (shown in SEQ ID No. 7).

Three of the

sites

102, 114, 152 showed the highest degree of binding. Subsequently, we constructed bioactive polypeptides, screened the optimal concentration for cell stimulation, and found that 10ug/ml was the optimal concentration by in vitro ALP results. Co-IP

experiments using polypeptides

102, 114, 152 to stimulate cells revealed that 152 was not effective in blocking the binding of KDM6B and WDR 5. To further exclude the effect of polypeptide-linked FITC fluorescence and the cell effects of the penetratin, we designed control polypeptides and Co-IP results showed that Peptide102, 114 could indeed block KDM6B/WDR5 complex formation. On the basis, the inventor guesses whether the blocking of the formation of a KDM6B/WDR5 protein complex can enhance the functional action of KDM6B so as to restore the regeneration capacity of dry cell tissue repair under the aging condition. Interestingly, our in vitro osteogenesis/odontoblastic differentiation experiments confirmed the above guessed results through aging experiments. KDM6B/

WDR5 blocking polypeptides

102 and 114 can enhance osteogenic/odontoblastic directional differentiation capability and anti-aging capability of bone marrow mesenchymal stem cells and apical tooth papilla stem cells. Moreover, KDM6B/

WDR5 blocking polypeptides

102 and 114 can rescue the osteogenic differentiation capacity of the aged mouse bone marrow mesenchymal stem cells and reduce the expression of aging markers beta-gal and P16. To further demonstrate that the 102, 114 sequences can block KDM6B/WDR5 protein complex formation. We transfect apical tooth papilla stem cells after removing the

sequences

102 and 114 from HA-tagged KDM6B plasmid, and then perform Co-IP experiments to verify that the mutant Peptide102 and 114 sequences block KDM6B/WDR5 complex formation. And both fragments enhance the capability of KDM6B in promoting bone/tooth differentiation of mesenchymal stem cells and resisting aging.

In summary, our studies suggest that WDR5 may be a co-binding protein that negatively regulates the function of histone demethylases KDM6B and MLL 1. Based on mechanism research, small molecule polypeptides are developed and utilized aiming at the sequence of a KDM6B and WDR5 binding region, and the function of mesenchymal stem cells is regulated by regulating the binding of a KDM6B/WDR5 complex. Finally, the function of the mesenchymal stem cells is recovered under the aging condition, thereby promoting the repair and regeneration of bone/tooth tissues. Further research shows that the small molecular polypeptide has the functions of preventing and treating osteoporosis and periodontitis.

Experimental cells:

root tip dental papilla Stem Cells (SCAPs) source: orthodontic reduction teeth or impacted third molars which are extracted by an outpatient clinic of oral, maxillofacial and alveolar surgery of Beijing oral hospital affiliated to university of capital medical science are selected and collected under the informed consent of patients (16-22 years old), and the patients are required to have no systemic diseases of the whole body, and the collected teeth have no tooth body and periodontal diseases, and primary cell culture is carried out.

Source of human mesenchymal stem cells (BMSCs): purchased from ScienCell corporation.

C57BL/6 mouse bone marrow mesenchymal stem cell source: c57BL/6 mice, 2 and 18 months old, were selected and subjected to primary cell culture.

293T cells were purchased from Jima Gene, Suzhou, Inc. The experimental SCAPs and BMSCs are the 3 rd-5 th generation cells. Experimental animals:

male C57BL/6 mice, 2 and 18 months of age, were purchased from Schbefu (Beijing) Biotechnology, Inc.

The main equipment is as follows:

the main reagents are as follows:

statistical analysis:

SPSS 19.0 statistical software is adopted for statistical analysis, two groups of measurement data are compared by adopting t test, a plurality of groups of measurement data are compared by adopting ANOVA analysis, and the statistical difference of P <0.05 is taken as the basis.

TABLE 1 abbreviations/symbolic description

English abbreviation English full name Chinese full name

ALP alkali phosphatase

BMSCs Bone mesenchymal cells Bone marrow MSCs

BSP Bone sialoprotein

Co-IP Co-Immunoprecipitation

DMSO Dimethyl sulfoxide

DSPP Dentin sialophoprotein

FBS total bone serum

KDM6B lysine (K) -specific demethylase 6B histone demethylase 6B

KDM3B lysine (K) -Specific Demethylase 3B histone Demethylase 3B

MSCs Mesenchymal stem cells

MLL1 Mixed line Leukemia 1 Mixed line Leukemia 1

OCN Osteocalcin Osteocalcin

OSX Osterix osteogenesis related transcription factor

OPN Osteopontin

PBS Phosphate buffered saline Phosphate buffer

RUNX2 Runt-related transcription factor 2 RUnt-related transcription factor 2

SCAPs Apical papillala stem cells

TERT Telomerase Reverse Transcriptase Transcriptase Telomerase Reverse Transcriptase

WDR5 WD repeat domain 5 WD repeat protein 5

Beta-gal beta-Galactosidase beta-Galactosidase

alpha-MEM alpha-minimal essential medium

In the polypeptide sequence of KDM6B and the regulation and control application of the function of the mesenchymal stem cells, the raw materials and reagents used in the method can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 mesenchymal Stem cell harvesting, isolation and culture

(1) Isolated culture of human root apical deciduous head stem cells

Under the informed condition of the patient, the orthodontic addictive teeth or the third molar of the patient are extracted under local anesthesia in a sterile manner, and the in vitro teeth are placed in a PBS sterile centrifuge tube which is prepared in advance and contains double antibodies. In the super clean bench, the papillary tissue at the apex of the tooth root is scraped by a sterile blade. After repeatedly washing the removed papilla tissues with a large amount of PBS containing double antibody, they were put into a 1:1 formulated digestive juice containing collagenase type I (3g/L) and Dispase (4g/L) and cut into pieces. After 40 minutes of digestion in an incubator at 37 ℃ the digestion was stopped by adding 2 volumes of medium and the cells were collected in 15mL sterile centrifuge tubes. Centrifuging at 1100rpm/min for 6min, removing supernatant, adding culture medium to suspend cell precipitate, blowing, mixing, inoculating in 60mm culture dish, and culturing at 37 deg.C with 5% CO₂And (5) culturing in an incubator. After 3 days of culture, the cells were observed under a microscope for growth, replaced with fresh medium, and when the cells had grown to about 80% confluency, they were digested with 0.25% trypsin and passaged at 1:2 in 100mm dishes.

(2) Isolation culture of mesenchymal stem cells of C57BL/6 mouse bone marrow

After the neck of a mouse is cut off, the mouse is soaked in 75% alcohol for 5 minutes, the skin of four limbs is peeled off in an ultra-clean bench, the muscle and fascia on the surfaces of tibia, femur and humerus are carefully removed, the joints at two ends of a long bone are cut off by an ophthalmic scissors, an alpha-MEM culture medium with the constant temperature of 37 ℃ is extracted by using a 1ml syringe, and the marrow in the long bone is flushed into a culture dish with the length of 5cm by matching with a No.2 needle. At 37 ℃ with 5% CO₂Culturing, and after cell clone formation is observed after 5 days, using 0.25% trypsin to transfer to a large dish, and changing the liquid every 2-3 days. The growth of the cells was observed daily under an inverted microscope. When the cells were grown to 80% confluence, the cells were trypsinized according to 1: 3, digestion passage.

Example 2 cell culture and osteogenic/odontoblastic Induction differentiation

Culturing mesenchymal stem cells in ScienCell mesenchymal stem cell culture medium, and culturing at 37 deg.C with 5% CO₂A cell incubator. Primary cellsThe culture medium was used for cell experiments after 3-5 passages. After the cells were prepared, the cells were digested into single cells with trypsin and counted by a cell counter at 3.0X 10⁵The cells were plated in 6-well plates. Osteogenic induction is performed after the cell number has increased to 80% -90%. The liquid is changed once every three days.

Example 3 cryopreservation and recovery of mesenchymal Stem cells

(1) Freezing and storing

Cells were replaced with cell culture medium one day prior to cryopreservation. And putting experimental articles such as used gun heads, pipettors, centrifuge tubes and the like into a super clean bench in advance for disinfection for 30 minutes. After washing the cells 2 times with PBS, they were digested with 0.25% trypsin at 37 ℃ for 2 minutes, and after the cells were observed to float and become single cells under an inverted microscope, 3 volumes of medium were added to stop the digestion. The digested cells were suspended by blowing, mixed well and transferred to a 15mL centrifuge tube and centrifuged at 1100rpm/min for 6 minutes. Removing supernatant from the centrifuge tube, adding the freezing solution, mixing the cells uniformly, subpackaging in the freezing tube, placing in a refrigerator at-80 deg.C overnight, and storing in a liquid nitrogen tank for a long time. The cell names, the numbers and the dates are marked in the freezing tube.

(2) Resuscitation

And putting experimental articles such as used gun heads, pipettors, centrifuge tubes and the like into a super clean bench in advance for disinfection for 30 minutes. Taking out the culture medium from the refrigerator, and placing the culture medium in an incubator for preheating. The cells stored in liquid nitrogen were removed by wearing a mask and gloves, and rapidly placed in a 37 ℃ water bath and continuously shaken to melt them. Taking out the melted cells from the water bath, wiping with 75% alcohol for disinfection, opening the freezing tube in a super clean bench, sucking out the fine bubble suspension, transferring into a centrifuge tube containing the culture medium, and centrifuging at 1100rpm for 6 min. Removing supernatant, adding culture medium, blowing to mix cells, inoculating to culture dish, culturing at 37 deg.C with 5% CO₂Culturing in an incubator.

Example 4 viral packaging and cell transfection

(1) Construction of viral plasmids

Through NCBI database platform https// www.ncbi.nlm.nih.gov/query gene sequences of KDM6B, WDR5 and MLL1, SiRNAs of KDM6B, WDR5 and MLL1 are designed by using a program provided by Whitehead, inserted into shRNA vector pLKO.1 of lentivirus, sequenced and identified to finally construct plasmids of KDM6B shRNA, WDR51shRNA and MLL1 shRNA. The full length of KDM6B gene with surface label HA tag and the full length of WDR5 gene of Myc tag are obtained by gene synthesis method, and connected to the expression vector of retrovirus PQCXIN, and sequenced and identified to construct over-expression plasmid of KDM6B and WDR 5. The full length of KDM6B gene with surface label HA tag is obtained by gene synthesis method, 102 and 114 site sequences are removed, then the full length is connected to an expression vector of retrovirus PQCXIN, sequencing and identification are carried out, and finally overexpression plasmid of KDM6B with

mutation

102 and 114 sites is constructed. PQCXIN served as empty vector control.

(2) Packaging viruses

Control Scramble shRNA (Scramsh), KDM6B shRNA (KDM6Bsh), WDR5 shRNA (WDR5sh), MLL1shRNA (MLL1sh) and corresponding packaging plasmid (VSVG and dv-8.2)293T cells were transfected, and 48 hours after transfection, supernatants were collected for virus titer identification, and stored in-80 ℃ refrigerators after split charging. Retroviral control empty plasmids PQCXIN, PQCXIN-HA-KDM6B, PQCXIN-HA-KDM6B-mut102, PQCXIN-HA-KDM6B-mut114, PQCXIN-Myc-WDR5 and the corresponding packaging plasmids (VSVG and GPZ) were transfected into 293T cells; and collecting supernatant 72 hours after transfection, performing virus titer identification, subpackaging and storing in a refrigerator at 80 ℃ below zero.

(3) Establishment of stably transfected cells

The cells were seeded in a petri dish and after the cells had grown to a density of 50% -60%, 6mL of medium was replaced and 6 μ g/mL Polybrene was added. Cells were transfected with Scaramsh, KDM6Bsh, WDR5sh, MLL1sh virus, respectively, and the cell culture medium was changed after 12 hours of transfection. After 48 hours of transfection, puromycin was used for screening for 3 days to obtain stably transfected cells knocked out by the control genes of Scamsh, KDM6B, WDR5 and MLL1, and the knocking-out effect was detected at protein and RNA levels. The cells transfected by the control plasmids PQCXIN, PQCXIN-HA-KDM6B, PQCXIN-HA-KDM6B-mut102, PQCXIN-HA-KDM6B-mut114 and PQCXIN-Myc-WDR5 viruses are screened by G418 for 7 days after 48 hours after transfection, and the expression of exogenous KDM6B and WDR5 is detected and identified at protein and RNA levels to obtain the stably transfected cells over-expressed by KDM6B, KDM6B-mut102, KDM6B-mut114 and WDR 5.

Example 5Western Blot to detect changes in protein expression

(1) Total protein extraction

After the cell culture time is up, preparing experiment reagent and experiment material in advance, and preparing ice in advance. The culture medium in the dish was discarded and 5mL of 4 ℃ pre-chilled PBS was used to rinse the cells 3 times. Preparing a lysate, mixing RIPA, PMSF and PIC according to a ratio of 100:1:1, determining the adding amount according to specific conditions (a 10cm culture dish is usually 500 mu L), placing the culture dish in a refrigerator at 4 ℃ for incubation for 20min, and shaking the lysate in the culture dish every 5min to cover the bottom of the dish. The scraped cells were transferred to a 1.5mL centrifuge tube. Centrifuge at 14000rpm for 15min at 4 ℃. The supernatant was pipetted into a 1.5mL EP tube, labeled and stored at-80 ℃.

(2) Protein concentration measurement (Bradford method)

Taking a protein sample stored at-80 ℃, quickly melting on ice, adding 200 mu L of 1 Xcoomassie brilliant blue (Biobad) and 1 mu L of the protein sample (the protein loading amount is determined according to the color change) into a 96-well plate, uniformly mixing, discharging bubbles, and detecting the OD value on a machine. Drawing a standard curve, loading equal-volume equal-mass samples, loading according to 25 mu g (calculating protein volume, diluting to 20 mu L with PBS + PMSF + PIC, adding 5 mu L of 5 × loading buffer, performing protein denaturation at 95-100 ℃ for 10min, performing ice preservation for 10min, and storing at-20 ℃ after denaturation.

(3) Electrophoresis

Taking out the pre-formed gel, drawing out the bottom insulating strip, correctly placing the gel and the back plate (the front faces of the fonts face the experimenter), adding an electrophoresis buffer, adding the recyclable electrophoresis solution into the periphery according to the scales, and pulling out the comb after the mixture is filled. And drawing out the pre-gel comb (with gentle motion), adding samples in each pore channel, adding a Marker beside the pore channel of the protein sample, and adding 8 mu L of the Marker. Running the concentrated glue at 80V and 40min and running the gradient glue at 120V until the Marker runs to the bottom black line, and closing the switch.

(4) Transfer film (Do not touch water)

Taking out the pre-formed gel, cutting off the concentrated gel and the bottom part gel, covering the PVDF film on the surface (marking 1 position), putting the PVDF film, the gel and the filter paper on a rotary template in the order of filter paper-PVDF film-gel-filter paper, exhausting bubbles, covering a rotary membrane box cover, and screwing down. The membrane was transferred (constant pressure 1.3V, 7min), the power was turned off, the PVDF membrane was taken out (quick action), washed three times with 1 XTSST, 5min each time. And (3) sealing: 5% skimmed milk powder dissolved in 1 XTBST is prepared, the PVDF membrane is placed in the confining liquid, incubated for 1 hour at room temperature, and washed for 10min each time by a shaking table of 1 XTBST.

(5) Incubation of primary antibody and secondary antibody

Determining an incubation antibody mode according to the molecular weight difference between the internal reference and the target protein: cutting off PVDF membrane incubation antibody with difference larger than 5 Kda; antibodies were incubated in two aliquots with a difference of less than 5 Kda. The membrane was placed in TBST milk containing the appropriate concentration of primary antibody (including internal references such as GAPDH, HSP90, etc., which may require incubation after membrane shearing) and shaken overnight at 4 ℃. The primary coated membrane was washed 3 times with 1 × TBST for 5min each. The secondary antibody is prepared from the following components in percentage by weight: diluting at 2000 deg.C, and shaking for 1h at room temperature. PVDF membrane was washed three times with 1 XTSST, 5min each time.

(6) Development

The luminescent liquid was prepared in advance and disposed in a 1:1 mixing darkroom. The PVDF film was placed in a cassette and the cut film was recovered. And (3) the luminescent liquid drops are excited on the PVDF membrane (the area where the protein is located) by red light for 2-3min, and the image is formed in a BIO-RAD imaging system.

EXAMPLE 6 protein Co-immunoprecipitation (Co-immunoprecipitation, Co-IP)

(1) Extraction of Total cellular protein

And (3) extracting proteins of the knockout group cells and the overexpression group cells by using the IP lysate. Knocking out group cells SCAPs-Scamsh and SCAPs-KDM6Bsh, SCAPs-Scamsh and SCAPs-WDR5sh, respectively adding KDM6B antibody, WDR5 antibody and IgG antibody into BMSCs-Scamsh and BMSCs-WDR5sh, respectively, rotating a shaker overnight at 4 ℃, respectively adding protein A/G beads on the second day, rotating the shaker for 2 hours at 4 ℃ to form a protein-antibody-agarose bead compound, and detecting whether the KDM6B and the WDR5 form a protein compound or not through WB; the overexpression group cells SCAPs-Vector, SCAPs-HA-KDM6B, SCAPs-HA-KDM6B-mut102 and SCAPs-HA-KDM6B-mut114, the SCAPs-Vector and SCAPs-Myc-WDR5 are added with protein A/G beads with HA antibodies and protein A/G beads with Myc antibodies respectively and incubated overnight to form a protein-antibody-agarose bead compound, and WB is used for detecting whether the KDM6B and the WDR5 form a protein compound.

(2) Grouping: input group (25 μ g load); antibody panel (800 μ g loading, 2 μ g antibody); IgG group (800. mu.g of 2. mu.g antibody loaded) and the samples diluted with lysis buffer.

(3) Preparation of protein A/G beads

After 4 washes with PBS, 2000G for 2min, the supernatant was carefully aspirated, PBS was added to achieve a protein A/G beads content of 50%, 30. mu.L of protein A/G beads were added to each tube, and the tubes were spun overnight at 4 ℃.

(4)4 degrees 5000rpm, 30s, PBS wash 4-5 times upside down to get final product, last centrifugation to remove supernatant, adding 30 u L2 × loading buffer, 100 degrees boiling for 5 min.

(5) Taking the supernatant and carrying out electrophoresis.

Example 7 osteogenic/odontoblastic Induction experiments

(1) Osteogenic medium, purchased from Invitrogen, usa.

(2) Quantitative determination of alkaline phosphatase (ALP) Activity

1) After 3 days of osteogenesis induction, the culture medium is discarded, and PBS is used for washing twice;

2) adding 500. mu.L of lysine buffer, incubating at 37 ℃ for 15 min;

3) scraping the cells, transferring the cells into a 1.5ml EP tube, centrifuging the tube for 10 minutes in a 4-degree centrifuge at 14000rpm, and transferring the supernatant into a new EP tube (measuring the protein concentration, the same procedure as Western blot);

4) adding 5mL of distilled water into a capsule (Stock substrate Sol) in an alkaline phosphatase kit, and violently shaking to fully dissolve and uniformly mix the capsule;

5) adding 50 mu L of ALP buffer solution into 50 mu L of Stock substrate Sol, adding 100 mu L of sample into each hole in a 96-hole plate, adding 10 mu L of sample, uniformly mixing, arranging a blank hole additionally, not adding the sample, and adjusting zero;

6) incubate at 37 ℃ for 15min, and measure OD at 405 nm.

Y18.904X-0.2817 (OD value)

Results ═ Y/incubation time/protein concentration

(3) Alizarin red staining

1) After the osteogenesis of the cells is induced for two weeks, removing the culture medium, rinsing the cells for 3 times by using 4-degree precooled PBS, fixing the cells by using 70% ethanol, and carrying out 1 hour at 4 ℃;

2) washing with double distilled water for 2 times, dyeing with 40mM alizarin red solution (pH4.2) at room temperature for 10min, and observing with naked eyes;

3) washed 3 times with double distilled water.

(4)Ca²⁺Concentration determination

To quantitatively determine the calcium content, 10% cetylpyridinium chloride was added to dissolve for 30 minutes at room temperature. Measuring OD value at 562nm by using a spectrophotometer, calculating the calcium ion concentration of the sample by using a standard calcium ion concentration curve, and taking the total protein concentration as an internal reference.

Example 8 cell senescence-specific beta-galactosidase staining experiment

Before the experiment, the GENMED staining solution (Reagent E) in the kit is taken out from a refrigerator at the temperature of-20 ℃ and placed in an ice tank to wait for melting. Then 9.5 ml of GENMED diluent (Reagent D) is transferred to a 15ml conical centrifuge tube, 500 microliters of GENMED staining solution (Reagent E) is added, mixed evenly and placed in a 37 ℃ constant temperature water tank for preheating, and the mixture is marked as GENMED staining working solution. The following operations were then carried out:

(1) carefully remove the culture medium from the 24-well cell culture plate;

(2) adding 500 microliters of GENMED cleaning solution (Reagent A) into each well, and cleaning the surfaces of growing cells;

(3) carefully removing the cleaning solution from each well;

(4) 500 microliters of GENMED fixative (Reagent B) was added to each well, covering the entire growth surface;

(5) incubation at room temperature for 5 minutes;

(6) carefully removing the fixative;

(7) adding 500 microliters of GENMED acidic solution (Reagent C) into each well, and cleaning the cell surface;

(8) carefully removing the acidic solution;

(9) repeat experiment steps 7 and 8 once;

(10) adding 400 microliters of preheated GENMED staining working solution into each hole to cover the whole cell surface;

(11) placing in an incubator at 37 ℃ and incubating for 3 to 16 hours, or the cells appear blue (note: avoid evaporation of liquid);

(12) observation and counting under an optical microscope: cells expressing senescence-specific beta-galactosidase were positive and appeared blue.

Example 9 telomerase reverse transcriptase ELISA assay

(1) Preparation before detection: calculating a standard curve by using the concentration gradients of the standard substances, namely 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 0 ng/mL;

(2) add 100. mu.L of standard and sample per well. Cover with the supplied sealing plate membrane and incubate at 37 ℃ for 2 hours. Recording the name of the sample on the surface of the sealing plate film;

(3) discarding and spin-drying samples in each hole without cleaning;

(4) mu.L of Biotin-antibody (1X) was added to each well. Covered with a new shrouding membrane and incubated at 7 ℃ for 1 hour. The Biotin-antibody (1x) may be turbid, heated to room temperature, and gently stirred until the dissolution disappears;

(5) samples from each well were discarded and washed with Wash Buffer, and this procedure was repeated twice for a total of three washes. The cleaning process uses a discharging gun, 200 mu L of Wash Buffer is added each time, and the standing is carried out for 2 minutes each time, and the key of good performance is that liquid is completely removed in each step. After the last Wash, discard the Wash Buffer and invert the 96-well plate and blot it with a clean paper towel.

(6) Add 100. mu.L of HRP-avidin (1X) per well, cover the microtiter plate with a new sealing plate, incubate for 1 hour at 37 ℃;

(7) repeating the liquid-discarding/washing process of step 6 for 5 times;

(8) add 90. mu.L of TMB Substrate per well. Incubate at 37 ℃ for 15-30 minutes, and protect from light. Add 50. mu.L of Stop Solution to each well and tap the plate gently to ensure adequate stirring.

(9) The optical density of each well was determined within 5 minutes using a microwell reader set at 450 nm.

Example 10 detection of binding reaction of polypeptide microarray with recombinant protein

(1) Polypeptide chip synthesis: synthesizing a polypeptide chip according to the sequence of KDM6B protein, and designing by overlaying, wherein the total number of the two arrays is.

(2) Polypeptide array synthesis: the activated substrate chip membrane is placed on a full-automatic polypeptide chip synthesizer, and the Fmoc-amino acid solution is automatically transferred to a specific position on the activated membrane to react with the membrane according to a program. And (3) sequentially immersing the membrane into a blocking solution I and a blocking solution II to carry out side chain blocking and washing the membrane by DMF. The membrane was placed in a deprotection solution for removal of the Fmoc protecting group at the amino terminus, after which the membrane was washed with DMF and then dried with ethanol. Repeating the steps until the polypeptide array is completely synthesized. After the synthesis, the side chain protecting group is removed by using a specific organic reagent, and the membrane is washed by using CH2Cl2, dried by using ethanol and used immediately or stored at the temperature of-20 ℃.

(3) And (3) sealing: activating the polypeptide microarray chip, adding a sealing liquid, oscillating and sealing at room temperature for 4 hours, and washing the chip;

(4) biotin labeling of the target protein: taking 1ml of protein sample WDR5 synthetic protein (concentration is 1.5mg/ml), and carrying out protein labeling by using EZ-link NHS-PEO4-Biotinylation kit (prod # 21455);

(5) and (3) incubating the labeled protein sample with the polypeptide chip: biotin-labeled WDR5 synthetic protein samples (final concentration 1ug/ml) diluted with blocking solution were mixed with 5ml polypeptide microarray chip and incubated overnight at 4 ℃ with control incubated with blocking solution;

(6) Streptavidin-HRP incubation: incubating a reaction reagent Streptavidin-HRP (High Sensitivity Streptavidin-HRP (prod #21133)), diluting a confining liquid (1:10000), incubating a polypeptide microarray chip with 5ml, shaking at room temperature for 2 hours, and washing the chip;

(7) color development: ECL luminescence reagent, Chempchemi digital imager, digital imaging was added.

(8) Chip scanning and color rendering point data analysis: 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.

Effect example 1 research on Effect and control mechanism of KDM6B/WDR5 on senescence and osteogenic differentiation of mesenchymal Stem cells

Research on influence and regulation mechanism of KDM6B on bone marrow mesenchymal stem cell senescence

1.2 WDR5 can form protein complex with KDM6B and MLL1 in bone marrow mesenchymal stem cells

To determine whether WDR5 and KDM6B, MLL1 could form protein complexes, we performed Co-IP experiments on mesenchymal stem cells stably knocked out WDR 5. Co-IP experimental results show that compared with a control group, WDR5 knockout bone marrow mesenchymal stem cells have reduced complex formation of WDR5, KDM6B and MLL1 proteins (figure 1).

1.3 binding of WDR5 to KDM6B and MLL1 in mesenchymal stem cells of aging mice was significantly increased

In order to clarify the regulation effect of KDM6B, WDR5 and MLL1 on mesenchymal stem cells under aging conditions, a Co-IP experiment is carried out on 2-month-old C57BL/6 mice and 18-month-old C57BL/6 mice to detect the formation condition of a protein complex. Co-IP experimental results show that compared with a control group, the marrow mesenchymal stem cells of aged C57BL/6 mice have increased complex formation of WDR5, KDM6B and MLL1 (figures 1-2).

Effect example 2 influence of KDM6B/WDR5 protein Complex on osteogenic differentiation Capacity of mesenchymal Stem cells and study on regulatory mechanism

3.1KDM6B/WDR5 has 7 binding sites, and

polypeptides

102 and 114 effectively block KDM6B/WDR5 protein complex formation

In order to deeply research the function control mechanism of KDM6B on bone marrow mesenchymal stem cells, a subject group applies a polypeptide microarray technology to discover a KDM6B/WDR5 protein complex binding site sequence. Polypeptide microarray technology found 7 positive binding sites to be present in WDR5 and KDM6B (fig. 3A). The selection of positive polypeptide binding sites follows: the spot optical density value exceeds 30% and the spot optical density value on the negative reaction film is less than 30%. Chip hybridization gray scale value analysis found that the binding degree of the three 102, 114, 152 among the 7 binding sites was the highest (FIG. 3B). Peptide102, peptide114 and peptide152 were synthesized by binding site analysis and sequence design.

peptide102：FITC-(Acp)-KEKSRRVLGNLDLQSYGRKKRRQRRR；

peptide114：FITC-(Acp)-ADLTISHCAADVVRAYGRKKRRQRRR；

peptide152：FITC-(Acp)-IVPMIHVSWNVARTVYGRKKRRQRRR。

Co-IP results show that only peptide102 and peptide114 in three

bioactive polypeptides

102, 114 and 152 can effectively open the binding of a KDM6B/WDR5 protein complex (FIG. 3C).

3.2

Polypeptides

102, 114 inhibit the senescence of mesenchymal stem cells and promote the osteogenic differentiation of mesenchymal stem cells

We simultaneously investigated the functional effects of peptide102 and peptide114 on bone marrow mesenchymal stem cells. Results of telomerase reverse transcriptase ELISA experiments show that peptide114 promotes expression of telomerase reverse transcriptase in mesenchymal stem cells of bone marrow compared with a control group (FIG. 4A). The results of beta-gal staining and quantitative analysis showed that peptide102 and peptide114 caused a significant decrease in the number of beta-gal positive cells in mesenchymal stem cells of bone marrow compared to the control (FIG. 4B, C). After 3 days of induction in osteogenic medium, we found that peptide102 and peptide114 promoted ALP activity of bone marrow mesenchymal stem cells compared to the control group (fig. 4D). Alizarin red staining and calcium quantitative analysis were performed after 2 weeks of culture in osteogenic induction medium. The results show that the mineralization capacity of mesenchymal stem cells of peptide102 and peptide114 groups is remarkably increased compared with the control group (fig. 4E, F).

TABLE 2 data for FIG. 4A

		SD
			Mock	0.011714	0.000928673
control peptide	0.014349	0.000934984
			102peptide	0.022363	0.000953919
114peptide	0.038878	0.002975362

TABLE 3 data of FIG. 4B

TABLE 4 data of FIG. 4D

Mock	3.435171	0.39624
			control peptide	3.262909	0.064275
102-peptide	4.217977	0.07766
			114-peptide	4.620564	0.191912

TABLE 5 data for FIG. 4F

		SD
			Mock	1.367242	0.042852
control peptide	1.368383	0.04055
			102peptide	1.584158	0.015308
114peptide	1.806783	0.063106

3.3

Polypeptides

102, 114 inhibit aging of bone marrow mesenchymal stem cells of aging mice, and promote osteogenic differentiation of aging bone marrow mesenchymal stem cells

To confirm that

bioactive polypeptides

102, 114 can exert a functional regulatory effect on senescent bone marrow mesenchymal stem cells, we performed senescence and differentiation function-related experiments. The beta-gal staining results indicated that peptide102 and peptide114 caused a significant decrease in the number of beta-gal positive cells of senescent bone marrow mesenchymal stem cells compared to the control group (FIG. 5A). After 3 days of induction in osteogenic medium, we found that peptide102 and peptide114 promoted ALP activity of senescent bone marrow mesenchymal stem cells compared to the control group (fig. 5B). Alizarin red staining and calcium quantitative analysis were performed after 2 weeks of culture in osteogenic induction medium. The results showed that the mineralization capacity of mesenchymal stem cells of aged bone marrow of peptide102 and peptide114 group was significantly increased compared to the control group (fig. 5C, D).

TABLE 6 data of FIG. 5B

		SD
			Mock	6.02553	0.264389
control peptide	5.763749	0.432205
			102-peptide	6.950772	0.156132
114-peptide	7.611731	0.242683

TABLE 7 data of FIG. 5D

Mock	1.082396	0.006429
			control peptide	1.103677	0.011295
102peptide	1.153978	0.012147
			114peptide	1.251294	0.009707

Effect example 3 study on Effect of WDR5 on mesenchymal Stem cell senescence and odontogenic differentiation Capacity and control mechanism

5.1 KDM6B can form a protein complex with WDR5 in the root apical papilla stem cells

In order to further study the regulation mechanism of KDM6B on the papilla of the root apices, we tested the binding of KDM6B and WDR5 by Co-IP experiments. Co-IP experiments showed that KDM6B and WDR5 binding was significantly reduced in the apical papilla stem cells of the KDM6B knockout group compared to the control group (FIG. 6A). To further confirm that KDM6B and WDR5 can form protein complexes, we performed Co-IP experiments on apical papilla stem cells stably knocked out WDR 5. Co-IP experimental results showed that KDM6B and WDR5 protein complex formation was reduced in cells of the knockout WDR5 group compared to the control group (FIG. 6B).

Effect example 4 Effect of KDM6B/WDR5 protein Complex on odontogenic differentiation ability of mesenchymal Stem cells and study of regulatory mechanism

7.1 mutation of

KDM6B sequence

102, 114 site, inhibiting root cusp deciduous cell senescence and promoting root cusp deciduous cell odontogenic differentiation

To further confirm that the 102, 114 sequences can block formation of the KDM6B/WDR5 protein complex, we performed Co-IP experiments on cells overexpressing KDM6B at the 102 and 114 sites of the transfection mutation. The experimental results showed that KDM6B and WDR5 binding was reduced in HA-KDM6B-mut102 group and HA-KDM6B-mut114 compared to HA-KDM6B group (FIG. 7A). The effect of overexpression of KDM6B at the

mutation

102 and 114 sites on root tip papilla stem cell senescence was subsequently examined. The results of the telomerase reverse transcriptase ELISA experiments show that compared with the control group, the HA-KDM6B-mut102 group and the HA-KDM6B-mut114 promote the telomerase reverse transcriptase expression of the papilla deciduous cells of the root tips (FIG. 7B). The results of the beta-gal staining and quantitative analysis showed that the HA-KDM6B-mut102 group and HA-KDM6B-mut114 group resulted in a significant decrease in the number of beta-gal positive cells in the papilla stem cells of the apical teeth compared to the control group (FIGS. 7C, D). After 3 days of induction in the tooth-forming medium, we found that the HA-KDM6B-mut102 group and the HA-KDM6B-mut114 promoted ALP activity of root apical papilla stem cells compared to the control group (FIG. 7E). Alizarin red staining and calcium quantitative analysis were performed after 2 weeks of culture in the adult tooth induction medium. The results showed a significant increase in mineralization capacity of the HA-KDM6B-mut102 and HA-KDM6B-mut114 groups compared to the control group (FIG. 7F, G).

TABLE 8 data of FIG. 7B

		SD
			Vector	0.120301	0.002321238
HA-KDM6B	0.151428	0.001219
			HA-KDM6B-mut102	0.148849	0.002428533
HA-KDM6B-mut114	0.167124	0.001247402

TABLE 9 data of FIG. 7D

	MEAN	SD
			Vector	29.44052	3.676117107
HA-KDM6B	18.2746	0.752269296
			HA-KDM6B-mut102	11.74683	1.302423965
HA-KDM6B-mut114	13.15876	0.433142542

TABLE 10 data for FIG. 7E

		SD
			Vector	0.116858	0.005575
HA-KDM6B	0.28432	0.069515
			HA-KDM6B-mut102	0.459554	0.082139
HA-KDM6B-mut114	0.751555	0.133336

TABLE 11 data of FIG. 7G

Vector	0.704162	0.004041
			HA-KDM6B	0.865251	0.008312
HA-KDM6B-mut102	1.235037	0.007506
			HA-KDM6B-mut114	1.212432	0.071305

7.2

Polypeptides

102, 114 inhibit the senescence of root apical papilla stem cells and promote the odontogenic differentiation of root apical papilla stem cells

On the basis of previous research, the function of the root tip tooth papilla stem cells is further regulated by regulating and controlling the combination of KDM6B/WDR5 complexes by aiming at sequences of a KDM6B and WDR5 binding region and developing and utilizing small-molecule bioactive polypeptides. We performed Co-IP experiments on bioactive polypeptide-treated root tip dental papilla stem cells. The experimental results showed that KDM6B and WDR5 binding was reduced in root apical dental papilla stem cells of peptide102 and peptide114 groups compared to the control group (fig. 8A). The effect of peptide102 and peptide114 on the senescence of root apical papilla stem cells was then examined. Results of telomerase reverse transcriptase ELISA experiments showed that peptide114 promoted telomerase reverse transcriptase expression in deciduous cells of apical teeth compared to control (FIG. 8B). The results of the β -gal staining and quantitative analysis showed that peptide102 and peptide114 resulted in a significant reduction in the number of β -gal positive cells in the papilla stem cells of the root tips compared to the control (fig. 8C, D). After 3 days of induction in the tooth-forming medium, we found that peptide102 and peptide114 promoted ALP activity of root apical deciduous papilla stem cells compared to the control group (fig. 8E). Alizarin red staining and calcium quantitative analysis were performed after 2 weeks of culture in the adult tooth induction medium. The results showed that root apex deciduous papilla stem cells of peptide102 and peptide114 groups had significantly increased mineralization compared to the control group (fig. 8F, G).

7.3 the presence of KDM6B and WDR5 co-regulates downstream target genes.

TABLE 12 data of FIG. 8B

		SD
			Mock	0.032608	0.001955173
control peptide	0.030542	0.002918559
			102peptide	0.033312	0.004895822
114peptide	0.051677	0.001020189

TABLE 13 data of FIG. 8D

		SD
			Mock	25.07861	2.419658
control peptide	24.20205	0.643601
			102peptide	7.29624	0.448604
114peptide	3.978156	0.35091

TABLE 14 data of FIG. 8E

		SD
			Mock	0.218303	0.087875
control peptide	0.285743	0.012709
			102peptide	0.396121	0.038258
114peptide	0.723759	0.003245

TABLE 15 data of FIG. 8G

Mock	1.28938	0.031225
			control peptide	1.209692	0.033382
102peptide	1.63736	0.016823
			114peptide	1.619093	0.032005

Effect example 5

1. New bone formation volume (mm) of small pig periodontitis model³) As shown in table 16 and fig. 9.

TABLE 16 data of FIG. 9

PBS	Control peptide	114peptide
			23.73	26.69	80.68
48.06	69.27	79.08
			21.96	47.63	80.48
23.9	38.3	76.48
			16.85	32.25	85.49
37.64	33.48	101.99

2. Clinical probing index of small pig periodontitis model

Establishing experimental periodontitis bone defect model of small-sized pig

The modeling method comprises the following steps: a method for manufacturing bone defects and ligature threads is adopted to establish a experimental periodontitis model of the miniature pigs. The specific method comprises the following steps: the method comprises the following steps of carrying out general anesthesia on the miniature pig, carrying out mouth disinfection, selecting the first constant molar of the lower jaw of the miniature pig as an experimental tooth, making an incision in a ditch and a vertical incision, turning over a periosteal flap, exposing the middle buccal root of the first constant molar of the lower jaw of the miniature pig by adopting a bone removal method, and making a bone defect with the size of 3mm multiplied by 5mm multiplied by 7 mm. Namely: removing alveolar bone of the mesial-adjacent surface by 3mm × 5mm × 7mm, removing the buccal side bone plate of the mesial-buccal root and the distal alveolar bone (3mm × 5mm, gingival depth flat mesial-adjacent surface); and (6) sewing in situ. And (5) ligating silk threads at the neck of the tooth. The bone defect can not be repaired by self after 4 weeks of operation, and a periodontitis bone defect model can be successfully formed. The establishment of a periodontitis bone defect model is verified by CT imaging and clinical examination results.

Bioactive polypeptide 114 for regenerating and repairing experimental periodontitis bone defect of miniature pig

12 Wuzhishan minipigs establish a periodontitis model, 24 sides are totally divided into 3 groups at random, and the injection dosage of each side is 60 ul: group 1: flap-over and scraping plus sterile saline injection (untreated control group) were performed after modeling; group 2: performing flap scraping and contrast polypeptide injection after modeling (contrast group); group 3: flap scraping + bioactive polypeptide 114 injection (experimental group) was performed after modeling.

4w after the modeling operation, the medicine injection treatment is carried out. The influence of the bioactive polypeptide 114 on the regeneration and repair capacity of the small-sized pig periodontitis defective tissue mediated by stem cells is observed by comparing clinical index examination and imaging examination 4 weeks (-4w) after molding, 0w) after no drug injection and 3 months (12w) after drug treatment.

Results of CT imaging, Geomagic Studio 12, mimics17 bone metrological analysis and clinical examination show that the bioactive polypeptide 114 promotes the regeneration of periodontal tissues mediated by mesenchymal stem cells.

Post-treatment observation index

Observation of clinical, imaging and histological indicators

Clinical indices (PD, AL, GR) and CT imaging examinations were performed before the experiment, after modeling (-4w) and 3 months after treatment (12w), respectively.

Periodontal Probing Depth (PD): and recording the peridental pocket depth of the mesial and buccal sides of the teeth at probing pressure of 20-25 g.

Gingival Recession (GR): the distance from the cementum junction to the gingival margin was measured using a periodontal probe. If the gum recedes, the enamel cementum boundary is exposed, and the gum margin is positioned at the root of the enamel cementum boundary, the distance between the gum margin and the enamel cementum boundary is recorded as a positive value; if the gum is not retracted and the gingival margin is located in the coronal direction of the enamel cementum junction, the value is recorded as negative.

Loss of Attachment (AL): the extent of adhesion loss is determined by subtracting GR from the bag depth. If the subtraction of the two numbers is zero or the cementum enamel boundary can not be detected, indicating that no attachment is lost, if the gum is retracted to enable the gum margin to be positioned at the root of the cementum enamel boundary, the two readings are added to obtain the degree of the attachment loss.

2.1 Probingdepth (PD): as shown in table 17 and fig. 10.

TABLE 17 data of FIG. 10

Wherein, "-": one experimental animal died, and therefore, one set of data was missing.

After the small-sized pig periodontitis model is injected with bioactive polypeptide for 12 weeks, periodontal probe measurement shows that the periodontal probing depth of the 114peptide group is obviously lower than that of the PBS group and the Control peptide group; the difference between the 114peptide group and the rest groups has significance; p is less than or equal to 0.05;

2.2Attachment Loss (AL): as shown in table 18 and fig. 11.

Table 18 data of fig. 11

2.3Gingival Recession (GR): as shown in table 19 and fig. 12.

After the small-sized pig periodontitis model is injected with bioactive polypeptide for 12 weeks, the attachment loss condition of the 114peptide group is obviously lower than that of the PBS group and the Control peptide group through periodontal probe measurement; the difference between the 114peptide group and the rest groups has significant significance; p is less than or equal to 0.05;

table 19 data of fig. 12

After the small-sized pig periodontitis model is injected with bioactive polypeptide for 12 weeks, the gingival recession degree of the 114peptide group is obviously lower than that of the PBS group and the Control peptide group through periodontal probe measurement; the difference between the 114peptide group and the rest groups has significance; p is less than or equal to 0.05;

CBCT three-dimensional modeling As shown in FIG. 13, the examined miniature pig was lying on the CBCT scanner bed and taken from the natural bites

And fixing the head, continuously scanning to obtain CBCT sectional images and performing three-dimensional reconstruction on the DICOM images by using Mimics17.0 image processing software. CBCT data are acquired before, after and after modeling respectively for post-treatment assessment; the postoperative three-dimensional reconstruction model shows that new bones are formed in the PBS group/Control peptide group/114 peptide; the forming effect of the new bone of the 114peptide group is better than that of the Control peptide group and the PBS blank Control group;

as shown in FIG. 14, after 12 weeks of bioactive polypeptide injection, the wounds of PBS group/Control peptide group/114 peptide group in bone defect area of small pig periodontal tissue were healed in the small pig periodontitis model, and no infection or necrosis tissue was observed; the soft tissue repair effect of the 114peptide group is better than that of the Control peptide group and the PBS group.

Effect example 6

1. Defect of mucous membrane of palate

Balb/c8 month-old male mice, 15, were randomly assigned to PBS, control, and 114 groups of 5 mice each. Before the defect is established, 4% chloral hydrate is injected into the patient with the concentration of 0.2mL/20g for anesthesia and skin preparation, a circular full-cortical defect with the diameter of 6mm in the center of the back is established by a skin drill, and PBS, control polypeptide with the concentration of 10ul/mL and 114 polypeptide with the concentration of 100ul are injected into the defect edge at 2mm positions at four equidistant sites respectively on the day of establishing the defect, 3 days, 7 days, 10 days, 14 days, 17 days and 21 days.

The area of the defect area was calculated by Image-Pro plus software by taking a photograph of the defect site before each injection, and the healing rate was calculated as the healing rate (0 day defect area-14/21 day defect area)/0 day defect area. The T test p is less than 0.05, and the skin defect healing rate is shown to have statistical significance compared with the control group in 14 days and 21 days.

The results are shown in FIGS. 15 to 16 and Table 20.

Table 20 data of fig. 16

14-pbs	14-control	14-114	21-pbs	21-control	21-114
						8.553957	8.083566	5.781627	4.259305	2.598592	2.259207
7.139076	7.187624	7.253595	2.066862	2.159928	0.3319243
						7.393813	7.306628	8.230819	3.930576	2.134038	1.963786
7.693449	7.503132	5.520400	3.418914	5.362377	2.329762
						7.695074	7.520237	6.400598	3.418914	3.063734	2.213450

By counting the areas of unhealed palatine mucosa defects of mice on days 14 and 21 in the pbs group, the Controlpeptide group and the 114peptide group, the 114peptide group is found to have significant statistical difference compared with the pbs blank control group and the control peptide group; p is less than or equal to 0.05.

2. Skin defect

The results are shown in FIGS. 17 to 18 and Table 21.

TABLE 21 data of FIG. 18

By counting the healing rate of the skin defect on the back of the mouse 10 days and 14 days after the injection of the medicament in the pbs group, the control peptide group and the 114peptide group, the 114peptide group is found to have significant statistical difference compared with the pbs group and the control peptide group; p is less than or equal to 0.05, P is less than or equal to 0.01, P is less than or equal to 0.001;

3. prevention of bone loss in OVX mouse osteoporosis model

25 mice, 3 months old C57BL/6, were purchased in Wintolite and randomized into sham, OVX, PBS, control and 114peptide groups of 5 mice each. OVX group, PBS group, control peptide group and 114peptide group were used to construct osteoporosis model by removing bilateral ovaries, and sham group was used without ovaries. The mice were each injected intraperitoneally 6 weeks after ovariectomy at a dose of 10mg/kg for control peptide and 114 peptide. Sacrificed after 3 months of continuous injection. For the distal femur, the left distal femur of each mouse was scanned ex vivo using a micro-CT system. And selecting each section of trabecula bone for segmentation, performing three-dimensional reconstruction, and calculating BMD.

The results are shown in fig. 19 and table 22.

TABLE 22 data of FIG. 19B

sham	OVX	PBS	control peptide	114peptide
					0.1551	0.1060	0.0191	0.0158	0.1440
0.1848	0.0355	0.0612	0.0600	0.1242
					0.1490	0.0863	0.1043	0.0894	0.1416
0.2242	0.0203	0.0659	0.0978	0.1071
					0.1421	0.0143	0.0415	0.0400	0.1137

micro-CT analysis is carried out on femoral shaft dirty ends of mice, and the mice injected with PBS and Control peptide are found to have no obvious difference between the femoral bone trabecular bone density and the mice in OVX group; when the OVX group, the PBS group, the Control peptide group and the Sham group are compared, the trabecular bone density is obviously reduced; bone trabecular bone density of mice in the 114peptide group is obviously increased compared with that of mice in the OVX group, the PBS group and the Control peptide group.

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

Claims

1. The protein complex is used as a target for preparing a reagent or a medicament for inhibiting the aging of the mesenchymal stem cells and promoting the osteogenic differentiation or the odontogenic differentiation of the mesenchymal stem cells;

the first protein complex comprises WDR5 and KDM 6B;

the second protein complex comprises KDM6B, WDR5 and MLL 1.

2. A biologically active peptide, characterized in that it has:

(I) the amino acid sequence shown as SEQ ID No.1 or 2;

3. Use of the bioactive peptide of claim 2 in the preparation of an agent or medicament for aging mesenchymal stem cells, promoting osteogenic differentiation of mesenchymal stem cells and/or promoting odontogenic differentiation of deciduous head stem cells at the root tip.

4. The use of the biologically active polypeptide according to claim 2 for the preparation of a reagent or a medicament for the prevention and/or treatment of osteoporosis, periodontitis and/or the repair of mucosal and skin defects.

5. An agent or medicament comprising the biologically active peptide of claim 2 and a pharmaceutically acceptable excipient.