Polypeptide for promoting proliferation and differentiation of osteoblasts and application thereof

Abstract

The invention discloses a polypeptide for promoting proliferation and differentiation of osteoblasts and application of the polypeptide, and relates to the technical field of biology. The invention provides three polypeptides GM-15, AG-17 and GN-18 capable of promoting osteoblast proliferation and improving differentiation activity by taking gelatin of fish scales and fish skin of tilapia mossambica as a raw material and by means of a protease digestion technology, LC-MS / MS peptide fragment identification, computer simulation screening combined with molecular docking auxiliary screening and the like. The cell proliferation promoting activity of the three polypeptides is high, and the maximum proliferation rate can reach 124%. Meanwhile, the three polypeptides show relatively strong effects of promoting differentiation and mineralization of osteoblasts. The three polypeptides provided by the invention have the characteristics of good water solubility, no toxicity, no anaphylaxis and strong activity of promoting osteoblast proliferation and differentiation, and have better application prospects in the fields of osteoblast proliferation promoting related orthopedic drugs, health care products, functional foods and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide promoting osteocyte proliferation and differentiation and an application thereof. Background Art

[0002] Osteoporosis (OP) is a chronic bone disease characterized by osteopenia and bone microarchitecture degradation, which often leads to increased bone fragility and increased fracture risk. Its pathogenesis is complex and involves the disruption of bone metabolism balance, especially the imbalance of osteoblast and osteoclast function. The occurrence of osteoporosis is closely related to aging. With the acceleration of the global aging process, it is expected that by 2050, the proportion of the global elderly population will rise sharply, and the incidence of osteoporotic fractures will also increase accordingly, becoming one of the important issues affecting public health. At present, although drugs such as bisphosphonates and calcitonin are widely used in the treatment of osteoporosis in clinical practice, their long-term use may cause side effects such as gastrointestinal discomfort and osteonecrosis, and there is a potential risk of toxicity. Therefore, natural active substances based on food sources have gradually become a research hotspot. At present, many studies aim to find safer and more effective treatment approaches in order to provide better intervention options for patients with osteoporosis.

[0003] In recent years, with the in-depth study of the mechanism of osteoporosis, bioactive peptides that promote osteoblast proliferation and differentiation have gradually attracted attention. These peptides are usually derived from the enzymatic hydrolysis products of food proteins and have the potential to promote osteoblast proliferation, differentiation and mineralization. Studies have shown that some bioactive peptides can accelerate the maturation of osteoblasts by activating the Wnt / β-catenin signaling pathway, promoting ALP activity and the synthesis of type I collagen. In addition, active peptides can also enhance the deposition of calcium ions in the bone matrix, which helps to increase bone density. Compared with traditional drugs, these food-derived peptide molecules not only have good biocompatibility in the body, but also have low toxicity and few side effects, showing broad application prospects in the prevention and treatment of osteoporosis. Future research will continue to explore the molecular mechanisms of these peptides and their potential in clinical applications, providing a scientific basis for nutritional intervention in osteoporosis.

[0004] Tilapia (Oreochromis niloticus) is a freshwater fish that is widely farmed around the world. Its high yield is accompanied by the production of a large number of by-products, such as fish skin and fish scales. These by-products are often discarded or used for low-value-added products in traditional processing, resulting in a huge waste of resources. However, studies have found that tilapia skin and fish scales are rich in collagen and gelatin, which not only have excellent biocompatibility, but also have the potential to promote osteoblast proliferation and differentiation. Through appropriate enzymatic treatment, high-quality collagen peptides can be extracted from these by-products, which can promote the repair and regeneration of bone tissue. Therefore, the use of tilapia by-products for the extraction of bioactive peptides not only provides potential functional ingredients for the treatment of osteoporosis, but also provides a new way for the efficient utilization of aquatic resources, which is in line with the concept of resource recycling and sustainable development. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide that promotes osteoblast proliferation and differentiation and its application to solve the problems existing in the above-mentioned prior art. The polypeptide provided by the present invention has the characteristics of good water solubility, non-toxicity, non-allergy, and strong activity in promoting osteoblast proliferation and differentiation, and has good application prospects in the fields of orthopedic drugs, health products, functional foods, etc. related to promoting osteoblast proliferation.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a polypeptide GM-15 for promoting osteocyte proliferation and differentiation, and the amino acid sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides a polypeptide GN-18 for promoting osteocyte proliferation and differentiation, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] The present invention also provides a polypeptide AG-17 that promotes osteocyte proliferation and differentiation, and the amino acid sequence is shown in SEQ ID NO.3.

[0010] The present invention also provides the use of the above-mentioned polypeptide GM-15, polypeptide GN-18 and / or polypeptide AG-17 in the preparation of drugs for preventing and / or treating osteoporosis.

[0011] The present invention also provides the use of the above-mentioned polypeptide GM-15, polypeptide GN-18 and / or polypeptide AG-17 in the preparation of products for improving bone density;

[0012] The product is a health product or a functional food.

[0013] The present invention also provides a drug for preventing and / or treating osteoporosis, wherein the active ingredient comprises at least one of the above-mentioned polypeptide GM-15, polypeptide GN-18 and polypeptide AG-17.

[0014] The present invention also provides a health product or functional food for improving bone density, wherein the active ingredient comprises at least one of the above-mentioned polypeptide GM-15, polypeptide GN-18 and polypeptide AG-17.

[0015] The present invention also provides a method for preparing the polypeptide GM-15, comprising the step of synthesizing the polypeptide GM-15 by solid phase synthesis.

[0016] The present invention also provides a method for preparing the polypeptide GN-18, comprising the step of synthesizing the polypeptide GN-18 by solid phase synthesis.

[0017] The present invention also provides a method for preparing the polypeptide AG-17, comprising the step of synthesizing the polypeptide AG-17 by solid phase synthesis.

[0018] The present invention discloses the following technical effects:

[0019] The present invention uses gelatin from tilapia scales and skin as raw materials, and provides three polypeptides GM-15, AG-17 and GN-18 that promote osteoblast proliferation and improve differentiation activity by means of protease digestion technology, LC-MS / MS peptide identification, computer simulation screening combined with molecular docking assisted screening. The three polypeptides have high cell proliferation promoting activity, and the maximum proliferation rate can reach 124%. At the same time, the three polypeptides show strong effects of promoting osteoblast differentiation and mineralization.

[0020] The three polypeptides provided by the present invention have the characteristics of good water solubility, non-toxicity, non-allergy, and strong activity in promoting osteoblast proliferation and differentiation. They have good application prospects in the fields of orthopedic drugs, health products, functional foods, etc. related to promoting osteoblast proliferation. 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the total ion current diagram of fish scale gelatin alkaline protease enzymatic hydrolysate;

[0023] Figure 2 This is the total ion current of fish scale gelatin trypsin hydrolysate;

[0024] Figure 3 This is the total ion current diagram of fish skin gelatin trypsin hydrolysate;

[0025] Figure 4 This is an analysis of the proportion of peptides with different molecular weights in three enzymatic hydrolysates identified by LC-MS / MS;

[0026] Figure 5 Schematic diagram of 2D interaction between GM-15 and EGFR molecules docking;

[0027] Figure 6 Schematic diagram of the 3D interaction between GM-15 and EGFR molecules;

[0028] Figure 7 Schematic diagram of 2D interaction between GN-18 and EGFR molecules;

[0029] Figure 8 Schematic diagram of the 3D interaction between GN-18 and EGFR molecules;

[0030] Fig. 9 Schematic diagram of 2D interaction between AG-17 and EGFR molecules;

[0031] Fig.10 Schematic diagram of the 3D interaction between AG-17 and EGFR molecules;

[0032] Fig.11 is the secondary mass spectrum of GM-15;

[0033] Fig.12 is the secondary mass spectrum of AG-17;

[0034] Fig.13 is the secondary mass spectrum of GN-18;

[0035] Fig.14 is the HPLC chromatogram of GM-15;

[0036] Fig.15 is the high performance liquid chromatogram of AG-17;

[0037] Fig.16 is the high performance liquid chromatogram of GN-18;

[0038] Fig.17 It is a statistical diagram of the proliferation activity of Saos-2 cells under the action of different concentrations of GM-15, GN-18 and AG-17;

[0039] Fig.18Figures are ALP staining images and ALP activity statistics of Saos-2 cells under the action of different concentrations of GM-15, GN-18 and AG-17; A and B are ALP staining images and ALP activity statistics of Saos-2 cells under the action of different concentrations of GM-15; C and D are ALP staining images and ALP activity statistics of Saos-2 cells under the action of different concentrations of GN-18; E and F are ALP staining images and ALP activity statistics of Saos-2 cells under the action of different concentrations of AG-17; in A, C and E, before is the cell morphology before staining under the microscope, and after is the cell morphology after staining under the microscope;

[0040] Fig.19 The mineralization staining images and mineralization degree statistics of Saos-2 cells under the action of different concentrations of GM-15, GN-18 and AG-17; A and B are the mineralization staining images and mineralization degree statistics of Saos-2 cells under the action of different concentrations of GM-15; C and D are the mineralization staining images and mineralization degree statistics of Saos-2 cells under the action of different concentrations of GN-18; E and F are the mineralization staining images and mineralization degree statistics of Saos-2 cells under the action of different concentrations of AG-17;

[0041] Fig. 20 The expression statistics of osteogenesis-related genes and Wnt / β-catenin-related genes under the influence of GM-15, GN-18 and AG-17; among them, AI are ALP, IBSP, OCN, OSX, Runx2, Coll-α, β-catenin, LRP5 and GSK3β, respectively. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] Example 1

[0048] 1. Extraction of fish skin gelatin and fish scale gelatin

[0049] (1) Experimental Materials

[0050] Tilapia skin and scales were sourced from the local seafood market after processing fresh fish.

[0051] (2) Experimental methods

[0052] Fish skin gelatin extraction process: Tilapia skin was washed three times with distilled water to remove impurities and then air-dried naturally. The dried fish skin was chopped and soaked in 0.05 mol / L NaOH solution for 45 minutes at a solid-liquid ratio of 1:10 to remove the non-collagen part. The residual NaOH on the fish skin was washed with distilled water, and then stirred and soaked in 0.05 mol / L HCl solution for 15 minutes at a solid-liquid ratio of 1:10 to swell the fish skin. Then it was washed with distilled water 7 to 8 times until the pH value was close to neutral. The wet fish skin was mixed with distilled water at a ratio of 1:10 (m / v) and heated in a 60℃ water bath for 6 hours. Large impurities were filtered out with non-woven fabric, and then centrifuged at 10,000g for 15 minutes at room temperature. The supernatant was collected and freeze-dried using a freeze dryer for further study.

[0053] The extraction process of tilapia scale gelatin has the same steps as the above-mentioned fish skin gelatin extraction process, and also requires the following steps to be added after the NaOH soaking: decalcification by soaking in 0.4 mol / L HCl solution at a solid-liquid ratio of 1:10 for 3 hours.

[0054] 2. Preparation of enzymatic hydrolysate

[0055] (1) Experimental Materials

[0056] Trypsin and alkaline protease.

[0057] (2) Experimental methods

[0058] Fish skin and fish scale gelatin were hydrolyzed by alkaline protease and trypsin respectively according to the enzyme addition amount (mass ratio: 2%), the optimum temperature was 37°C (trypsin), 50°C (alkaline protease), the optimum pH was 7.5 (trypsin), 8.0 (alkaline protease), and the enzymolysis time was 6h. The optimal pH and temperature were selected for both proteases. During the hydrolysis process, 0.05mol / LNaOH and 0.05mol / LHCl were used to adjust and maintain the required pH value through a pH meter. After the hydrolysis was completed, the hydrolyzate was heated in a boiling water bath for 10 minutes to inactivate the protease. Afterwards, the hydrolyzate was centrifuged at 10000g for 10 minutes at room temperature, and the supernatant was collected and freeze-dried for further use.

[0059] 3. LC-MS / MS identification and computer-assisted screening of enzymatic hydrolysates

[0060] Experimental method: First, the peptides identified by LC-MS / MS were deduplicated and then summed. Peptides modified by phosphorylation or dephosphorylation at different amino acid positions were regarded as the same peptide, and their relative content was calculated according to their intensity. In order to study the main role in the hydrolysate, machine learning was used to screen peptides with a content percentage of more than 0.01% (fish skin trypsin hydrolysate: 578, accounting for about 55.52% of the total; fish scale alkaline protease hydrolysate: 486, accounting for about 49.79% of the total; fish scale trypsin hydrolysate: 594, accounting for about 61.74% of the total). See the LC-MS / MS identification results in the figure. Figure 1-4 .

[0061] The PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / ) tool was used to calculate the scores of the peptides in the three enzymatic hydrolysates. The peptide scores given by PeptideRanker range from 0 to 1, with the highest score (1) indicating the most likely bioactive peptide, and the higher the value, the greater the possibility of bioactivity.

[0062] Toxicity of peptides is one of the important concerns in the development of gelatin functional food ingredients. Therefore, computer prediction of toxicity of gelatin-derived peptides with PeptideRanker scores greater than 0.5 was investigated using ToxinPred (http: / / www.imtech.res.in / raghava / toxinpred / index.html). A SVM (support vector machine) based prediction method with a threshold of 0.0 was selected to separate toxic and non-toxic peptides.

[0063] GRAVY Calculator (https: / / www.gravy-calculator.de / ) can be used to calculate the hydrophilicity of proteins or peptides based on the amino acid sequence. A value less than zero is considered hydrophilic, and a larger absolute value represents a stronger hydrophilicity (hydrophobicity). The hydrophilicity of peptides identified and screened by LC-MS / MS can be obtained based on computer prediction, which can reflect the difficulty of their synthesis.

[0064] One of the key issues in developing gelatin-derived functional food ingredients is the allergenicity of peptides. Therefore, AllerTop1.0 (https: / / ddg-pharmfac.net / AllerTOP / index.html) is used to predict the allergenicity of the screened peptides, and "NON-ALLERGEN" means non-allergenic, indicating that the peptide can be used for subsequent experiments.

[0065] 4. Molecular docking

[0066] Experimental methods: The crystal structure of EGFR was obtained from RCSB PDBk (PDB ID: 1IVO, ), first remove the same structure of the homodimer, retain only one of the chains, then eliminate the ligands and water molecules that come with the protein, check the selected protein, fix its end, and add hydrogen, and finally minimize the energy to complete the preparation of the protein receptor. For the structural preparation of the polypeptide ligand, ChemDraw 20.0 is used to draw the polypeptide sequence, and the saved cdxml file is directly imported into the ChemDraw 3D software and the PDB file of the polypeptide is exported. The PDB file of the polypeptide is hydrogenated and the energy is minimized in the Discovery Studio 2019 software to complete the structural preparation of the polypeptide ligand. The position of the EGF factor in the EGFR crystal complex is used as the center to confirm the docking box, and its size is adjusted to completely wrap the position of EGF binding, that is, to complete the confirmation of the docking box. In order to study the interaction between the peptide identified by LC-MS / MS and EGFR, the present invention defines EGFR as a receptor and the polypeptide as a ligand. Molecular docking adopts the CDOCKER semi-flexible docking method of Discovery Studio 2019 software.

[0067] Analysis results: According to the results of the above screening and docking, the peptides with CDOCKER less than -90 were selected as having good binding efficacy for the next step of screening. Among them, the peptides with a content greater than 0.1% were identified as being present in large quantities in the enzymatic hydrolysate, so the last six peptides were selected for analysis, and comprehensive consideration was given to the binding sites, the number of hydrogen bonds, and other scores. The results showed that the interconnected amino acids of GMPGERGAAGLPGLR (SEQ ID NO.1) and EGFR include Lys13, Thr15, Gln16, and Tyr45. They also participate in the interaction sites between EGFR and EGF. GNPGAAGSAGPQGPIGPR (SEQ ID NO.2) includes Lys13, Thr15, and Gln16. The two peptides mainly rely on hydrogen bonds and hydrophobic forces to maintain the structure of the complex. Relative to the number of hydrogen bonds, the number of hydrogens in these two peptides is 8 and 6, respectively. The results showed that the peptides GMPGERGAAGLPGLR and GNPGAAGSAGPQGPIGPR that bind to EGFR may play a role in promoting osteoblast proliferation activity at the active site, so these two peptides were selected for subsequent synthesis experiments. At the same time, considering that AGPPGPPGLGGNFAAQY (SEQ ID NO.3) exists in large quantities in the fish skin trypsin hydrolysate and has a good molecular docking score of about 90.7113, three peptides were selected from the fish scale alkaline protease hydrolysate, fish scale trypsin hydrolysate, and fish skin trypsin hydrolysate, namely GMPGERGAAGLPGLR (GM-15), GNPGAAGSAGPQGPIGPR (GN-18), and AGPPGPPGLGGNFAAQY (AG-17). The secondary mass spectra of the three peptides were obtained at the same time (see Figure 11-13 ). Schematic diagram of 2D and 3D interactions of GM-15, GN-18 and AG-17 with EGFR molecules is shown in Figure 5-Figure 10 .

[0068] 5. Artificial peptide synthesis (solid phase synthesis)

[0069] Experimental method: The sequences of the three peptides were sorted according to "sequence: N-terminal-A1A2A3...An-C-terminal". The resin was activated first. Starting from the C-terminal An, the resin Fmoc-An-Resin was weighed according to the required amount and placed in the reaction column for 30 minutes. After the resin was soaked for 30 minutes, the solution was removed, and then deprotection was carried out with piperidine for 30 minutes. Within 30 minutes of deprotection, the amount of amino acid required for each step and the amount of condensation agent NMM reaction were calculated according to the amount made, and then the next amino acid Fmoc-An-1 was weighed. After 30 minutes of deprotection, the piperidine was removed and washed with DMF 6 times. After washing 6 times, the deprotection color was detected and recorded. After washing, deprotection and detection, the weighed materials were added in sequence according to the order of each peptide, and then a small amount of reaction solution was added, and then alkali and NMM were added. The gas was adjusted evenly, and the resin stuck to the inner wall of the reaction column was washed away with DCM, and then the reaction time was recorded. The reaction lasted for 30 minutes. After 30 minutes of reaction, the reaction solution was removed, washed with DMF 3 times, and then tested to check whether the reaction was complete. After the test was connected, the above steps were repeated to connect the next amino acid. If the test failed, it was soaked in DCM, and then without deprotection, the amino acid of this step was continued to be connected until it was connected.

[0070] GM-15, AG-17 and GN-18 were purified by HPLC technology, and the chromatograms of GM-15, AG-17 and GN-18 after high performance liquid chromatography analysis were obtained ( Figure 14-16 ). After high performance liquid chromatography analysis, GM-15, AG-17 and GN-18 were separated according to the separation conditions and the main peaks were collected. After freeze drying, the purity of the sample was above 95%.

[0071] 6. GM-15, AG-17, and GN-18 promote osteoblast proliferation

[0072] Experimental method: Saos-2 cells were grown in McCoy's 5A medium (antibiotics: 100 U / mL penicillin and 100 μg / mL streptomycin) containing 1% antibiotics and 10% fetal bovine serum in an incubator at 37°C and a humidified environment containing 5% CO2. The cells were subcultured every 2-3 days after being digested with 0.25% trypsin. If the subculture density was not reached, the medium was changed.

[0073] CCK8 assay was used to detect the proliferation effects of GM-15, AG-17 and GN-18 on Saos-2 cells. 3The cells were inoculated into 96-well plates at a density of 100 μg / mL and cultured at 37 ° C and 5% CO2 for 24 hours. Then, the complete medium was replaced with a basal medium (McCoy's 5A basal medium, with 1% double antibody added, without serum added) for 4 hours to keep the cells in a uniform state. Then, the basal medium was replaced with a complete medium containing GM-15, AG-17 or GN-18 at a concentration of 0 μg / mL (blank control), 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, respectively, and incubated for 24 hours. Subsequently, the culture medium was discarded and gently rinsed with PBS. CCK8 solution (v:v=10% in complete medium) was added to each well and incubated in the dark in a cell culture incubator for 1 hour until the corresponding absorbance was reached. Finally, the absorbance at 450 nm was measured using a microplate reader.

[0074] Analysis results: Fig.17 As shown in the figure, compared with the blank control group, GM-15 and GN-18 showed good osteoblast proliferation activity with increasing concentrations, among which GM-15 showed the best proliferation activity at a concentration of 10μg / mL, and the proliferation effect of GN-18 continued to increase with increasing concentrations. The proliferation effect of AG-17 was not obvious, and only the concentration of 50μg / mL showed good proliferation activity.

[0075] 7. Effects of GM-15, AG-17 and GN-18 on ALP activity and staining

[0076] Experimental methods:

[0077] ALP activity: The cells were seeded in 6-well plates at a density of 1 × 10 6 cells / well. When the cell density grew to about 80%, the cells were treated with differentiation medium containing different concentrations of osteogenic peptides GM-15, AG-17 or GN-18 (0, 1, 10 and 50 μg / mL) for 7 days. The differentiation medium was changed every two days. After 7 days, the protein concentration and ALP activity of the cells were determined using BCA protein assay and ALP assay kit, respectively.

[0078] ALP staining: Cells were seeded in 12-well plates at a density of 1 × 10 5cells / well. When the cell density grows to about 80%, it is treated with differentiation medium containing different concentrations of osteogenic peptides GM-15, AG-17 or GN-18 (0, 1, 10 and 50 μg / mL) for 7 days. The differentiation medium was changed every two days. Alkaline phosphatase (ALP) in Saos-2 cells after 7 days of differentiation was stained using an ALP staining kit. The cells were fixed with 4% paraformaldehyde at 37°C for 15 minutes, and the ALP staining procedure was started. The sample was incubated with a solution containing nitro blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt for 30 minutes until the desired color depth was reached, and micrographs were captured using an inverted optical microscope.

[0079] Result analysis: Fig.18 As shown in the figure, after induction with differentiation medium added with GM-15, AG-17 and GN-18, the intensity of staining was positively correlated with the concentration, and the best differentiation-promoting effect was shown at the highest concentration of 50 μg / mL, and the three osteogenic peptides could significantly improve the differentiation degree of Saos-2. At the same time, the results of the three enzymatic hydrolysates on the induction of differentiation of Saos-2 cells were compared. The enzymatic hydrolysate at the same concentration (400 μg / mL) was not as effective as the osteogenic peptide in promoting differentiation of Saos-2 cells, but according to the results of the ALP alkaline phosphatase detection kit, it can be seen that the enzymatic hydrolysate at this concentration also played a positive role in the differentiation of Saos-2 cells.

[0080] 8. Effects of GM-15, AG-17 and GN-18 on mineralization and Alizarin red staining

[0081] Experimental methods:

[0082] Mineralization degree: The cells were seeded in 12-well plates at a density of 3 × 10 6 cells / well. When the cell density grows to about 80%, the cells are treated with differentiation medium containing different concentrations of osteogenic peptides GM-15, AG-17 or GN-18 (0, 1, 10 and 50 μg / mL) for 21 days. The differentiation medium is replaced every two days, and the medium is replaced by half-change in the middle and late stages of differentiation to prevent the calcium nodules from being washed away.

[0083] Mineralization staining: The calcium nodules in Saos-2 cells after 21 days of differentiation were stained using an Alizarin Red staining kit to compare their mineralization degree. The cells were fixed with 4% paraformaldehyde at 37°C for 30 minutes and the Alizarin Red staining procedure was started. The stained calcium nodules were then dissolved with a 10% (w / v) cetylpyridinium chloride solution for 30 minutes, and the extracted stained calcium nodules were subsequently measured at 562 nm using a microplate reader.

[0084] Result analysis: Mineralization is the last step of osteoblast differentiation and can directly reflect the degree of bone formation. Alizarin red chelates with calcium ions to produce a deep red or purple complex, which is usually used to observe calcium nodules during osteoblast mineralization. Fig.19 As shown in the figure, the number of mineralized nodules in the blank control group was the lowest. After treatment with GM-15, AG-17, GN-18 and three kinds of enzymatic hydrolysates, the number of mineralized nodules increased significantly, and the intensity of alizarin red staining also increased significantly. However, the degree of mineralization and staining results did not show concentration dependence, indicating that mineralization is a long-term and complex system and process. However, as long as GM-15, AG-17 or GN-18 collagen peptides are added, the degree of mineralization can be significantly improved and the degree of staining can be deepened.

[0085] 9. GM-15, AG-17 and GN-18 qPCR analysis

[0086] Experimental method: Saos-2 cells were cultured at 1×10 6 The cells were seeded at a density of 10 cells / well in a 6-well plate. When the cells grew to 80% density, they were treated with differentiation medium containing 50 μg / mL osteogenic peptides GM15, AG17 or GN18 for 7 days, and the differentiation medium was changed every two days. The total RNA sample obtained was reverse transcribed into cDNA using the oligo(dT) primer of the PrimScript cDNA Synthesis Kit. GADPH was used as the internal reference gene to determine the relative expression levels of ALP, IBSP, OCN, RUNX2, COL1A1, OSX, β-catenin, GSK-3Beta and LRP5. 2 -ΔΔCt Methods Calculate fold change.

[0087] Result analysis:

[0088] Osteogenesis-related genes (6): In order to confirm the above speculation, the present invention detected the expression of genes related to osteogenic activity. Since the incubation time was 7 days, the concentration with the best ALP activity (50μg / mL) was selected for qPCR analysis. ALP is an early marker of differentiation, IBSP appears in the middle stage of osteogenic differentiation, and OCN is mainly synthesized and secreted by mature osteoblasts and is the main non-collagen protein in the bone matrix. It promotes the calcification of mineral deposition in normal bone tissue and its regulation of bone, and its production time is in the late stage of differentiation. The Coll-α gene (i.e., collagen α chain gene) is mainly responsible for encoding collagen, a class of structural proteins widely present in connective tissue. Runx2 has been described as a key osteogenic transcription factor involved in the differentiation and maturation of osteoblasts, which is key to bone tissue formation and mineralization. OSX is a direct downstream of the transcription regulator Runx2 and is also a marker of bone formation. Such as Fig. 20As shown in Figures AF, compared with the blank control group, osteogenic peptides and enzymatic hydrolysate both showed enhanced expression of osteogenic related genes, among which GN-18 showed good significance in all six genes, proving that it regulates the entire osteoblast proliferation and differentiation process, AG-17 significantly upregulated the RUNX-2 gene by more than 600%, indicating that it may promote osteogenesis by participating in RUNX-2 related regulatory factors and pathways, and GM-15 played a significant role in upregulating the ALP gene, proving that it regulates osteogenesis by regulating the ALP gene in the early stage of osteogenic differentiation. Through the above invention, it can be concluded that all collagen peptides can promote the expression of related genes in osteoblasts, but the expression through what regulatory method still needs to be explored in subsequent experiments.

[0089] Wnt / β-catenin pathway-related genes (3): There are some signaling pathways that regulate the proliferation and differentiation of osteoblasts, including the OPG / RANKL / RANK signaling pathway, the BMPs signaling pathway, the MAPK signaling pathway, and the Wnt / β-catenin signaling pathway. Among these pathways, the Wnt / β-catenin signaling pathway is the most commonly used pathway for studying osteogenesis. The present invention speculates that these three collagen peptides may promote the differentiation of Saos-2 cells by activating the Wnt / β-catenin signaling pathway through docking with EGFR to screen the mRNA and protein expression of Wnt / β-catenin-related factors, and determine the signaling pathways including β-catenin, LRP5 and GSK3β to verify the hypothesis of the present invention. All three collagen peptides significantly downregulated the expression of the GSK3β gene. The overexpression of GSK3β inhibits Wnt / β-catenin, indicating that GM-15, AG-17, and GN-18 can express the expression of the Wnt / β-catenin pathway by inhibiting GSK3β ( Fig. 20 β-catenin is an important factor in this pathway. AG-17 showed an expression of more than 200% of β-catenin, while the expression of the other two peptides was not significant, indicating that AG-17 is most likely to regulate the proliferation and differentiation of osteoblasts through the Wnt / β-catenin pathway.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide GM-15 that promotes osteoblast proliferation and differentiation, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A polypeptide GN-18 that promotes osteoblast proliferation and differentiation, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. A polypeptide AG-17 that promotes osteoblast proliferation and differentiation, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

4. Use of the polypeptide GM-15 according to claim 1, the polypeptide GN-18 according to claim 2 and / or the polypeptide AG-17 according to claim 3 in the preparation of a medicament for preventing and / or treating osteoporosis.

5. Use of the polypeptide GM-15 according to claim 1, the polypeptide GN-18 according to claim 2 and / or the polypeptide AG-17 according to claim 3 in the preparation of a product for improving bone density; The product is a health product or a functional food.

6. A drug for preventing and / or treating osteoporosis, characterized in that: The active ingredient comprises at least one of the polypeptide GM-15 according to claim 1, the polypeptide GN-18 according to claim 2, and the polypeptide AG-17 according to claim 3.

7. A health product or functional food for improving bone density, characterized in that: The active ingredient comprises at least one of the polypeptide GM-15 according to claim 1, the polypeptide GN-18 according to claim 2, and the polypeptide AG-17 according to claim 3.

8. A method for preparing the polypeptide GM-15 as claimed in claim 1, characterized in that: The method comprises the step of synthesizing the polypeptide GM-15 by solid phase synthesis method.

9. A method for preparing the polypeptide GN-18 as claimed in claim 2, characterized in that: The method comprises the step of synthesizing the polypeptide GN-18 by a solid phase synthesis method.

10. A method for preparing the polypeptide AG-17 according to claim 3, characterized in that: The method comprises the step of synthesizing the polypeptide AG-17 by solid phase synthesis method.

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