Neuropeptide and application thereof in promoting growth and development of mandible

Abstract

The invention relates to the field of polypeptides, in particular to neuropeptide and application thereof in promoting growth and development of mandible. In-vivo and in-vitro experiments verify that the novel 5-Peptide promotes the osteogenic differentiation function of MSCs so as to promote the growth and development of the mandible, and explains the molecular action mechanism that the 5-Peptide is combined with CNR2 to regulate and control an AKT / ERK1 / 2 / STAT3 signal channel; clinical application basis is provided for treating oral diseases such as small mandibular deformity and malocclusion caused by hypoplasia of mandible by using 5-Peptide.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, in particular to neuropeptides and applications thereof in promoting the growth and development of mandibular bones. Background Art

[0002] As human evolution continues, the dietary structure has gradually transitioned from chewing hard food to a modern, more refined diet, which has led to a significant reduction in the size of human jaws and teeth, which in turn causes oral diseases such as micrognathia, malocclusion, and tooth-bone imbalance, which seriously affect the patient's occlusal function, pronunciation, aesthetics, gastrointestinal function, and physical and mental health. Therefore, without changing the modern human dietary strategy, it is of great significance to promote the normal growth and development of the mandible and restore the normal occlusal relationship. At present, the main clinical treatment for micrognathia and malocclusion is orthodontic treatment through wearing braces and orthognathic surgery, but there are problems such as high treatment costs, long treatment cycles, and large surgical trauma.

[0003] In recent years, studies have shown that regulating the use of autologous or allogeneic MSCs to promote malocclusion caused by insufficient mandibular development is an emerging treatment method. However, the clinical application of MSCs is limited due to limited sources of MSC seed cells, high costs, and ethical issues.

[0004] In recent years, the latest evidence shows that in addition to sensing stimuli such as pain, itch, temperature, taste and smell, sensory nerves can also produce various neuropeptides, such as substance P, calcitonin gene-related peptide (CGRP) and vasoactive peptides, which play an important role in maintaining bone homeostasis. The nerves that innervate the mandible originate from the mandibular nerve, a branch of the trigeminal nerve, including the inferior alveolar nerve (IAN), buccal nerve, lingual nerve and mental nerve, among which the inferior alveolar nerve is a sensory nerve. The basic unit of bone formation is active osteoblasts, which originate from MSCs. Mandibular osteogenesis mainly includes intramembranous ossification and endochondral ossification, in which MSCs play an important role. Our previous study found that under the action of chewing force, POMC precursor peptide is produced in the trigeminal ganglion of mice, which can promote the growth and development of the mandible by regulating the osteogenic differentiation function of MSCs. Our previous study found that POMC derived from sensory nerves can regulate the osteogenic differentiation function of MSCs. POMC is a precursor peptide that can be specifically cleaved into a variety of neuropeptides in different environments, such as adrenocorticotropic hormone (ACTH), α-, β-, γ-melanotropin (α-, β-, γ-MSH), ACTH-like intermediate peptide, endogenous opioid β-endorphin (β-endorphin), γ-lipotropin (γ-LPH) and met-enkephalin. These peptides are packaged in large vesicles and released to play a role in a range of biological activities, including pigmentation, adrenal cortex function, energy metabolism, sexual behavior and immune regulation. However, whether these cleaved neuropeptides can regulate MSCs function and the molecular mechanism of action needs to be explored. Summary of the invention

[0005] In view of this, the present invention uses a new neuropeptide (5-Peptide) to promote the growth and development of the mandible by regulating the osteogenic differentiation function of mesenchymal stem cells (MSCs), so as to solve oral diseases such as mandibular underdevelopment, micrognathia, malocclusion, and imbalance of tooth and bone mass caused by refined diet in modern humans.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a polypeptide having:

[0008] (I), the amino acid sequence shown in SEQ ID No.241;

[0009] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0010] (III) An amino acid sequence having 90% or more identity with the amino acid sequence described in (I) or (II).

[0011] In a second aspect, the present invention also provides the use of the polypeptide in the preparation of a drug for promoting the growth and development of mandibular bones or treating oral diseases.

[0012] In some specific embodiments of the present invention, the polypeptide is a receptor to which α-MSH and / or β-MSH binds.

[0013] In some specific embodiments of the present invention, the receptor of the polypeptide is CNR2.

[0014] In some specific embodiments of the present invention, the oral disease includes one or more of mandibular underdevelopment, micrognathia, malocclusion, tooth imbalance or bone imbalance.

[0015] In some specific embodiments of the present invention, the polypeptide promotes the osteogenic differentiation of MSCs by regulating AKT / ERK1 / 2 / STAT3 by binding to CNR2 on the surface of BMMSCs.

[0016] In some specific embodiments of the present invention, the polypeptide promotes the growth and development of the mandibular bone by binding to the CNR2 receptor and regulating the osteogenic differentiation function of MSCs.

[0017] In some specific embodiments of the present invention, the polypeptide inhibits ALP activity and mineralization ability after knockout of CNR2 in BMMSCs.

[0018] In some specific embodiments of the present invention, the polypeptide inhibits the expression of osteogenesis-related transcription factors OCN, RUNX2 and OSX after knocking out CNR2 in BMMSCs.

[0019] In a third aspect, the present invention also provides an agent or a medicine, comprising the polypeptide.

[0020] The present invention verifies the function of the novel 5-Peptide in promoting the osteogenic differentiation of MSCs and thus promoting the growth and development of the mandible through in vivo and in vitro experiments, and explains the molecular mechanism of action of 5-Peptide in regulating the AKT / ERK1 / 2 / STAT3 signaling pathway by binding to CNR2. The use of 5-Peptide to treat oral diseases such as micrognathia and malocclusion caused by insufficient mandibular development provides a clinical application basis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0022] Figure 1 Schematic diagram showing the effects of different neuropeptides produced by POMC provided in the embodiments of the present invention on the osteogenic differentiation function of BMMSCs and possible receptors; wherein, A shows the expression of alkaline phosphatase activity (ALP) after α-MSH, β-MSH, γ-MSH, ACTH, γ-LPH, β-endorphin, β-LPH, CLIP, and met-enkephalin acted on BMMSCs, indicating that α-MSH and β-MSH can significantly promote the alkaline phosphatase activity of BMMSCs; B shows the detection of α-MS by Alizarin red staining (ARS). H, the effects of β-MSH, γ-MSH, ACTH, γ-LPH, β-endorphin, CLIP, and met-enkephalin on the osteogenic mineralization ability of BMMSCs, showing that α-MSH and β-MSH can significantly promote the formation of calcium nodules in BMMSCs; C shows the knockdown efficiency of CNR1 and CNR2 in BMMSCs detected by RT-PCR; D shows the expression of alkaline phosphatase activity (ALP) in BMMSCs after α-MSH and β-MSH knockdown of CNR1 and CNR2, showing that the knockdown of CNR1 and CNR2 After knocking down CNR1 and CNR2, α-MSH and β-MSH inhibited the alkaline phosphatase activity of BMMSCs; E showed the effect of α-MSH and β-MSH on the osteogenic mineralization ability of BMMSCs after knocking down CNR1 and CNR2, which showed that α-MSH and β-MSH inhibited the calcium nodule formation of BMMSCs after knocking down CNR1 and CNR2; FG showed the content of α-MSH (F) and β-MSH (G) in mouse mandibular samples under different chewing forces detected by ELISA; H showed the bioinformatics analysis found that α-MSH The receptors that α-MSH and β-MSH bind to are MC1R, MC3R, MC4R and MC5R. RT-PCR was used to detect the knockdown efficiency of MC1R, MC3R, MC4R and MC5R in BMMSCs. I shows the expression of alkaline phosphatase activity (ALP) in BMMSCs after α-MSH and β-MSH knocked down MC1R, MC3R, MC4R and MC5R. It shows that after knocking down MC5R, α-MSH and β-MSH inhibited the alkaline phosphatase activity of BMMSCs, indicating that MC5R may be the receptor that binds to α-MSH and β-MSH.

[0023] Figure 2Schematic diagram of in vitro and in vivo experiments provided in the embodiments of the present invention showing that the novel pentapeptide (5-Peptide) promotes the growth and development of the mandible by regulating the osteogenic differentiation function of MSCs by binding to the CNR2 receptor; wherein, A shows that α-MSH and β-MSH have a common 5 amino acid sequence (EHFRW) and are synthesized into the novel pentapeptide 5-Peptide; B shows the use of alkaline phosphatase activity (ALP) and Alizarin red staining (ARS) to detect the osteogenic differentiation ability of 5-Peptide on BMMSCs, and the results show that 5-Peptide promotes the ALP activity and mineralization ability of BMMSCs; C shows the use of RT- PCR detection of the effect of 5-Peptide on the maintenance of BMMSCs stemness showed that 5-Peptide promoted the expression of stemness genes SOX2, OCT4, and NANOG in BMMSCs; D shows an in vivo experiment using 4-week-old mice fed soft food + injected with 5-Peptide, which lasted for 8 weeks and ended after 9 weeks, and the mice were killed to obtain mandibular samples for analysis; EF shows gross photographs (E) showing that the height and length of the mandible (F) in the soft food + 5-Peptide injection group were higher than those in the soft food group, and close to those in the hard food group, with no statistical difference between the two; GH shows micro-CT analysis showing that soft food + 5-Peptide injection The cortical bone density (G), bone mineral density (BMD), bone volume fraction (BV / TV) and trabecular thickness (H) of the mandible in the ide group were higher than those in the soft food control group, and close to those in the hard food group, with no statistical difference between the two groups; IJ shows the results of modified Masson's trichrome staining (I) and Sirius red staining (J), which showed that the distribution of collagen fibers related to bone formation in the mandible of the soft food + 5-Peptide injection group was higher than that in the soft food group, and close to that in the hard food group; K shows the use of peptide chip array technology to detect the binding of 5-Peptide with CNR1, CNR2 and MC5R, showing that 5-Peptide has 4 binding sites with MC5R, There are 4 binding sites with CNR1 and 2 binding sites with CNR2; L shows that the protein molecular docking method is used to further verify the peptide chip array binding sites of 5-Peptide with CNR1, CNR2 and MC5R, showing that -Peptide binds to CNR2; by predicting the complexes of CNR1, CNR2, MCR5 and peptide EHERW, it is found that the pLDDT of the three proteins is generally high, reflecting the high quality of structural prediction of several proteins, poor conservation of the ends, mostly irregular curling, which is consistent with the current cryo-electron microscopy results. It is difficult to obtain the structure of this part, so the pLDDT value is also relatively low. In addition, since the three proteins are all GPCR proteins, it can be clearly found that the three proteins have 7 obvious high-quality peaks, which is closely related to the 7 transmembrane of GPCR. The linkers adjacent to the transmembrane region are all irregularly curled, so the pLDDT value is also relatively low.From the predicted pLDDT value of the peptide EHFRW, it is relatively low in the interaction prediction with the three proteins, and its value is generally below 40. After in-depth comparison of the 5 models of each system, it is found that in the prediction of the CNR2-EHFRW complex, its pLDDT value is relatively high compared with that of the other two proteins; therefore, it has a large interaction potential and needs to be further analyzed in combination with the predicted model; M shows the use of alkaline phosphatase activity (ALP) and Alizarin red staining (ARS) to detect the osteogenic differentiation ability of 5-Peptide after knocking out CNR2 in BMMSCs. The results show that 5-Peptide inhibits the ALP activity and mineralization ability after knocking out CNR2 in BMMSCs; N shows the use of RT-PCR to detect the expression of osteogenic genes in BMMSCs after knocking out CNR2 after 1 week (1W) and 2 weeks (2W) of osteogenic induction. The results show that 5-Peptide inhibits the expression of osteogenic-related transcription factors OCN, RUNX2 and OSX after knocking out CNR2 in BMMSCs;.

[0024] Figure 3 The in vivo experiment provided in the embodiment of the present invention shows that the growth and development of the mandible is inhibited by using the CNR2 gene knockout tool mouse; wherein, A shows that 4-week-old wild-type control mice (WT) and CNR2 knockout mice (CNR2- / -) were fed soft food and hard food, respectively, and the feeding lasted for 8 weeks. The mice were killed in the 9th week to obtain mandibular samples for analysis. The gross photos showed that the mandible of the CNR2- / - group mice was underdeveloped under different chewing force stimulations, which was close to that of the soft food group, with no statistical difference. The mandibles of the CNR2- / - group and the soft food group were lower than those of the hard food WT group; B showed the gross measurement of the height and length of the mandible of each group, showing that the height and length of the mandible of the CNR2- / - group mice were poorly developed under different chewing force stimulations. The mandibular height and length of the CNR2- / - group and the soft diet group were lower than those of the hard diet WT group. CD showed that the mandibular cortical bone density (C), bone mineral density (BMD), bone volume fraction (BV / TV) and trabecular thickness (C) of each group were further analyzed by micro-CT, showing that the mandibular cortical bone density and bone mineral density (BMD), bone volume fraction (BV / TV) and trabecular thickness of the CNR2- / - group mice were close to those of the soft diet group, and lower than those of the hard diet WT group. EF showed that the distribution of osteogenic collagen fibers in the mandibular bones of each group was detected by modified Masson's trichrome staining and Sirius red staining, showing that the distribution of osteogenic collagen fibers in the mandibular bones of the CNR2- / - group mice was close to that of the soft diet group, and lower than that of the hard diet WT group.

[0025] Figure 4The figure shows a schematic diagram of the 5-Peptide provided in the embodiment of the present invention regulating AKT / ERK1 / 2 / STAT3 to promote the osteogenic differentiation of MSCs by binding to CNR2 on the surface of BMMSCs. The changes of AKT / ERK1 / 2 / STAT3 signaling pathway in BMMSCs of WT group and CNR2- / - group under different chewing forces were detected by Western blot, showing that the phosphorylated AKT / ERK1 / 2 / STAT3 of BMMSCs of CNR2- / - group was close to that of BMMSCs of soft food group, and both were lower than those of WT group of hard food, and β-actin was used as an internal reference;

[0026] Figure 5-6 A schematic diagram showing that the common binding receptors of α-MSH and β-MSH provided by the embodiment of the present invention are MC1R, MC3R, MC4R and MC5R, the string protein interaction database is used to query the proteins interacting with α-MSH and β-MSH, and the common binding receptors of α-MSH and β-MSH are further screened out as MC1R, MC3R, MC4R and MC5R in combination with the reported literature;

[0027] Figure 7 The schematic diagram shows the prediction ability of the EHFRW amino acid sequence of 5-Peptide provided by the embodiment of the present invention and the polypeptide interaction in CNR1 and CNR2 proteins; through prediction, it is found that the prediction ability of the interaction between polypeptides is generally not high, among which the polypeptide (No. 151) from CNR1 has the best pLDDT of more than 75%. The pLDDT of polypeptides No. 81 and 82 of CNR1 can only reach about 60%, and No. 146 of CNR1 is even lower, only about 40%. For the conformation prediction of EHFRW, its pLDDT is the highest at about 50% (prediction of interaction with 146 and 151 of CNR1), among which the pLDDT of EHFRW predicted to interact with polypeptides No. 81 and No. 82 can only reach about 40%. The conformational pLDDT of peptides 190 and 202 of CNR2 is only about 50%, which predicts that this region may be a l0op region with poor conservation and greater flexibility. However, the pLDDT of peptide EHFRW is higher than that of CNR2 in a certain area. The interaction between the 5-peptide and the two peptides of CNR2 still needs to be further analyzed in combination with conformation.

[0028] Figure 8The schematic diagram of the binding conformation of the EHFRW amino acid sequence of 5-peptide provided by the embodiment of the present invention interacting with the two polypeptides of CNR2 is shown; according to the prediction, the interaction conformations of the two polypeptides of CNR2 and EHFRW show that multiple conformations have shown the result of the two polypeptides approaching each other. Only a few conformations do not show the interaction phenomenon, which is similar to the interaction between this protein and FHFRW. Combined with the performance of nDDT, the possibility of the interaction between the two polypeptide segments of CNR2 and EHFRW is very high, and there is a certain degree of electrostatic complementarity;

[0029] Fig. 9 The binding site of the EHFRW amino acid sequence of the 5-peptide provided by the implementation example of the present invention and the interaction conformation of CNR2 is shown; from the interaction analysis between CNR2 and EHERW, the polypeptide Trp5 and CNR2 have more interactions, among which there is a sigma-pi interaction with Cys134, and a Cation-pi interaction is formed with Arg302, and the terminal carboxyl group of the polypeptide Trp5 forms an ionic interaction with Arg242. Phe3 of the polypeptide can form a Cation-pi interaction with Arg236, and the side chain carboxyl group of Glu1 forms a strong ionic interaction with Lys142; from these interactions, EHFRW and CNR5 have more electrostatic complementary interactions, and are in the pocket of CNR2. Based on the above analysis, it is more likely that EHFRW interacts with CNR2;

[0030] Fig. 10A The present invention provides an example of implementation Figure 2 H further supplements the quantitative statistical analysis of trabecular bone thickness; Fig. 10B The present invention provides an example of implementation Figure 2 H further supplements the quantitative statistical analysis of bone density; Fig. 10C The present invention provides an example of implementation Figure 2 H further supplements the quantitative statistical analysis of bone volume fraction. DETAILED DESCRIPTION

[0031] The present invention discloses neuropeptides and their application in promoting the growth and development of mandibular bones. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] In the present invention, the applicant artificially synthesized 9 neuropeptides, namely α-MSH, β-MSH, γ-MSH, ACTH, γ-LPH, β-endorphin, β-LPH, CLIP, and met-enkephalin, and stimulated bone marrow mesenchymal stem cells (BMMSCs) with appropriate concentrations, respectively, showing that α-MSH and β-MSH can significantly promote the osteogenic differentiation function of BMMSCs. Further, CNR1, CNR2 and MC5R were knocked down in BMMSCs through in vitro cell experiments, and the results showed that α-MSH and β-MSH inhibited the osteogenic differentiation function of BMMSCs. In order to explore why α-MSH and β-MSH can regulate the function of BMMSCs through common receptors, bioinformatics analysis found that the two have the same 5 amino acid sequences (EHFRW), and then artificially synthesized 5-Peptide, referred to as a new pentapeptide. Through peptide array technology and protein binding site analysis, it was found that the receptor bound by 5-Peptide is CNR2. In vitro cell experiments found that 5-Peptide inhibited the osteogenic differentiation function of BMMSCs by knocking down CNR2 in BMMSCs. Further in vivo animal experiments found that local injection of 5-Peptide can promote the growth and development of mouse mandibles, and CNR2 gene knockout mice were used to further verify this result. In-depth study of the molecular mechanism of action showed that 5-Peptide regulates AKT / ERK1 / 2 / STAT3 by binding to CNR2 on the surface of BMMSCs to promote the osteogenic differentiation function of MSCs, thereby promoting the growth and development of mandibles and bone formation.

[0033] At present, the main clinical treatment for micrognathia and malocclusion is orthodontic treatment by wearing appliances and orthognathic surgery, but there are problems such as high cost, long treatment cycle and large surgical trauma. The present invention verifies the function of the new 5-Peptide in promoting the osteogenic differentiation of MSCs and thus promoting the growth and development of the mandible through in vivo and in vitro experiments, and explains the molecular mechanism of 5-Peptide regulating the AKT / ERK1 / 2 / STAT3 signaling pathway by binding to CNR2. By using 5-Peptide to treat oral diseases such as micrognathia and malocclusion caused by insufficient mandibular development, a clinical application basis is provided.

[0034] In the neuropeptide provided by the present invention and its application in promoting the growth and development of mandibular bones, the raw materials and reagents used can be purchased from the market.

[0035] Manufacturer of soft and hard feed: Sibeifu (Beijing) Biotechnology Co., Ltd.

[0036] Item No.: Hard food, SPF-F02-001; Soft food: SPF-F02-003

[0037] The formula of soft and hard feeds is exactly the same: crude protein, crude fat, crude fiber, crude ash, calcium, and total phosphorus.

[0038] Primary culture medium: α-MEM medium (Invitrogen 2276722); 5% high-quality fetal bovine serum (Invitrogen A5256701); 100 μ / mL double antibody (streptomycin, penicillin) (Invitrogen 15070067); L-glutamine (Invitrogen A2916801).

[0039] The mineralization induction fluid was purchased from Saiye Company, with the catalog number (HUXXC-90021).

[0040] Alkaline phosphatase activity kit: (Invitrogen EEA002).

[0041] The present invention will be further described below in conjunction with embodiments:

[0042] Example 1

[0043] (1) Animal experiments:

[0044] The animal ethics committee of Beijing Stomatological Hospital, Capital Medical University was followed. Four-week-old male mice were fed with soft and hard diets with the same nutritional composition for 8 weeks. The mice were killed by cervical dislocation at the 9th week, and the mandibular specimens were taken for photographing under a microscope, and the mandibular height and length were measured, and micro-CT scanning was performed for analysis.

[0045] (2) Cell culture:

[0046] Male C57BL / 6 mice aged 6-8 weeks were killed by cervical dislocation and placed in a container containing 75% alcohol for 1 minute. The femur of the mouse was removed with sterile instruments in the clean bench, and repeatedly rinsed with PBS buffer plus PS. The femoral bone marrow was flushed out by aspirating the primary culture medium with a 1mL syringe, and repeatedly rinsed until the bone marrow cavity turned white. The cells were placed in a 15mL centrifuge tube, and the cells were collected and centrifuged at 1100 rpm for 6 minutes. The prepared primary culture medium was used to resuspend the cells into a single cell suspension, and the cells were inoculated in a 10cm cell culture dish and cultured at 37°C and 5% CO2. The medium was changed every 3 days. The cell growth was observed under a microscope. When the cells grew to 80% confluence, they were digested and passaged at a ratio of 1:2 using 0.25% trypsin.

[0047] (3) Viral transfection:

[0048] CNR1, CNR2, MC1R, MC3R, MC4R and MC5R knockdown lentiviruses were purchased from Shanghai Gene Gene Co., Ltd. The knockdown group (CNR1sh, CNR2sh, MC1Rsh, MC3Rsh, MC4Rsh, MC5Rsh) and the control group (Consh empty vector) lentiviruses were transfected into BMMSCs for 12 hours under the action of 6μg / ml polybrene. After 48 hours of transfection, 1ug / ml puromycin was used to select for 3 days to obtain stably transfected cells. The knockdown efficiency was detected at the mRNA level by RT-PCR, and the knockdown group (CNR1sh, CNR2sh, MC1Rsh, MC3Rsh, MC4Rsh, MC5Rsh) and the control group (Consh) cell lines with stable transfection were obtained.

[0049] (4) Alkaline phosphatase, Alizarin red staining and RT-PCR:

[0050] Alkaline phosphatase activity was determined using mineralization induction solution + knockdown

[0051] BMMSCs were co-induced with CNR1sh / CNR2sh / MC1Rsh / MC3Rsh / MC4Rsh / MC5Rsh for 3 days, and alkaline phosphatase activity was detected according to the instructions of the alkaline phosphatase activity detection kit. Alizarin red staining was used to detect the in vitro mineralization ability of cells. After 2 weeks of induction, BMMSCs were fixed with 70% ethanol and stained with 2% alizarin red. The calcium ion concentration was quantitatively determined by fading alizarin red with 10% cetylpyridinium chloride dissolved in sodium phosphate at room temperature for 30 minutes, and the calcium ion concentration was measured by measuring the absorbance at 562nm and converted using a standard curve. In the RT-PCR experiment, total cell RNA was extracted with Trizol, and cDNA was obtained by reverse transcription. Real-time PCR was used to quantitatively measure the expression of osteogenic / odontogenic transcription factors OSX, OCN, and RUNX2.

[0052] RT-PCR method

[0053] The primer sequences are as follows:

[0054] Table 1

[0055] Gene name Sequence number Primer sequence (5'-3') GAPDH-F SEQ ID No.225 GAAGATATGGGCACAGGGGA GAPDH-R SEQ ID No.226 CAAGAAGATGCGGCTGTCTC SOX2-F SEQ ID No.227 CGGCACAGATGCAACCGAT SOX2-R SEQ ID No.228 CCGTTCATGTAGGTCTGCG OCT4-F SEQ ID No.229 AGAGGATCACCTTGGGGTACA OCT4-R SEQ ID No.230 CGAAGCGACAGATGGTGGTC NANOG-F SEQ ID No.231 TCTTCCTGGTCCCCACAGTTT NANOG-R SEQ ID No.232 GCAAGAATAGTTCTCGGGATGAA OCN-F SEQ ID No.233 CAGACAAGTCCCACACAGCA OCN-R SEQ ID No.234 CTTGGCATCTGTGAGGTCAG OSX-F SEQ ID No.235 TCCCTGGATATGACTCATCCCT OSX-R SEQ ID No.236 CCAAGGAGTAGGTGTGTGTTGCC RUNX2-F SEQ ID No.237 GACTGTGGTTACCGTCATGGC RUNX2-R SEQ ID No.238 ACTTGGTTTTTCATAACAGCGGA

[0056] RNA extraction

[0057] 1) Add 1 mL of Trizol to each large dish of cells and let it rest for 5 minutes, then transfer to a 1.5 mL EP tube;

[0058] 2) Add 200uL chloroform to every 1mL Trizol, vortex 15S, and incubate at room temperature for 5min;

[0059] 3) Centrifuge at 14000 rpm for 15 min at 4°C;

[0060] 4) Transfer the supernatant water phase to a new EP tube, add an equal volume of isopropanol, mix by inversion (vortex), and incubate at room temperature for 10 min;

[0061] 5) Centrifuge at 14000 rpm for 10 min at 4°C;

[0062] 6) Discard the supernatant, wash once with 1 mL of 75% ethanol, mix by inversion, and centrifuge at 9500 rpm for 10 minutes at 4°C;

[0063] 7) Discard the supernatant and dry at room temperature for about 10-20 minutes;

[0064] 8) Dissolve in dd H2O, 20-40 μL;

[0065] 9) Measure RNA concentration. First calibrate the instrument with 1 μL RNase free water. An ideal value is 260 / 280 between 1.8 and 2.0.

[0066] Reverse transcription PCR (Takara kit)

[0067] Genomic DNA removal reaction (10 μL)

[0068] 1) 5X DNA Eraser Buffer 2μL

[0069] 2) gDNAErase 1 μL

[0070] 3)RNase Free dd H2O up to 10μL

[0071] 4) Total RNA XμL

[0072] Step 1) and step 2) can be mixed to prepare several total RNA mixes; put on the reverse transcription machine for about 5 minutes. Reverse transcription reaction (10μL) needs to be performed on ice

[0073] 1) 5X primerScriptBuffer 4μL

[0074] 2)primer ScriptEnzyme MixI 1μL

[0075] 3) RT primer mix 1 μL

[0076] 4) RNase free water 4μL

[0077] 5) 10 μL of the reaction solution after the first step on the machine

[0078] Get on the machine, about 30 minutes

[0079] 6) After the reaction, cDNA is stored at -20℃

[0080] Realtime PCR

[0081] Prepare Realtime PCR reaction (20 μL system)

[0082]

[0083] The real-time PCR reaction system is as follows:

[0084] Table 2

[0085]

[0086] Sirius red staining (G1470, Solebol):

[0087] 1) The tissue was fixed in 10% formalin and routinely dehydrated and embedded;

[0088] 2) The slices were 6 μm thick and routinely dewaxed to water;

[0089] 3) Sirius red dye drop dyeing;

[0090] 4) Rinse with running water to remove the dye on the surface of the slice;

[0091] 5) Mayer's hematoxylin staining solution was used to stain the cell nucleus;

[0092] 6) Rinse with running water for 10 minutes;

[0093] 7) Conventional dehydration and transparency, sealing with neutral gum.

[0094] Staining results:

[0095] Type L collagen fibers: tightly arranged, showing strong birefringence, and appearing as yellow or red fibers.

[0096] Type II collagen fibers: show weak birefringence and are distributed in a loose network of various colors.

[0097] Type III collagen fibers: show weak birefringence and appear as green thin fibers.

[0098] Type LV collagen fibers: basement membrane showing weak birefringence, pale yellow.

[0099] According to the staining results, the collagen fibers of the mandible in the soft diet + 5-Peptide group and the hard diet group were yellow or red, indicating an increase in the collagen that forms the bone; while the collagen fibers in the soft diet group were green, indicating fewer collagen fibers.

[0100] Modified Masson's trichrome staining (G1346, Solebro):

[0101] 1) The tissue was fixed in 10% formalin and routinely dehydrated and embedded;

[0102] 2) The slices were 4 μm thick and routinely dewaxed to water;

[0103] 3) The slices are immersed in the mordant solution and left at room temperature overnight or placed in a 57-60°C incubator for 1 hour for mordant dyeing, and then rinsed with running water for 10 minutes;

[0104] 4) Dye with lapis lazuli blue dye for 2-3 minutes and then rinse twice, 10-15 seconds each time;

[0105] 5) Dye with Mayer's hematoxylin solution for 2-3 minutes and wash twice with distilled water, each time for 10-15 seconds;

[0106] 6) Differentiate in acidic differentiation solution for a few seconds, wash with water to terminate differentiation, and rinse with distilled water for 10 minutes;

[0107] 7) Dye with Ponceau fuchsin solution for 10 min, and wash twice with distilled water, 10-15 s each time;

[0108] 8) Treat with phosphomolybdic acid solution for about 10 minutes;

[0109] 9) Pour off the supernatant, add aniline blue staining solution directly to the slices without washing them, and stain for 5 minutes;

[0110] 10) After washing away the aniline blue solution with a weak acid solution, continue to drip weak acid working solution to cover the slices for 2 minutes;

[0111] 11) Dehydration with 95% ethanol for 30 seconds, dehydration with anhydrous ethanol twice, the first time for 30 seconds, the second time for 1 minute;

[0112] 12) Transparent the slides twice with xylene, each time for 1-2 min, and seal the slides with neutral gum.

[0113] Staining results:

[0114] Collagen fiber: blue

[0115] Muscle fibers, cytoplasm, cellulose, keratin, and red blood cells: varying degrees of red

[0116] Nucleus: blue-brown

[0117] According to the results of section staining: the collagen fibers of the mandible in the soft diet + 5-Peptide group and the hard diet group were blue, indicating an increase in the collagen that forms the bone; while the collagen fibers in the soft diet group were red, indicating fewer collagen fibers.

[0118] (5) Western blot analysis:

[0119] The cells were lysed with RIPA lysis buffer, and the protein concentration was determined by Bradford method. The samples were separated by 10% SDS polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer membrane device. The transferred PVDF membrane was blocked with 5% skim milk powder for 1 hour and placed on a shaker for shaking. The primary antibody was incubated and shaken at 4°C overnight. The filter was placed in the secondary antibody diluted in 5% skim milk powder, and the immune complex was incubated with rabbit or mouse immunoglobulin G antibody and visualized with chemiluminescent substrate reagent, and imaged in the BIO-RAD chemiluminescence imaging system. The expression of AKT, P-AKT, ERK1 / 2, P-ERK1 / 2, Stat3, and P-Stat3 was measured.

[0120] Primary antibodies: anti-AKT anti-p-AKT (1:1000Cell Signaling Technology) anti-ERK1 / 2 anti-p-ERK1 / 2 (1:1000Cell Signaling Technology) anti-stat3 anti-p-stat3 (1:1000Cell Signaling Technology), β-actin (1:2000Abcam).

[0121] Secondary antibodies: anti-mouse IgG (1:2000 Abcam), anti-rabbit IgG (1:5000 Abcam).

[0122] (6) POMC precursor peptide can synthesize different peptides, including α-MSH and β-MSH, in different tissues and environments. Amino acid sequences of α-MSH, β-MSH and 5-peptide: The amino acid sequences of α-MSH and β-MSH were searched by informatics analysis technology. α-MSH, β-MSH peptides and 5-peptide were synthesized by Hubei Qiangyao Biotechnology Co., Ltd.

[0123] α-MSH sequence (NC): SYSMEHFRWGKPV (as shown in SEQ ID No. 239);

[0124] β-MSH sequence (NC): DDGPYRVEHFRWSNPPKD (as shown in SEQ ID No. 240);

[0125] 5-Peptide sequence (NC): EHFRW (as shown in SEQ ID No. 241).

[0126] (7) Peptide chip array technology:

[0127] According to MC5R_MOUSE 325 amino acids, CNR1_MOUSE 473 amino acids, CNR2_MOUSE 347 amino acids, one chip is divided into 224 strips every 15AA and one chip every 5AA, of which 1-63 peptide spots are from MC5R_MOUSE protein, 64-156 peptide spots are from CNR1_MOUSE protein, and 157-224 peptide spots are from CNR2_MOUSE protein.

[0128] Two array chips were synthesized. One array was used for interaction study with biotin-labeled 5-Peptide (EHFRW); the other array was used for negative control experiments. The peptide array chip SOP was prepared and synthesized. The peptide chip was hybridized with biotin EHFRW peptide and imaged with FX-7 digital imaging analyzer. 5-Peptide (EHFRW) was hybridized with the peptide array chip derived from MC5R_MOUSE protein, and 4 obvious color spots were found on the peptide chip. 5-Peptide (EHFRW) was hybridized with the peptide array chip derived from CNR1_MOUSE protein, and 4 obvious color spots were found on the peptide chip. 5-Peptide (EHFRW) was hybridized with the peptide array chip derived from CNR1_MOUSE protein, and 2 obvious color spots were found on the peptide chip.

[0129] The corresponding relationship between the color spots on the peptide chip and the peptide array sequence is: Figure 2 K-list.

[0130] The peptide array sequence information is shown in Table 3:

[0131] Table 3

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Example 2 ELISA (α-MSH β-MSH) The content of α-MSH in the mandibular sample was determined using an ELISA kit (ml037968, enzyme-linked). The specific experimental steps are as follows:

[0139] 1) Mouse mandibular specimen: After cutting the specimen, weigh 0.1g of mandibular bone. Add 200μL of PBS, pH7.4. Quickly freeze with liquid nitrogen for later use. After the specimen is thawed, keep the temperature at 2-8℃. Add 100μL of PBS (PH7.4) and homogenize the specimen by hand or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. Aliquot one portion for testing and freeze the rest for later use.

[0140] 2) Adding standard samples: set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well;

[0141] 3) Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μl of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0142] 4) Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank well.

[0143] 5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0144] 6) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use.

[0145] 7) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0146] 8) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and color development at 37°C in the dark for 15 minutes.

[0147] 9) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0148] 10) Measurement: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0149] Table 5

[0150]

[0151] Statistical results: Figure 1 As shown in F.

[0152] Example 3 The content of β-MSH in the mandibular bone sample was determined using an ELISA kit (ml037968-J, enzyme-linked). The specific experimental steps are as follows:

[0153] 1) Mouse mandibular specimen: After cutting the specimen, weigh 0.1g of mandibular bone. Add 200μL of PBS, pH7.4. Quickly freeze with liquid nitrogen for later use. After the specimen is thawed, keep the temperature at 2-8℃. Add 100μL of PBS (PH7.4) and homogenize the specimen by hand or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. Aliquot one portion for testing and freeze the rest for later use.

[0154] 2) Adding standard samples: set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well;

[0155] 3) Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μl of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0156] 4) Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank well.

[0157] 5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0158] 6) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use.

[0159] 7) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0160] 8) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and color development at 37°C in the dark for 15 minutes.

[0161] 9) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0162] 10) Measurement: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0163] Table 6

[0164]

[0165] Statistical results: Figure 1 As shown in G.

[0166] Example 4

[0167] Bioinformatics analysis revealed that α-MSH and β-MSH co-bind with MC1R, MC3R, MC4R and MC5R

[0168] Bioinformatics method: The string protein interaction database was used to query proteins that interact with α-MSH and β-MSH. The results are as follows: Figure 5 , Figure 6 shown.

[0169] The common binding receptors of α-MSH and β-MSH were screened as MC1R, MC3R, MC4R and MC5R.

[0170] Example 5

[0171] Experimental steps: Detect the degree of binding of 5-Peptide to the three receptor proteins CNR1CNR2 and MC5R. First, query the amino acid sequences of the three proteins CNR1CNR2 and MC5R of mice.

[0172] CNR1:>sp|P47746|CNR1_MOUSE Cannabinoid receptor 1OS=Mus musculus OX=10090GN=Cnr1 PE=1SV=1 (as shown in SEQ ID No.242)

[0173] MKSILDGLADTTFRTITTDLLYVGSNDIQYEDIKGDMASKLGYFPQKFPLTSFRGSPFQEKMTAGDNSPLVPAGDTTNITEFYNKSLSSFKENEDNIQCGENFMDMECFMILNPSQQLAIAVLSLTLGTFTVLENLLVLCVILHSRSLRCRPSYHFIGSLAVADLLGSVIFVYSFVDFHVFHRKDSPNVFLFKLGGVTASFTASVGSLFLTAIDRYISIHRPLAYKRIVTRPKAVVAFCLMWTIAIVIAVLPLLGWNCKKLQSVCSDIFPLIDETYLMFWIGVTSVLLLFIVYAYMYILWKAHSHAVRMIQRGTQKSIIIHTSEDGKVQVTRPDQARMDIRLAKTLVLILVVLIICWGPLLAIMVYDVFGKMNKLIKTVFAFCSMLCLLNSTVNPIIYALRSKDLRHAFRSMFPSCEGTAQPLDNSMGDSDCLHKHANNTASMHRAAESCIKSTVKIAKVTMSVSTDTSAEAL

[0174] CNR2: >sp|P47936|CNR2_MOUSE Cannabinoid receptor 2 OS=Mus musculus OX=10090 GN=Cnr2 PE=1 SV=1 (as shown in SEQ ID No. 243)

[0175] MEGCRETEVTNGSNGGLEFNPMKEYMILSSGQQIAVAVLCTLMGLLSALENMAVLYIILSSRRLRRKPSYLFISSLAGADFLASVIFACNFVIFHVFHGVDSNAIFLLKIGSVTMTFTASVGSLLLTAVDRYLCLCYPPTYKALVTRGRALVALCVMWVLSALISYLPLMGWTCCPSPCSELFPLIPNDYLLGWLLFIAILFSGIIYTYGYVLWKAHRHVATLAEHQDRQVPGIARMRLDVRLAKTLGLVLAVLLICWFPALALMGHSLVTTLSDQVKEAFAFCSMLCLVNSMVNPIIYALRSGEIRSAAQHCLIGWKKYLQGLGPEGKEEGPRSSVTETEADVKTT

[0176] MC5R:>sp|P41149|MC5R_MOUSE Melanocortinreceptor 5OS=Mus musculus OX=10090GN=Mc5r PE=2SV=1 (as shown in SEQ ID No.244)

[0177] MNSSSTLTVLNLTLNASEDGILGSNVKNKSLACEEMGIAVEVFLTLGLVSLLENILVIGAIVKNKNLHSPMYFFVGSLAVADMLVSMSNAWETVTIYLLNNKHLVIADTFVRHIDNVFDSMICISVVASMCSLLAIAVDRYITIFYALRYHHIMTARRSGVI IACIWTFCISCGIVFIIYYESKYVIICLISMFFTMLFFMVSLYIHMFLLARNHVKRIAASPRYNSVRQRTSMKGAITLTMLLGIFIVCWSPFFLHLILMISCPQNVYCSCFMSYFNMYLILIMCNSVIDPLIYALRSQEMRRTFKEIVCCHGFRRPCRLLGGY

[0178] 5-Peptide: EHFRW (as shown in SEQ ID No. 241)

[0179] After the protein is prepared, active site prediction is required to find the approximate docking pocket location; a docking box is established with the center of the predicted pocket location as the coordinate origin; after the above steps are determined, the final docking parameters need to be set and the docking accuracy needs to be selected; the structure file and parameter file need to be checked, and the server resources are called to start the docking program.

[0180] The results are as follows:

[0181] Effect Example 1

[0182] (1) Cell culture:

[0183] Male C57BL / 6 mice aged 6-8 weeks were killed by cervical dislocation and placed in a container containing 75% alcohol for 1 minute. The femur of the mouse was removed with sterile instruments in the clean bench, and repeatedly rinsed with PBS buffer plus PS. The femoral bone marrow was flushed out by aspirating the primary culture medium with a 1mL syringe, and repeatedly rinsed until the bone marrow cavity turned white. The cells were placed in a 15mL centrifuge tube, and the cells were collected and centrifuged at 1100 rpm for 6 minutes. The prepared primary culture medium was used to resuspend the cells into a single cell suspension, and the cells were inoculated in a 10cm cell culture dish and cultured at 37°C and 5% CO2. The medium was changed every 3 days. The cell growth was observed under a microscope. When the cells grew to 80% confluence, they were digested and passaged at a ratio of 1:2 using 0.25% trypsin.

[0184] (2) Virus transfection:

[0185] CNR1, CNR2, MC1R, MC3R, MC4R and MC5R knockdown lentiviruses were purchased from Shanghai Gene Gene Co., Ltd. The knockdown group (CNR1sh, CNR2sh, MC1Rsh, MC3Rsh, MC4Rsh, MC5Rsh) and the control group (Consh empty vector) lentiviruses were transfected into BMMSCs for 12 hours under the action of 6μg / ml polybrene. After 48 hours of transfection, 1ug / ml puromycin was used to select for 3 days to obtain stably transfected cells. The knockdown efficiency was detected at the mRNA level by RT-PCR, and the knockdown group (CNR1sh, CNR2sh, MC1Rsh, MC3Rsh, MC4Rsh, MC5Rsh) and the control group (Consh) cell lines with stable transfection were obtained.

[0186] (3) Alkaline phosphatase, Alizarin red staining and RT-PCR:

[0187] 1 μg / mL of α-MSH, β-MSH, γ-MSH, ACTH, γ-LPH, β-endorphin, β-LPH, CLIP, and met-enkephalin were used to induce BMMSCs in CNR1sh / CNR2sh / MC1Rsh / MC3Rsh / MC4Rsh / MC5Rsh for 3 days. Alkaline phosphatase activity was detected according to the instructions of the alkaline phosphatase activity detection kit. Alizarin red staining was used to detect the in vitro mineralization ability of cells. After 2 weeks of induction, BMMSCs were fixed with 70% ethanol and stained with 2% alizarin red.

[0188] (4) ELISA (α-MSH β-MSH) The α-MSH content in the mandibular samples was determined using an ELISA kit (ml037968, enzyme-linked immunosorbent assay). The specific experimental steps are as follows:

[0189] 1) Mouse mandibular specimen: After cutting the specimen, weigh 0.1g of mandibular bone. Add 200μL of PBS, pH7.4. Quickly freeze with liquid nitrogen for later use. After the specimen is thawed, keep the temperature at 2-8℃. Add 100μL of PBS (PH7.4) and homogenize the specimen by hand or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. Aliquot one portion for testing and freeze the rest for later use.

[0190] 2) Adding standard samples: set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well;

[0191] 3) Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μl of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0192] 4) Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank well.

[0193] 5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0194] 6) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use.

[0195] 7) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0196] 8) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and color development at 37°C in the dark for 15 minutes.

[0197] 9) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0198] 10) Measurement: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0199] Table 5

[0200]

[0201] Statistical results: Figure 1 As shown in F.

[0202] (5) The ELISA kit (ml037968-J, enzyme-linked immunosorbent assay) was used to determine the content of β-MSH in the mandibular bone samples. The specific experimental steps are as follows:

[0203] 1) Mouse mandibular specimen: After cutting the specimen, weigh 0.1g of mandibular bone. Add 200μL of PBS, pH7.4. Quickly freeze with liquid nitrogen for later use. After the specimen is thawed, keep the temperature at 2-8℃. Add 100μL of PBS (PH7.4) and homogenize the specimen by hand or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. Aliquot one portion for testing and freeze the rest for later use.

[0204] 2) Adding standard samples: set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well;

[0205] 3) Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μl of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0206] 4) Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank well.

[0207] 5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0208] 6) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use.

[0209] 7) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0210] 8) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and color development at 37°C in the dark for 15 minutes.

[0211] 9) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0212] 10) Measurement: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0213] Table 6

[0214]

[0215] Statistical results: Figure 1As shown in G.

[0216] Figure 1 Schematic diagram showing the effects of different neuropeptides produced by POMC provided in the embodiments of the present invention on the osteogenic differentiation function of BMMSCs and possible receptors; wherein, A shows the expression of alkaline phosphatase activity (ALP) after α-MSH, β-MSH, γ-MSH, ACTH, γ-LPH, β-endorphin, β-LPH, CLIP, and met-enkephalin acted on BMMSCs, indicating that α-MSH and β-MSH can significantly promote the alkaline phosphatase activity of BMMSCs; B shows the detection of α-MSH, β-MSH by alizarin red staining (ARS) -MSH, γ-MSH, ACTH, γ-LPH, β-endorphin, CLIP, and met-enkephalin act on the osteogenic mineralization ability of BMMSCs, showing that α-MSH and β-MSH can significantly promote the formation of calcium nodules in BMMSCs; C shows the knockdown efficiency of CNR1 and CNR2 in BMMSCs detected by RT-PCR; D shows the expression of alkaline phosphatase activity (ALP) in BMMSCs after α-MSH and β-MSH act on CNR1 and CNR2, showing that knockdown of CNR1 and CNR 2, α-MSH and β-MSH inhibited the alkaline phosphatase activity of BMMSCs; E shows the effect of α-MSH and β-MSH on the osteogenic mineralization ability of BMMSCs after knocking down CNR1 and CNR2, which showed that α-MSH and β-MSH inhibited the calcium nodule formation of BMMSCs after knocking down CNR1 and CNR2; FG shows the ELISA detection of the contents of α-MSH (F) and β-MSH (G) in mouse mandibular samples under different chewing forces; H shows the bioinformatics analysis found that α-MSH and β-MSH co-binding The combined receptors are MC1R, MC3R, MC4R and MC5R. The knockdown efficiency of MC1R, MC3R, MC4R and MC5R in BMMSCs was detected by RT-PCR, and the knockdown efficiency was higher than 50%; I shows the expression of alkaline phosphatase activity (ALP) in BMMSCs after α-MSH and β-MSH knocked down MC1R, MC3R, MC4R and MC5R, showing that α-MSH and β-MSH inhibited the alkaline phosphatase activity of BMMSCs after knocking down MC5R, indicating that MC5R may be a receptor bound to α-MSH and β-MSH.

[0217] Effect Example 2

[0218] (1) Alkaline phosphatase, Alizarin red staining and RT-PCR:

[0219] α-MSH and β-MSH synthesized 5-Peptide through their common amino acid sequence (EHERW). BMMSCs were induced with 1 μg / mL 5-Peptide for 3 days. Alkaline phosphatase activity was detected according to the instructions of the alkaline phosphatase activity detection kit. Alizarin red staining was used to detect the in vitro mineralization ability of cells. After 2 weeks of induction, BMMSCs were fixed with 70% ethanol and stained with 2% alizarin red.

[0220] (2) RT-PCR method

[0221] The primer sequences are as follows:

[0222] Table 1

[0223] Gene name Sequence number Primer sequence (5'-3') GAPDH-F SEQ ID No.225 GAAGATATGGGCACAGGGGA GAPDH-R SEQ ID No.226 CAAGAAGATGCGGCTGTCTC SOX2-F SEQ ID No.227 CGGCACAGATGCAACCGAT SOX2-R SEQ ID No.228 CCGTTCATGTAGGTCTGCG OCT4-F SEQ ID No.229 AGAGGATCACCTTGGGGTACA OCT4-R SEQ ID No.230 CGAAGCGACAGATGGTGGTC NANOG-F SEQ ID No.231 TCTTCCTGGTCCCCACAGTTT NANOG-R SEQ ID No.232 GCAAGAATAGTTCTCGGGATGAA

[0224] RNA extraction

[0225] 1) Add 1 mL of Trizol to each large dish of cells and let it rest for 5 minutes, then transfer to a 1.5 mL EP tube;

[0226] 2) Add 200uL chloroform to every 1mL Trizol, vortex 15S, and incubate at room temperature for 5min;

[0227] 3) Centrifuge at 14000 rpm for 15 min at 4°C;

[0228] 4) Transfer the supernatant water phase to a new EP tube, add an equal volume of isopropanol, mix by inversion (vortex), and incubate at room temperature for 10 min;

[0229] 5) Centrifuge at 14000 rpm for 10 min at 4°C;

[0230] 6) Discard the supernatant, wash once with 1 mL of 75% ethanol, mix by inversion, and centrifuge at 9500 rpm for 10 minutes at 4°C;

[0231] 7) Discard the supernatant and dry at room temperature for about 10-20 minutes;

[0232] 8) Dissolve in dd H2O, 20-40 μL;

[0233] 9) Measure RNA concentration. First calibrate the instrument with 1 μL RNase free water. An ideal value is 260 / 280 between 1.8 and 2.0.

[0234] Reverse transcription PCR (Takara kit)

[0235] Genomic DNA removal reaction (10 μL)

[0236] 1) 5X DNA Eraser Buffer 2μL

[0237] 2) gDNA Erase 1 μL

[0238] 3)RNase Free dd H2O up to 10μL

[0239] 4) Total RNA XμL

[0240] Step 1) and step 2) can be used to mix several total RNAs and prepare several mixes; put them on the reverse transcription instrument for about 5 minutes.

[0241] Reverse transcription reaction (10 μL), to be performed on ice

[0242] 1) 5X primerScript Buffer 4μL

[0243] 2) Primer Script Enzyme MixI 1μL

[0244] 3) RT primer mix 1 μL

[0245] 4) RNase free water 4μL

[0246] 5) 10 μL of the reaction solution after the first step on the machine

[0247] Get on the machine, about 30 minutes

[0248] 6) After the reaction, cDNA is stored at -20℃

[0249] Realtime PCR

[0250] Prepare Realtime PCR reaction (20 μL system)

[0251] 1) SYBR premix TaqII (2X) 10 μL

[0252] 2) PCR Forward primer 0.8 μL

[0253] PCR Reverse primer 0.8μL

[0254] 3) cDNA 1 μL

[0255] 4) dd H2O 7.4μL

[0256] The real-time PCR reaction system is as follows:

[0257] Table 2

[0258]

[0259] (3) In vivo experiments, mice aged 4 weeks were fed a soft diet and injected with 1 μg / mL 5-Peptide locally into the mandible for 8 weeks. The mice were killed at the 9th week and the mandible samples were analyzed. The mandible specimens were photographed under a microscope, the mandibular height and length were measured, and micro-CT scanning was performed for analysis.

[0260] (4) Sirius red staining (G1470, Solebol):

[0261] 1) The tissue was fixed in 10% formalin and routinely dehydrated and embedded;

[0262] 2) The slices were 6 μm thick and routinely dewaxed to water;

[0263] 3) Sirius red dye drop dyeing;

[0264] 4) Rinse with running water to remove the dye on the surface of the slice;

[0265] 5) Mayer's hematoxylin staining solution was used to stain the cell nucleus;

[0266] 6) Rinse with running water for 10 minutes;

[0267] 7) Conventional dehydration and transparency, sealing with neutral gum.

[0268] Staining results:

[0269] Type L collagen fibers: tightly arranged, showing strong birefringence, and appearing as yellow or red fibers.

[0270] Type II collagen fibers: show weak birefringence and are distributed in a loose network of various colors.

[0271] Type III collagen fibers: show weak birefringence and appear as green thin fibers.

[0272] Type LV collagen fibers: basement membrane showing weak birefringence, pale yellow.

[0273] According to the staining results, the collagen fibers of the mandible in the soft diet + 5-Peptide group and the hard diet group were yellow or red, indicating an increase in the collagen that forms the bone; while the collagen fibers in the soft diet group were green, indicating fewer collagen fibers.

[0274] (5) Modified Masson's trichrome staining (G1346, Solebol):

[0275] 1) The tissue was fixed in 10% formalin and routinely dehydrated and embedded;

[0276] 2) The slices were 4 μm thick and routinely dewaxed to water;

[0277] 3) The slices are immersed in the mordant solution and left at room temperature overnight or placed in a 57-60°C incubator for 1 hour for mordant dyeing, and then rinsed with running water for 10 minutes;

[0278] 4) Dye with lapis lazuli blue dye for 2-3 minutes and then rinse twice, 10-15 seconds each time;

[0279] 5) Dye with Mayer's hematoxylin solution for 2-3 minutes and wash twice with distilled water, each time for 10-15 seconds;

[0280] 6) Differentiate in acidic differentiation solution for a few seconds, wash with water to terminate differentiation, and rinse with distilled water for 10 minutes;

[0281] 7) Dye with Ponceau fuchsin solution for 10 min, and wash twice with distilled water, 10-15 s each time;

[0282] 8) Treat with phosphomolybdic acid solution for about 10 minutes;

[0283] 9) Pour off the supernatant, add aniline blue staining solution directly to the slices without washing them, and stain for 5 minutes;

[0284] 10) After washing away the aniline blue solution with a weak acid solution, continue to drip weak acid working solution to cover the slices for 2 minutes;

[0285] 11) Dehydration with 95% ethanol for 30 seconds, dehydration with anhydrous ethanol twice, the first time for 30 seconds, the second time for 1 minute;

[0286] 12) Transparent the slides twice with xylene, each time for 1-2 min, and seal the slides with neutral gum.

[0287] Staining results:

[0288] Collagen fiber: blue

[0289] Muscle fibers, cytoplasm, cellulose, keratin, and red blood cells: varying degrees of red

[0290] Nucleus: blue-brown

[0291] According to the results of section staining: the collagen fibers of the mandible in the soft diet + 5-Peptide group and the hard diet group were blue, indicating an increase in the collagen that forms the bone; while the collagen fibers in the soft diet group were red, indicating fewer collagen fibers.

[0292] Figure 2Schematic diagram of in vitro and in vivo experiments provided in the embodiments of the present invention showing that the novel pentapeptide (5-Peptide) promotes the growth and development of the mandible by regulating the osteogenic differentiation function of MSCs by binding to the CNR2 receptor; wherein, A shows that α-MSH and β-MSH have a common 5 amino acid sequence (EHFRW) and are synthesized into the novel pentapeptide 5-Peptide; B shows the use of alkaline phosphatase activity (ALP) and Alizarin red staining (ARS) to detect the osteogenic differentiation ability of 5-Peptide on BMMSCs, and the results show that 5-Peptide promotes the ALP activity and mineralization ability of BMMSCs; C shows the use of RT-PCR to detect the effect of 5-Peptide on BMMSCs s stemness maintenance, the results showed that 5-Peptide promoted the expression of stemness genes SOX2, OCT4, and NANOG in BMMSCs; D showed an in vivo experiment using 4-week-old mice fed soft food + injected with 5-Peptide, which lasted until 8 weeks, and the mice were killed at the 9th week to obtain mandibular samples for analysis; EF showed gross photographs (E) showing that the height and length (F) of the mandible in the soft food + 5-Peptide injection group were higher than those in the soft food group, and close to those in the hard food group, with no statistical difference between the two; GH showed micro-CT analysis showing the cortical bone density (G) and bone mineral density (BMD), bone volume fraction (BV / TV) and The trabecular thickness (H) was higher than that of the soft food control group, and was close to that of the hard food group, with no statistical difference between the two groups; IJ showed the results of modified Masson's trichrome staining (I) and Sirius red staining (J), showing that the distribution of collagen fibers related to bone formation in the mandible of the soft food + 5-Peptide injection group was higher than that of the soft food group, and was close to that of the hard food group; K showed the use of peptide chip array technology to detect the binding of 5-Peptide with CNR1, CNR2 and MC5R, showing that 5-Peptide had 4 binding sites with MC5R, 4 binding sites with CNR1, and 2 binding sites with CNR2; L showed the use of protein molecular docking method to further verify the binding of 5-Peptide with CNR1, CNR2, and MC5R. The peptide chip array binding sites of R2 and MC5R show that -Peptide binds to CNR2; M shows the use of alkaline phosphatase activity (ALP) and Alizarin red staining (ARS) to detect the osteogenic differentiation ability of 5-Peptide after knocking out CNR2 in BMMSCs, and the results show that 5-Peptide inhibits the ALP activity and mineralization ability after knocking out CNR2 in BMMSCs; N shows the use of RT-PCR to detect the expression of osteogenic genes in BMMSCs after knocking out CNR2 by 5-Peptide, and the results show that 5-Peptide inhibits the expression of osteogenic-related transcription factors such as OCN, RUNX2 and OSX after knocking out CNR2 in BMMSCs.

[0293] in, Figure 2G: After micro-CT scanning, the mandibular specimens of each group were reconstructed in 3D using 3D-slicer software and the cortical bone was analyzed from the sagittal, coronal, and horizontal planes. The cortical bone thickness was automatically generated based on the defined ROI.

[0294] Figure 2 H and quantitative statistical analysis after scanning, the specific data are as follows:

[0295] Table 7

[0296]

[0297] like Fig. 10A shown.

[0298] Table 8

[0299]

[0300] like Fig. 10B shown.

[0301] Table 9

[0302]

[0303] like Fig. 10C shown.

[0304] Effect Example 3

[0305] (1) In vivo experiments were performed using CNR2 knockout mice. Four-week-old wild-type control mice (WT) and CNR2 knockout mice (CNR2- / -) were fed soft food and hard food, respectively, for 8 weeks. At the 9th week, the mice were killed and the mandibular bone samples were taken for analysis. The mandibular specimens were photographed under a microscope, the mandibular height and length were measured, and micro-CT scanning was performed for analysis.

[0306] (2) Sirius red staining (G1470, Solebol):

[0307] 1) The tissue was fixed in 10% formalin and routinely dehydrated and embedded;

[0308] 2) The slices were 6 μm thick and routinely dewaxed to water;

[0309] 3) Sirius red dye drop dyeing;

[0310] 4) Rinse with running water to remove the dye on the surface of the slice;

[0311] 5) Mayer's hematoxylin staining solution was used to stain the cell nucleus;

[0312] 6) Rinse with running water for 10 minutes;

[0313] 7) Conventional dehydration and transparency, sealing with neutral gum.

[0314] Staining results:

[0315] Type L collagen fibers: tightly arranged, showing strong birefringence, and appearing as yellow or red fibers.

[0316] Type II collagen fibers: show weak birefringence and are distributed in a loose network of various colors.

[0317] Type III collagen fibers: show weak birefringence and appear as green thin fibers.

[0318] Type LV collagen fibers: basement membrane showing weak birefringence, pale yellow.

[0319] According to the staining results, the distribution of osteogenic collagen fibers in the mandible of CNR2- / - group mice was similar to that of the soft diet group, but lower than that of the hard diet WT group.

[0320] (3) Modified Masson's trichrome staining (G1346, Solebol):

[0321] 1) The tissue was fixed in 10% formalin and routinely dehydrated and embedded;

[0322] 2) The slices were 4 μm thick and routinely dewaxed to water;

[0323] 3) The slices are immersed in the mordant solution and left at room temperature overnight or placed in a 57-60°C incubator for 1 hour for mordant dyeing, and then rinsed with running water for 10 minutes;

[0324] 4) Dye with lapis lazuli blue dye for 2-3 minutes and then rinse twice, 10-15 seconds each time;

[0325] 5) Dye with Mayer's hematoxylin solution for 2-3 minutes and wash twice with distilled water, each time for 10-15 seconds;

[0326] 6) Differentiate in acidic differentiation solution for a few seconds, wash with water to terminate differentiation, and rinse with distilled water for 10 minutes;

[0327] 7) Dye with Ponceau fuchsin solution for 10 min, and wash twice with distilled water, 10-15 s each time;

[0328] 8) Treat with phosphomolybdic acid solution for about 10 minutes;

[0329] 9) Pour off the supernatant, add aniline blue staining solution directly to the slices without washing them, and stain for 5 minutes;

[0330] 10) After washing away the aniline blue solution with a weak acid solution, continue to drip weak acid working solution to cover the slices for 2 minutes;

[0331] 11) Dehydration with 95% ethanol for 30 seconds, dehydration with anhydrous ethanol twice, the first time for 30 seconds, the second time for 1 minute;

[0332] 12) Transparent the slides twice with xylene, each time for 1-2 min, and seal the slides with neutral gum.

[0333] Staining results:

[0334] Collagen fiber: blue

[0335] Muscle fibers, cytoplasm, cellulose, keratin, and red blood cells: varying degrees of red

[0336] Nucleus: blue-brown

[0337] According to the results of section staining: the distribution of osteogenic collagen fibers in the mandible of CNR2- / - group mice was similar to that of the soft diet group, but lower than that of the hard diet WT group (which was blue).

[0338] Figure 3 The in vivo experiment provided in the embodiment of the present invention shows that the growth and development of the mandible is inhibited by using the CNR2 gene knockout tool mouse; wherein, A uses 4-week-old wild-type control mice (WT) and CNR2 knockout mice (CNR2- / -) to feed soft food and hard food respectively, which lasts for 8 weeks. At the 9th week, the mice were killed to obtain mandibular samples for analysis. The gross photos show that the mandible of the CNR2- / - group mice is underdeveloped under different chewing force stimulations, which is close to that of the soft food group, with no statistical difference. The mandibles of the CNR2- / - group and the soft food group are lower than those of the hard food WT group; B shows the gross measurement of the height and length of the mandible of each group, showing that the height and length of the mandible of the CNR2- / - group mice are poorly developed under different chewing force stimulations. The mandibular height and length of the CNR2- / - group and the soft diet group were lower than those of the hard diet WT group. CD showed that the mandibular cortical bone density (C), bone mineral density (BMD), bone volume fraction (BV / TV) and trabecular thickness (C) of each group were further analyzed by micro-CT, showing that the mandibular cortical bone density, bone mineral density (BMD), bone volume fraction (BV / TV) and trabecular thickness of the CNR2- / - group mice were close to those of the soft diet group, and lower than those of the hard diet WT group. EF showed the distribution of osteogenic collagen fibers in the mandible of each group detected by modified Masson's trichrome staining and Sirius red staining, showing that the distribution of osteogenic collagen fibers in the mandible of the CNR2- / - group mice was close to that of the soft diet group, and lower than that of the hard diet WT group.

[0339] in, Figure 3 B quantitative data unit: mm:

[0340] Table 10

[0341]

[0342] Figure 3 C-3D quantitative data:

[0343] Table 11

[0344]

[0345] Effect Example 4

[0346] Western blot analysis:

[0347] Western blot was used to detect the changes of AKT / ERK1 / 2 / STAT3 signaling pathway in BMMSCs of WT group and CNR2- / - group under different chewing forces. Cells were lysed with RIPA lysis buffer, protein concentration was determined by Bradford method, samples were separated by 10% SDS polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membrane using semi-dry transfer membrane device, the transferred PVDF membrane was blocked with 5% skim milk powder for 1h and placed on a shaker for shaking, incubated with primary antibody and shaken at 4℃ overnight. The filter was placed in the secondary antibody diluted with 5% skim milk powder, the immune complex was incubated with rabbit or mouse immunoglobulin G antibody and visualized with chemiluminescent substrate reagent, and imaged in BIO-RAD chemiluminescence imaging system. The expression of AKT, P-AKT, ERK1 / 2, P-ERK1 / 2, Stat3, and P-Stat3 was determined.

[0348] Primary antibody: anti-AKT anti-p-AKT (1:1000Cell Signaling Technology) anti-ERK1 / 2

[0349] anti-p-ERK1 / 2 (1:1000Cell Signaling Technology) anti-stat3 anti-p-stat3 (1:1000Cell Signaling Technology), β-actin (1:2000Abcam).

[0350] Secondary antibodies: anti-mouse IgG (1:2000 Abcam), anti-rabbit IgG (1:5000 Abcam).

[0351] Figure 4The figure shows a schematic diagram of the 5-Peptide provided by the embodiment of the present invention regulating AKT / ERK1 / 2 / STAT3 to promote the osteogenic differentiation of MSCs by binding to CNR2 on the surface of BMMSCs. Western blot was used to detect the changes in the AKT / ERK1 / 2 / STAT3 signaling pathway in BMMSCs of the WT group and the CNR2- / - group under different chewing forces, showing that the phosphorylated AKT / ERK1 / 2 / STAT3 of BMMSCs in the CNR2- / - group was close to that of BMMSCs in the soft food group, and both were lower than those of the hard food WT group, and β-actin was used as an internal reference.

[0352] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that It has: (I), the amino acid sequence shown in SEQ ID No.241; (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) An amino acid sequence having 90% or more identity with the amino acid sequence described in (I) or (II).

2. Use of the polypeptide as claimed in claim 1 in the preparation of a drug for promoting the growth and development of mandibular bones or treating oral diseases.

3. The use according to claim 2, characterized in that The polypeptide is a receptor to which α-MSH and / or β-MSH binds.

4. The use according to claim 2, characterized in that The receptor of the polypeptide is CNR2.

5. The use according to claim 2, characterized in that The oral diseases include one or more of mandibular underdevelopment, micrognathia, malocclusion, tooth imbalance or bone imbalance.

6. The use according to claim 2, characterized in that The polypeptide regulates AKT / ERK1 / 2 / STAT3 by binding to CNR2 on the surface of BMMSCs to promote the osteogenic differentiation of MSCs.

7. The use according to claim 2, characterized in that The polypeptide promotes the growth and development of the mandibular bone by binding to the CNR2 receptor and regulating the osteogenic differentiation function of MSCs.

8. The use according to claim 7, characterized in that The polypeptide inhibits the ALP activity and mineralization ability of BMMSCs after knocking out CNR2.

9. The use according to claim 7, characterized in that The polypeptide inhibits the expression of osteogenic-related transcription factors OCN, RUNX2 and OSX after CNR2 is knocked out in BMMSCs.

10. A reagent or a drug, characterized in that Comprising the polypeptide as claimed in claim 1.

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