Secretory peptide P1DC derived from human dental pulp stem cell supernatant

Abstract

The invention discloses a secretory peptide and a secretory peptide P1DC derived from human dental pulp stem cell supernate. The amino acid sequence of the secretory peptide is as shown in SEQ ID NO.1, and the amino acid sequence of the secretory peptide P1DC is as shown in SEQ ID NO.3; according to the invention, supernate of human dental pulp stem cells is used as a raw material, enzymolysis is carried out to obtain a peptide fragment, high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) is adopted to analyze and identify the amino acid sequence of the peptide after step-by-step purification, a target peptide is artificially synthesized, and in-vitro experiments show that the polypeptide has good activity of promoting cells; the secretory peptide disclosed by the invention has the characteristics of simple structure, strong activity and the like and is easy to artificially synthesize, P1DC can be applied to preparation of medicines for promoting dental pulp injury repair and can be used for treating caries and dental pulp periapical diseases, and the discovery possibly provides a basis for developing new medicines for treating dental pulp diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of dental pulp damage repair, in particular to a secretory peptide derived from supernatant of human dental pulp stem cells and related uses thereof. Background Art

[0002] Dental caries is a common oral disease. Due to its high incidence and wide distribution, it has become one of the common chronic diseases in humans. The global prevalence of dental caries in children is about 60-90%, and the prevalence of dental caries in adults is about 100% (Pitts NB, Zero DT, Marsh PD, et al. Dental caries [J]. Nat Rev Dis Primers, 2017, 3: 17030). When caries reaches the deep layer of dentin, bacteria and toxic substances produced by their metabolism can enter the pulp tissue through the dentinal tubules, causing varying degrees of damage to the pulp tissue (Martignon S, Roncalli AG, Alvarez E, et al. Risk factors for dental caries in Latin American and Caribbean countries [J]. Braz Oral Res, 2021, 35 (suppl 01): e53). In mild cases, symptoms include dentin hypersensitivity, spontaneous pain, night pain, and occlusal pain. In severe cases, maxillofacial swelling may occur. If not effectively treated, it may even cause inflammation of the alveolar bone and jaw, eventually leading to tooth loss, seriously affecting the patient's chewing function and facial aesthetics, and significantly reducing the patient's quality of life (Innes N, Chu CH, Fontana M, et al. A Century of Change towards Prevention and Minimal Intervention in Cariology [J]. JDent Res, 2019, 98 (6): 611-617).

[0003] A large number of studies have shown that mesenchymal stem cells (MSCs) are a type of multipotent stem cells derived from the early developmental mesoderm. They are one of the most studied and important adult stem cells. During the culture process, they secrete more than 500 protein components including chemokines and growth factors, which can significantly drive the biological processes of metabolism, defense response and tissue regeneration. They are widely defined as conditioned medium (culture media, CM) (Giovannelli L, Bari E, Jommi C, Tartara F, Armocida D, Garbossa D, Cofano F, Torre ML, Segale L. Mesenchymal stem cell secretome and extracellular vesicles for neurodegenerative diseases: Risk-benefit profile and next steps for the market access. Bioact Mater. 2023 Jun 28; 29: 16-35. doi: 10.1016 / j.bioactmat.2023.06.013). In the field of trauma treatment, preparations developed based on the MSC secretome are very likely to become new therapies to replace MSC therapy and have broad development prospects.

[0004] Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells derived from dental pulp tissue. They have strong growth ability and multidirectional differentiation potential. Compared with mesenchymal stem cells of other origins, they have unique advantages in the regeneration of dental pulp-dentin complex, including non-invasive sampling, rich content, avoidance of ethical issues, and lower immunogenicity. More and more studies have shown that primary extracted human dental pulp stem cells can differentiate into odontoblast-like cells under the action of mineralization induction fluid in vitro, can effectively combine with biomaterials, and can form reparative dentin in vivo (Calabrese EJ, Agathokleous E, Dhawan G, et al. Human dental pulp stem cells and hormesis [J]. Ageing Res Rev, 2022, 73: 101540), thereby promoting the repair of dental pulp damage. Therefore, hDPSCs are an important cell model for studying dental pulp damage and repair. Whether they can specifically and effectively differentiate into odontoblast-like cells (odontogenic differentiation) is the key to the success of dental pulp regeneration.

[0005] Many scholars have conducted careful studies on the components of hDPSCs supernatant, and the results show that hDPSCs can secrete IL-6, IL-8, IL-4, IL-10, transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor (TNF-α), etc. These factors are very important in angiogenesis, anti-inflammatory processes, and immune regulation (Bar JK, Lis-NawaraA, Grelewski P G. Dental Pulp Stem Cell-Derived Secretome and Its Regenerative Potential [J]. Int J Mol Sci, 2021, 22 (21).). It can be seen that the composition of hDPSCs-CM is complex, and the components secreted by cells have various biological activities under different cell physiological states and the environment in which the cells are located. At present, the research on hDPSCs supernatant is still in a relatively early stage and further research is needed. Summary of the invention

[0006] The object of the present invention is to provide a secretory peptide derived from the supernatant of human dental pulp stem cells, a secretory peptide P1DC and applications thereof, so as to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a secretory peptide derived from the supernatant of dental pulp stem cells. The amino acid sequence of the secretory peptide is shown in SEQ ID NO.1, SEQ ID NO.1ASGVAVSDGVIKVF.

[0009] The present invention also provides a method for preparing secretory peptides derived from the supernatant of dental pulp stem cells, comprising the following steps: isolating human dental pulp tissue primary cells from in vitro dental pulp tissue, identifying dental pulp stem cells on cells extracted from the dental pulp tissue primary cells, and confirming them as dental pulp stem cells; culturing the dental pulp stem cells to 80%, changing to a basic DMEM culture medium without serum and any growth factors, and collecting the cell supernatant after 48 hours; culturing the cells to 80%, mineralizing for 14 days, changing to a basic DMEM culture medium without serum and any growth factors, and collecting the cell supernatant after 48 hours; centrifuging the collected cell supernatants before and after differentiation at 1000 rpm for 10 minutes at 4°C to remove cell debris; filtering through a 0.22 μm pore size sterile filter, centrifuging at 5000 rpm for 40 minutes at 4°C, and storing at -80°C. Finally, the secreted peptides in the culture supernatant of hDPSCs before and after differentiation were detected by LC-MS / MS. The mass spectrometry results showed that a total of 1,349 peptides were identified, and 90 differential peptides were finally screened, including 66 up-regulated peptides and 24 down-regulated peptides. Bioinformatics was used to analyze the physicochemical properties of the secreted peptides, and combined with cell experiments, peptides with obvious differences and stable physicochemical properties were screened. The polypeptide was the secreted original peptide shown in SEQ ID NO.1, SEQ ID NO.1ASGVAVSDGVIKVF.

[0010] The present invention also provides a secretory peptide P1DC, which is formed by adding a cell-penetrating peptide to the N-terminus of the amino acid sequence shown in SEQ ID NO.1 and performing amidation modification on the C-terminus. The sequence of the cell-penetrating peptide is shown in SEQ ID NO.2, SEQ ID NO.2GRKKRRQRRRPPQQ.

[0011] Furthermore, the secretory peptide P1DC has an amino acid sequence as shown in SEQ ID NO.3, SEQ ID NO.3GRKKRRQRRRPPQQASGVAVSDGVIKVF.

[0012] The present invention also provides a use of the secretory peptide P1DC as described above in the preparation of a drug for promoting the proliferation activity and / or differentiation of human dental pulp stem cells.

[0013] The present invention also provides a use of the secretory peptide P1DC as described above in the preparation of a medicine for treating dental pulp diseases.

[0014] Furthermore, the effective concentration of the secretory peptide P1DC is 2.5-10 uM.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The secretory peptide shown in the sequence SEQ ID NO.1 disclosed in the present invention is obtained by separating the cells of dental pulp tissue from the primary culture, collecting the culture fluid of hDPSCs before and after differentiation, sending it to mass spectrometry after 30kD ultrafiltration, and screening the peptides obtained from the mass spectrometry data. Combined with bioinformatics analysis and cytological experiments for functional verification, the secretory peptide P1DC that can promote cell growth is obtained, which is derived from the precursor protein CFL1. Bioinformatics analysis and experimental results show that: ① P1DC is derived from CFL1 protein, and this region also exists on CFL1 proteins such as mice, rats and cattle, so P1DC is highly conserved in mammals. ② Protparam online analysis found that the original peptide of P1DC has an instability coefficient of -2.84, which belongs to a stable polypeptide, a fat coefficient of 125, and an average hydrophilicity of 1.279, which is hydrophobic and lipophilic; ③ In the process of cell culture, treatment with P1DC can significantly promote the proliferation of hDPSCs and regulate the odontogenic differentiation ability of hDPSCs. Therefore, P1DC can be used in the preparation of drugs to promote the repair of dental pulp damage and can be used to treat caries and dental pulp and periapical diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the steps for extracting the secretory peptide of the present invention;

[0018] Figure 2 The culture identification pictures of primary extracted hDPSCs, where a is cells crawling out of the tissue block (×40), b is rapid proliferation, and after the third generation, the cells are uniformly long spindle-shaped (×100), c is mineral nodules (×100, Alizarin red staining), and d is lipid droplet formation (×100, Oil Red O staining);

[0019] Figure 3 is the screening result of peptides;

[0020] Figure 4 To screen the optimal concentration of secreted peptide P1DC to promote the proliferation of hDPSCs;

[0021] Figure 5 This is the Edu experiment to analyze the effect of secreted peptide P1DC on the proliferation activity of hDPSCs, where a, b, and c are control groups, and d, e, and f are P1DC groups;

[0022] Figure 6 The effect of secreted peptide P1DC on ALP activity, where a is the control group; b is the P1DC group;

[0023] Figure 7 The effect of secreted peptide P1DC on alizarin red nodules, where a is the control group; b is the P1DC group;

[0024] Figure 8Q-PCR was used to detect the effect of secreted peptide P1DC on odontogenesis-related genes during the differentiation process of hDPSCs. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] Primary extraction and identification of hDPSCs and preparation of hDPSC-CM before and after differentiation

[0028] hDPSCs were isolated and cultured from the dental pulp tissue of patients with tooth extraction, and the hDPSCs were induced to differentiate into osteoblasts and adipocytes, and the multidirectional differentiation ability of hDPSCs was tested for verification. Figure 2 As shown, the primary cells extracted are small, most of them are spindle-shaped, and they are basically full after about one week of primary culture ( Figure 2 (a, b) After 21 days of mineralization induction, the cells showed obvious mineral nodules stained with alizarin red ( Figure 2 Middle c). Cells cultured in adipogenic induction medium for 21 days were stained with Oil Red O. Under a microscope, small red, shiny lipid droplets were observed between cells ( Figure 2 (d) This result is consistent with the characteristics of mesenchymal stem cells reported in the literature.

[0029] After the cells were cultured to 80% confluence, they were replaced with basal DMEM medium without serum and any growth factors, and the cell supernatant was collected after 48 hours. After the cells were cultured to 80% confluence and mineralized for 14 days, they were replaced with basal DMEM medium without serum and any growth factors, and the cell supernatant was collected after 48 hours. The collected cell supernatants before and after differentiation were centrifuged at 1000 rpm for 10 minutes at 4°C to remove cell debris. The hDPSCs-CM before and after differentiation were prepared by filtration with a 0.22 μm pore size sterile filter and centrifuged at 5000 rpm for 40 minutes at 4°C, and stored at -80°C.

[0030] Example 2

[0031] Peptideomics analysis by LC-MS / MS and characterization by bioinformatics

[0032] The hDPSCs-CM samples prepared in Example 1 before and after differentiation were stored in dry ice and transported to BGI for LC-MS / MS analysis. The results showed that a total of 1349 secretory peptides were detected in the supernatant of hDPSCs before and after differentiation. The ratio of the average concentration of the two groups of peptides was fold change. If the differentiated hDPSCs-CM group was greater than the undifferentiated hDPSCs-CM group (fold change ≥ 1.5 and p-value < 0.05), and only the peptides identified in the differentiated hDPSCs-CM group were defined as the differentially expressed peptide up-regulated group; on the contrary, it was defined as the differentially expressed peptide down-regulated group. A total of 90 differentially expressed peptides were finally screened, including 66 up-regulated peptides and 24 down-regulated peptides.

[0033] Based on the differentially expressed peptides, we conducted an in-depth bioinformatics analysis of their precursor proteins, and then used the UniProt database to explore the location of the differentially expressed peptides in the precursor proteins and the functions of the precursor proteins, and screened out 4 secretory peptides that may be closely related to the proliferation and differentiation of hDPSCs. In order to further optimize the molecular performance of these 4 polypeptides and enable them to better enter the cells to function, we added a transmembrane peptide fragment to its N-terminus, and while maintaining the acetylation modification of the N-terminus, we amidated its C-terminus. We commissioned Shanghai Kepeptide Biotechnology Co., Ltd. to synthesize these 8 polypeptides using the solid phase method, and the above synthesized polypeptides were dissolved in ultrapure water to 100μM and stored at -80℃ for later use.

[0034] Example 3

[0035] Peptide screening results

[0036] We tested the effects of 8 peptides on the proliferation of hDPSCs by CCK8 to further study the functions of differentially expressed peptides. Cells were treated with different concentrations of peptides (0, 1, 10uM), and 5 replicate wells were set for each concentration gradient. After 24h, 10μL CCK-8 reaction solution was added to each well and incubated at 37℃ for 1 hour. The absorbance was detected by microplate reader and the relative proliferation activity of cells was calculated.

[0037] The results are as follows Figure 3 It was shown that Peptide-1 (Ac-GRKKRRQRRRPPQQASGVAVSDGVIKVF-NH2), derived from the precursor protein CFL1 and named P1DC (peptide 1 derived from CFL1 protein), can significantly increase the proliferation of hDPSCs.

[0038] Example 4

[0039] Synthesis of secretory peptide P1DC and analysis of biological parameters

[0040] The original peptide sequence of the polypeptide P1DC is ASGVAVSDGVIKVF (SEQ ID NO.1), which is a polypeptide molecule composed of 14 amino acids. The main biological parameters of the polypeptide obtained by the online polypeptide analysis tool are as follows: the isoelectric point (Theoretical pI) is 5.88, and the molecular weight (Molecular weight, Mw) is 1348.56. In order to further optimize the performance of the polypeptide molecule and enable it to enter the cell better to function, we added a transmembrane peptide fragment to its N-terminus: GRKKRRQRRRPPQQ (SEQ ID NO.2), and the polypeptide sequence was modified to: GRKKRRQRRRPPQQASGVAVSDGVIKVF (SEQ ID NO.3), named P1DC, containing 28 amino acids, while maintaining the acetylation modification of the N-terminus, the C-terminus was amidated.

[0041] Example 5

[0042] Effect of secreted peptide P1DC on the proliferation of hDPSCs

[0043] 1) Screening of the optimal concentration of secreted peptide P1DC to promote hDPSCs proliferation

[0044] In order to explore the effect of P1DC on the proliferation of hDPSCs, this study first used CCK-8 to determine the optimal concentration of P1DC to promote the proliferation of hDPSCs. The results showed that the optimal peptide concentration for in vitro culture of hDPSCs was 2.5-10uM. Figure 4 )

[0045] 2) Edu experiment to analyze the effect of secreted peptide P1DC on the proliferation activity of hDPSCs

[0046] In the EdU experiment, the nuclei of all cells are stained with Hoechst dye to show blue fluorescence, and the nuclei of cells in the DNA replication phase can be stained with EdU to show red fluorescence. By calculating the proportion of EdU positive cells, it can be seen that the EdU positive rate of P1DC group cells is higher than that of the control group cells. ( Figure 5 )

[0047] Example 6

[0048] Effect of secreted peptide P1DC on odontogenic differentiation of hDPSCs

[0049] hDPSCs with added peptides were given mineralization induction conditions, and the expression of the following indicators was detected on the 0th and 7th days: ① ALP kit was used to detect ALP activity and ALP staining; ② Alizarin red staining was used to evaluate the formation of mineralized nodules; ③ qPCR was used to detect differentiation markers such as dentin sialophosphoprotein gene (DSPP), dentin matrix protein 1 (DMP1), and osteocalcin (OCN) at the gene level. The effect of P1DC on the odontogenic differentiation of hDPSCs was evaluated by detecting the above indicators. The results are shown in Figure 6 , 7 , 8 showed that P1DC could significantly promote the differentiation of hDPSCs.

[0050] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. 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 all fall within the protection scope of the claims of the present invention.

Claims

1. A secretory peptide derived from the supernatant of dental pulp stem cells, characterized in that: The amino acid sequence of the secretory propeptide is shown in SEQ ID NO.

1.

2. A method for preparing secretory peptides derived from the supernatant of dental pulp stem cells, characterized in that: The following steps are involved: Human dental pulp tissue primary cells were isolated from dental pulp tissue in vitro, and dental pulp stem cells were identified from the cells extracted from the dental pulp tissue primary cells. After confirmation as dental pulp stem cells, the dental pulp stem cells were cultured to 80%, and then replaced with a basic DMEM medium without serum and any growth factors, and the cell supernatant was collected after 48 hours. After the cells were cultured to 80%, and after mineralization for 14 days, the basic DMEM medium without serum and any growth factors was replaced, and the cell supernatant was collected after 48 hours. The collected cell supernatants before and after differentiation were centrifuged at 1000 rpm at 4°C for 10 minutes to remove cell debris. Filtered with a 0.22 μm pore size sterile filter, centrifuged at 5000 rpm for 40 min at 4°C, and stored at -80°C; finally, a polypeptide with obvious differences and stable physicochemical properties was screened out, and the polypeptide was the secretory original peptide shown in SEQ ID NO.

1.

3. A secretory peptide P1DC, characterized in that The secretory peptide P1DC is formed by adding a transmembrane peptide to the N-terminus of the secretory original peptide amino acid sequence as shown in SEQ ID NO.1 and performing amidation modification on the C-terminus.

4. A secretory peptide P1DC according to claim 3, characterized in that: The sequence of the contained cell-penetrating peptide is shown as SEQ ID NO.

2.

5. A secretory peptide P1DC according to claim 3, characterized in that: The sequence of the secretory peptide P1DC is shown as SEQ ID NO.

3.

6. Use of the secretory peptide P1DC according to any one of claims 3 to 5 in the preparation of a drug for promoting the proliferation activity and / or differentiation of human dental pulp stem cells.

7. Use of a secretory peptide P1DC according to claim 6 in the preparation of a drug for promoting the proliferation activity and / or differentiation of human dental pulp stem cells, characterized in that: The effective concentration of the secretory peptide P1DC is 2.5-10 uM.

8. Use of the secretory peptide P1DC according to any one of claims 3 to 5 in the preparation of a drug for treating dental pulp diseases.

9. Use of the secretory peptide P1DC according to claim 8 in the preparation of a drug for treating dental pulp diseases, characterized in that: The effective concentration of the secretory peptide P1DC is 2.5-10 uM.

Images